On the Moral and Legal Status of Abortion Essay

On the Moral and Legal Status of Abortion - Essay Example Methods of induction have ranged from utilizing sharpened tools, herbal medicines and physical trauma. Opinions, both culturally and legally differ worldwide, with public debate over legal ramifications and ethics of abortion being a very emotive issue. Abortion and the debate ranging around it have birthed activism, debates and controversy in equal measure. It is in fact a norm for people to refer to themselves as pro-life or pro-choice. Personal beliefs touching on responsibility, morality, government role in public policy and religion all affect ones view on abortion. Mary-Anne Warren, an American philosopher, put forward in her article â€Å"On the Moral and Legal Status of Abortion†, the perspective that abortion is acceptable morally. This paper seeks to delve into her article, especially her arguments and form an objection to her work. Warren begins her argument by debating how permissibility of abortion morally is dependent on whether the subject fetus is indeed a pers on. She argues that while a satisfactory defense of the right of a woman has to an abortion without proof of a fetus not being a human being is not satisfactorily possible, this difficulty in conclusion of a fetus’s status should not make it impossibility in the production of a solution to the moral status of the abortion problem (Warren 2). Pro-abortionists, due to not coming to grips with issues surrounding abortion, have had most of their arguments fall flat, failing to weaken traditional arguments on antiabortion. Their arguments are of two sorts, they either state that denial of a woman’s right to abortion is a deprivation of her rights to have control over her own body, or that deaths induced by illegal abortions, especially by poor women, is as a consequence of this law. This is obviously flawed since the fact that access restriction to abortion has such tragic side effects is not a pointer to the unjustified restrictions, as murder is still wrong. She cites J. Noonan saying, â€Å"The fundamental question in the long history of abortion is; how you determine the humanity of a human being?† (Warren 1). She goes on to argue that once the assumption of a fetus’ moral rights is allowed in full, the question of whether abortion is justifiable becomes a difficult and complex question. Warren seeks to push her abortion agenda via discussion of the five characteristics she believes are central to being a person. Warren tends to view persons as entities who have consciousness, self motivation, reasoning, communication capacity and presence of self awareness and self concepts (Warren 2). The first issue she tackles is the Definition of a human. She argues that â€Å"human† has two meanings, distinct but not easily distinguishable. Since killing of innocent humans is wrong and fetuses are an example of humans that are innocent, killing them is wrong. She shoots down this argument by claiming that the usage of human in similar se nse in both conclusions, whichever use is meant of the two, one of them begs questions. If used in two senses that are different, then the conclusion is still wrong (Warren 5). She claims that the presence of the human genome in fetuses does not point to it being morally human. Her second characteristic deals with the definition of moral community. She asks if it is indeed possible to establish if moral humanity can be sufficiently defined by genetic humanity. She describes the moral community as all people and only people, rather than all human beings and only human beings. Her argument continues to debate what characteristics enable any entity qualify for consideration as a person. She puts these characteristics down as consciousness and capacity to detect pain, acting out of self motivation,

Advances in DNA Sequencing Technologies

Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t Advances in DNA Sequencing Technologies Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t

The Art of Manipulation in Homers Odyssey Essay -- Homer Odyssey

The Art of Manipulation in Homer's Odyssey    They sit, entranced in the magic of his words. He pauses. On the edge of their seats, they await in silence his next utterance. The one spoken of is not a bard or man refined in the art of song, but rather a warrior scarred and hardened through intense conflict. He has a special mastery of the spoken language that enraptures his audience and a gift that endows him to command and persuade them without physical force. This man is a manipulator of words, a subtle combatant. The proverbial "He" represents Odysseus in Homer's epic adventure The Odyssey. Youthful Athenian men gained wisdom and admonitions about the machination of words by studying Odysseus's shrewd intellect, and in contrast the use of persuasion by Eurylochus whose ignorance brought about the demise of their comrades. The art of manipulation is vital to the survival and prosperity of men and women throughout The Odyssey. Odysseus exemplifies this distinctive quality, learning through his adventures how to better meet his needs through cleverly chosen words rather than vehement combat. Odysseus first reveals this gift of the gods, when he used trivial flattery and an appearance of humble supplication in approaching the Princess Nausicaa on the isle of the Phaecians. "At [her] knees," he comes before Princess Nausicaa cleverly appealing to her with questioning disbelief of whether she was "some goddess or a mortal woman." He then proceeds to draw upon her desire to wed with words that left questioning his own marital status, and sounded as though he were envious of the "most blessed among [the Phaecians] who with his wedding gifts would win [her]," the awe inspiring Nausicaa (89). At this moment in his life, Odysseu... ...e, the ability to manipulate words for the means of persuasion do not always have positive results. In the hands of the ignorant and irrational, persuasion becomes an evil that plagues all those who come in contact with and conform to it, but when used by the knowledgeable and thoughtful, manipulation can provide for the betterment of a society, such as the peace that ensues Odysseus's vengeance when Athena persuades them to stop the futility. Homer teaches young Athenians to be aware of the dangers of manipulation, rhetoric, and persuasion, but he also shows that a man who can do such effectively is deemed a leader, and that those who cannot are mere followers. Works Cited and Consulted Crane, Gregory. Calypso: Backgrounds and Conventions of the Odyssey,   Frankfurt, Athenaeum 1988 Homer. The Odyssey. trans. Robert Fagles. Penguin Books. New York. 1996.

Sentencing

Thinking about the issue of punishment gives rise to a number of questions, the most fundamental of which is, why should offenders be punished? And what are the objectives for the punishment. Some of the objectives are deterrence, retribution, restitution, rehabilitation, and the reason for such punishment. Deterrence is most effective at stopping crime that is planned or premeditated. Sometimes the goal is to deter the individual from repeating the behavior; other times it is to deter others from engaging in a similar behavior. An â€Å"eye for an eye, tooth for a tooth† punishment applied with the belief that offenders should suffer similarly to their victims this is the retribution punishment. Restitution is applied with the belief that offenders should repay their victim’s loss in money or services. The offenders should pay back to the victim for crimes that he has made to change a person life. He has to see that he cannot get away with committing crimes. Rehabilitation is used more frequently with juveniles; it is applied with the hopes of helping the person resolve his disorder or disease that may contribute to crime. The punishment is their so that the person can choose more of a better life in which he decides to live, or he may choose a better path. The concept of punishment has been theorized by moral philosophers, social theorists, and criminologists, When a court imposes a punishment on an offender, it often tries to balance the sorts of reasons for punishment noted earlier, but sometimes certain purposes of punishment dominate other purposes The third perspective on punishment is offered by criminologists and policy makers, who focus on penalties for offenses and policy concerns relevant to the punishment of offenders. There are differences in the state and federal punishments laws of punishment such as with the federal laws the penalties and range from long or short prison sentences in federal prison to include fines. Federal laws are enforced by the United States Government Agencies and also passed by the United States Government Agencies. There are criminal laws involved, usually dealing with crimes against the government and laws that just provide fines. State laws are those that are passed and enforced by the state. They cannot contradict the Federal laws and apply only to the specific state. The state enforcement agencies also have a duty to insure that Federal laws are not being broken. Most criminal laws are state created and penalties include fines and short or long prison sentences. Probation is a sentence with certain conditions that must be followed. If any of the conditions, such as no drug use, are violated, your probation officer will notify the court or prosecutor. The size and cost of America’s prison system has skyrocketed during the last few decades, largely as a result of laws and policies that put more offenders behind bars and keep them there longer. Yet recidivism rates remain stubbornly high, and crime still is a major public concern. State policy makers across the nation are asking whether soaring prison budgets are the best path to public safety. The federal prison population has reached record levels, that a high proportion of prisoners are non-violent drug offenders, and that racial disparities in sentencing and the proportion of lower-level drug offenders are increasing. Sentencing disparities is sentencing offenders in which those committing the same crime receive different sentences. Sentencing disparities are usually based on race, gender, region, or socioeconomic status and there are some grapple with this problem that must be solve. Many of the studies concluded that race had a direct effect on the in-out decision (in other words, the decision concerning whether the offender should be punished in a penal institution or out in the community) and that this effect remained even after the inclusion of controls for prior record and crime seriousness. Benefits of sentence-reduction programs, such as good-time laws and early parole release, include promotion of discipline within prisons (because inmates are motivated to engage in good behavior in order to earn or avoid losing good time) and the reduction of prison overcrowding. It is said that most offenders are released from prison before serving their full sentences and that indeterminate sentences produce gross sentencing disparities because they allow judges too much discretion.

Understanding Distributed Leadership and Impact on Teaching

Distributed leading has been the topic of much research in the domain of instruction in recent old ages. This research study explores how it is understood in the context of the Irish station primary school that I am presently employed in, with some mention to its impact on instruction and acquisition in the school. Our school is a Dublin south interior metropolis Presentation all-girls Secondary School ( now under the Backing of the late formed trust organic structure CEIST ) with disadvantaged position. There are 28 members of the teaching staff: principal, deputy principal, 7 Assistant Principals ( including a Programme Coordinator station ) , 8 Particular Duties Teachers and 11 instructors with no formal leading place. The Board of Management manages the school on behalf of the Patron and must confer with with and maintain the Patron informed of any determinations, proposals and policy alterations. Staff voluntaries have ever been invited to take part on assorted undertaking groups and subcommittees in our school. These groups were seen as being really of import in the development or alteration of policies or curricular issues and their recommendations were by and large taken on board by both staff and the principal/deputy principal. These groups have had no deficit of voluntary members from both postholders and non-postholders, which would propose a ‘fundamental nucleus of values that all members of the organisation clasp ‘ ( E849 Study Guide, pg. 21 ) and besides highlighted the fact that distributed leading exists within the school. The purpose of this assignment is to research the construct of distributed leading and the influence leading patterns have on instruction and acquisition in my school. The overall purpose of this research is to back up the instruction staff to go more cognizant of their ain leading perceptual experiences and patterns, with mention to the possibilities offered by distributed leading to positively impact on instruction and acquisition within the school. Given the fact that this was a little graduated table survey that had to be conducted in a short clip frame merely one research inquiry was addressed in the research: How make the instructors, chief and deputy chief understand the construct of distributed leading and how does this nexus to instruction and acquisition within the school? The attack taken throughout this research begins with the premise that a instructor ‘s leading function begins in the schoolroom with the influence they have on their students but besides extends beyond the walls of the schoolroom to working collaboratively with co-workers ( learning or accessory ) . A instructor ‘s leading function may widen to their part to the school civilization. Teachers may keep a station of duty ( Adjunct Principal or Special Duties ) or an in agreement place outside the formal station construction, e.g. capable coordinator. The Post of Responsibility system is a construction whereby a figure of instructors are given extra wage to transport out specified undertakings, responsibilities and duties in the school. It is besides called the â€Å" in-school direction † system. There are two classs of station ; Assistant Principal and Particular Duties. The Assistant Chief station carries an extra salary allowance of about a‚ ¬9,000 per annum and the Particular Duties allowance is about a‚ ¬5,000 per annum. Teachers in reception of either of these allowances are required to carry through responsibilities and take duties in add-on to their full instruction hours. The responsibilities attached to the station are defined by the Board of Management following a audience procedure affecting all the staff. The audience procedure includes an analysis of the school demands, understanding on the precedences and the pulling up of a â€Å" Agenda of stations † to fit the in agreement precedences. Each school is allocated a specific figure of Particular Duties and Assistant Principal stations on the footing of school size, harmonizing to a expression based on the figure of whole-time instructors in the school. Appointment to a station of duty is by competitory interview among the instructors already employed in the school, whether full clip or portion clip, lasting or impermanent. Choice standards have been agreed at national degree and include recognition for the figure of old ages experience in that school and â€Å" the most senior suited † ( DES Circular Letter 05/98 www.education.ie ) campaigner. Therefore, in most instances, instructors keeping stations of duty are more likely to be the instructors who have been in the school for the longest figure of old ages. However, other instructors may besides keep no formal place but may be influential with co-workers. Leadership at this degree may hold a important and direct influence on instruction and on the general acquisition environment. Distributed leading has been interpreted in many different ways, but incorporates many of the constructs outlined supra such as instructors as scholars, influence over co-workers and part to school clime and civilization whether or non in formal places of leading.Literature ReviewResearch has shown that leading is one of the most of import factors in doing a school successful ( OECD 2008, Leithwood and Riehl 2003 ) . Where leading is effectual staff and students are better motivated, people know what is traveling on because communications are clear and frequent, and everyone feels they are drawing together and working towards shared ends ( Day, Sammons et al 2007 ) . Distributed leading is one signifier of leading that is outstanding in the current educational discourse. The thought of distributed leading has been in being for about three decennaries. Murgatroyd and Reynolds ( 1984 ) stressed that â€Å" leading can happen at a assortment of degrees in response to a assortment of state of affairss and is non needfully tied to ownership of a formal organizational function † ( cited in Law and Glover 2003 p.37 ) . This construct incorporates thoughts such as instructors working together in squads and instructors taking a assortment of duties within the school. On the positive side, it was considered good to learning and larning within schools if instructors discussed their pattern with co-workers, gave and accepted reviews of their work and were unfastened to larning from each other. Another position broadened the range of their leading to decision-making in the overall operation of the school. Hallinger and Heck ( 1996 ) found small grounds associating distributed leading to improved pupil results. Weiss and Cambone ( 1994 ) found that instructors ‘ engagement in whole-school alteration could take away from schoolroom instruction. On the other manus, Greenleaf ( 1996 ) found it led to positive effects on instructor efficaciousness and degrees of morale within schools. Spillane, Halverson and Diamond ( 2001 ) position distributed leading as being cardinal to the instruction and larning procedure in the school and agree that leading involves all members of the school community, non merely the principal and deputy principal. They argue that leading happens in a assortment of ways throughout the school and is centred in the interactions between people. â€Å" Depending on the peculiar leading undertaking, school leaders ‘ cognition and expertness may be best explored at the group or corporate degree instead than at the single leaders degree † ( Spillane, Halverson and Diamond 2001, p.25 ) â€Å" Peoples in officially designated places and those without any such appellations can and make take duty for taking and pull offing in the schoolhouse † ( Spillane and Diamond 2007 p.7 ) . Therefore, this distributed leading position recognises that leading functions are played by different people at different times. Distributed leading ( Gronn, 2000 ) ‘sees leading as a map which is widely dispersed through the administration instead than as a duty vested in an person ‘ ( Study Guide, pg.21 ) . The station of duty construction in Irish schools allows for some of the leading maps to be distributed throughout the designated station holders, though this still leaves the inquiry about how to affect all non-post holders. Distributed leading ‘assumes that there is an underlying values consensus that enables staff to work harmoniously towards shared intents and to hold on the bases by which the effectivity of their organisation is judged ‘ ( Study Guide, pg.21 ) . This, therefore, would look to presume a greater engagement by all staff in the determination procedure of the administration. One of the features of distributed leading is â€Å" an emergent belongings of a group or web of interacting persons † ( Woods et al 2004, p.441 ) . Gronn footings this pooling of energies ‘concertive action ‘ and suggests that it is about the extra moral force which is the merchandise of conjoint activity – where people work together in such a manner that they pool their enterprise and expertness, the result is a merchandise or energy which is greater than the amount of their single actions ( Gronn 2000 ) . This is comparable to Spillane ‘s definition of distributed leading as â€Å" the collective belongingss of the group of leaders working together to ordain a peculiar undertaking, taking to the development of a leading pattern that is potentially more than the amount of each person ‘s pattern † ( Spillane et al 2001 p.25 ) . Theories on teamwork portion the position that working together produces consequences over and above what would be expected from persons working entirely. The literature on teamwork frequently makes the differentiation between formal and informal squads but suggests that both types operate best in a civilization that fosters an unfastened clime and where relationships are based on trust, common protection and support ( Belbin 2000, Nias et Al 1989 ) . There can be given to be some tensenesss between ‘designated leaders and distributed leading ‘ ( E849 Study Guide, pg.146 ) . School Principals are accountable for school public presentation, supported by deputy principal and designated station holders. ‘On the other manus, much of the productive work of educational organisations takes topographic point in collaborative squads, characterized by professional norms and distributed leading, where those with relevant expertness take the lead, irrespective of formal functions ‘ ( E849 Study Guide, pg.146 ) . However, ‘the construct of distributed leading still assumes that persons will follow that lead when it is provided ‘ ( E849 Study Guide, pg. 21 ) . Teamwork is a cardinal component of distributed leading in that the nature and intent of distributed leading is â€Å" the ability of those within a school to work together, building significance and cognition jointly and collaboratively † ( Lambert 1998 p.5 ) . However, the being of structured squads entirely does non represent distributed leading. In fact, distributed leading patterns may non underscore the formal structured attack to teamwork but instead acknowledge that groups of instructors work together as appropriate in order to accomplish a peculiar aim at a given clip. Another typical feature of distributed leading ( Woods et al 2004 ) , is that the distribution of leading varies harmonizing to expertness. There is acknowledgment that assorted undertakings require different expertness and that all the expertness does non shack in one individual at the top. Schools presents are complex administrations and therefore it is excessively much to anticipate that they can be led by one individual. â€Å" The function of chief is now so complex and demanding, that it is unrealistic to believe that any one individual can dispatch the function without the aid of considerable figure of co-workers, both from the instruction and the support staff † ( Martin 2006 ) . This is peculiarly important in the context of leading for improved acquisition as it is recognised in the literature that the most important influence on pupil acquisition is the direct influence the instructor has in the schoolroom. The construct of trust emerges from the literature as being important ( Duignan 2006 ) . Teachers need to experience sure and supported by their principals and their co-workers. Trust is necessary if instructors are to experience motivated in their work and if they are to be allowed to originate an activity and take duty for decisionmaking. Along with being trusted in their work, people besides need support. Peoples want to speak about what they are making – back uping these conversations is an indispensable undertaking of the leader ( Wheatley 1999 ) . Trust, allied with support, is an underpinning value within the construct of distributed leading. Harris ( 2004 ) recognises that structural and cultural barriers operate within schools which could do it really hard for some instructors to demo leading. Cheating for power places in a school can make a clime which is non contributing to, for illustration, immature instructors showing their sentiment, particularly if it differs from the traditional or prevalent sentiment. Such action could be perceived as a menace to the position quo. Another construct that links distributed leading with acquisition is that of professional larning communities. Professional larning communities may be viewed as an extension of teacher leading. For illustration, Harris et Al ( 2003 p.79 ) identifies four dimensions of the teacher leading function that extends to the overall operation of the school. Teacher leaders: – 1. translate the rules of school betterment into the patterns of single schoolrooms ( a brokering function ) ; 2. aid other instructors to cling around a peculiar development and further a more collaborative manner of working ; 3. drama a mediating function in school betterment. They are an of import beginning of expertness and information ; 4. forge close relationships with single instructors where common acquisition takes topographic point. Schools with professional acquisition communitiess study important benefits for pupils, including lower rates of absenteeism and decreased dropout rates. pupils have besides exhibited academic additions in maths, scientific discipline, history and reading than in traditional schools. ( Hirsh and Hord 2008 p.27 ) . The direct nexus between leading and pupil results â€Å" is a rare event so in the research literature on educational leading and school betterment † ( Mulford, Silins and Leithwood 2003 p.3 ) However, Mulford et Al ‘s research found that what was of import was that staff are actively and jointly take parting in the school and experience that their parts are valued. This contributes to making a acquisition administration where instructors ‘ acquisition, every bit good as pupil acquisition, is valued ( p.6 ) .MethodologyDavies and Ellison ( 1999 ) argue that a assortment of data-gathering techniques should be used to develop a balanced position of the administration ‘s strategic place. For this ground, my chief research methodological analysiss involved the usage of a elaborate questionnaire distributed to all learning staff every bit good as a follow up focal point group meeting. These methods of probe have designed with the intent of better functioning the aims of the research. Mellon ( 1990, pg.49 ) states that the two chief inquiries to be addressed were: â€Å" who might hold the information you need and who is accessible † ? As highlighted by Patton ( 1990, pg.45 ) , â€Å" where the focal point is on persons, an inductive attack begins with the single experiences of those persons † . This multi-method attack allowed for triangulation, utilizing different methods of informati ons aggregation within the survey to guarantee that it is as full and balanced as is possible within the comparatively short clip graduated table. A mixed-method attack was decided on, through which a questionnaire would place relevant issues on distributed leading in order that these issues could be examined in more item in focal point groups. Strauss and Corbin ( 1998 ) highlight the function of literature reappraisal as a valuable beginning of experience that leaves the research worker with: better apprehension of the information needs on the field, aware of the spreads left by old surveies, and sensitive to the issues he/she might place in the information. Literature could be a secondary beginning of informations, and assist the research worker to explicate inquiries to be used in interviews and questionnaires, during the initial stairss of the research. It can besides corroborate findings, comparing the research consequences to past grounds. This will be really of import in this survey. My trust with all instructors involved is really of import to develop. ‘ As Bassey ( 1999 ) points out, research workers, in taking informations from people, should make so in a manner that recognizes those people ‘s initial ownership of the informations and that respects them as fellow human existences who are entitled to self-respect and privateness ‘ ( Study Guide, pg.55 ) . As worlds were evidently be the most of import constituent of this research, the issue of informed consent had to be addressed. Therefore, it was necessary to inform all interview participants about the survey, their function within the probe, and how the information they provided would be used. While, as argued by Miles and Huberman ( 1994, pg 291 ) it may be that genuinely informed consent is impossible in qualitative research the issue could non be dismissed, and, consequently all participants selected for the survey were informed of both the nature and intent of the research. They were besides given the chance to make up one's mind whether to take part in the study or to retreat at anytime. The chief rules of research moralss are: The individuality of participants ‘ must be protected in order that the published consequences of the survey do non mortify or harm them in any manner. Anonymity must hence be extended to all records, written or electronically recorded, that are collected during the survey. All participants must be treated with regard and informed of the research worker ‘s involvements. The participant must hold to take part in the survey. The research worker must non lie to the participants or record conversations on concealed mechanical devices. The research worker must do clear the footings of the research and abide by the footings of the understanding. The findings must be based on the informations and truthfully reported. ( Bogdan and Biklen, 1992 ) These form the chief push of my ethical considerations when carry oning the survey. For this research, a questionnaire was designed to guage instructors ‘ perceptual experiences of distributed leading and the patterns in the school that contribute to it. The findings were used to organize the footing for farther probe through a focal point group treatment. The questionnaire was chiefly an attitudinal one and hence used the Likert graduated table which places people ‘s replies on an attitude continuum ( May 2001 p.104 ) . In add-on, three unfastened inquiries were included. This allowed participants greater freedom in their replies. Responses to the questionnaires were used to clear up the research inquiries and place more specific issues which would be discussed within the focal point group. Focus groups have been defined as a group of persons selected and assembled by research workers to discourse and notice on, from personal experience, the subject that is the topic of the research. ( Powell et al 1996 p.499 ) They can be used to determine attitudes, feelings, cognition, perceptual experiences, thoughts and beliefs of participants, from the participants ‘ personal experience. As the literature suggests ( Gibbs 1997 and McNamara 2006 ) , the research worker plays an of import function which includes supplying a clear intent, assisting people to experience at easiness and easing interaction between group members. This was made slightly more hard, given that the research was conducted in my ain school. In hindsight, it may hold been more appropriate to carry on the probe in a neighbouring school. Questionnaires were distributed to all instructors in the school. The questionnaire was designed on the footing of findings from the literature on distributed leading. Part 1 of the questionnaire contained 15 statements refering to leading and participants were asked to rate their understanding or otherwise with these statements utilizing the Likert graduated table. Respondents ‘ tonss of 4 or 5 denote understanding or strong understanding. Tonss of 2 or 1 denote dissension or strong dissension severally and a evaluation of 3 is considered ‘neutral ‘ . Part 2 consisted of 15 statements refering to leading patterns in schools. In this subdivision, participants were asked to hit each point on the footing of how far along a spectrum their school was in implementing this pattern. A mark of 4 or 5 denotes a well-established pattern and a pattern which is being refined, severally. Tonss of 2 or 1 denote that the pattern is get downing or does non go on in the school severally. A evaluation of 3 denotes that advancement is being made in this pattern. By inquiring respondents to bespeak their grades of understanding with these statements, the research worker can determine the respondents ‘ perceptual experiences of distributed leading and place the key issues which they highlight as being cardinal to the pattern of distributed leading and its connexions to learning and larning. These can be analysed from a normative position, based on the literature findings. The questionnaire included three unfastened inquiries ; the first elicits farther penetrations into respondents ‘ apprehension of distributed leading, the 2nd asks them to place factors that support their work and the 3rd seeks to place factors that inhibit their work. Following treatment at a staff meeting, where I explained the intent of the research, staff agreed to take part and questionnaires were distributed to the 28 instructors on staff. A sum of 16 questionnaires were returned ( 57 % response rate ) . In order to reply the research inquiries it was necessary to garner farther informations from a focal point group comprised of instructors that have experience of different degrees of leading. Therefore, the group comprised of the principal and deputy principal, 3 instructors who hold stations of duty and 3 instructors who do non keep stations of duty. The focal point group was about one hr continuance.FindingssResponses to the first set of statements on the questionnaire identified a figure of issues perceived by the respondents to be associated with distributed leading. It was clear from the questionnaire responses that all instructors perceived themselves to be leaders. However, the context of that leading was in the schoolroom – they see themselves as leaders of their pupils within the schoolroom, holding a direct influence on their acquisition. Their position of themselves as leaders with influence beyond the schoolroom was instead limited. The questionnaire besides revealed that instructors believe that learning and acquisition is influenced positively when instructors work together and when they engage in professional development to better their cognition and accomplishments. Besides, instructors saw distributed leading as including their engagement in decision-making and in taking new enterprises in the school. However, they besides acknowledge the cardinal function the principal dramas, for illustration in guaranting that there is a shared vision among staff and that pastoral attention systems operate efficaciously for pupils. In response to the 2nd set of statements on the questionnaire, respondents highlighted a figure of leading patterns that are operational in the schools to a greater or lesser extent. These patterns were identified as: Monitoring and back uping pupil acquisition Working together as a staff Structures and systems such as capable sections and stations of duty Monitoring and back uping pupil acquisition included holding systems to back up pupil larning analyzing consequences of scrutinies and utilizing the information to reappraisal patterns all instructors playing a function in supervising pupil public presentation and four ) all school policies being designed with a focal point on heightening, bettering and developing a high quality larning environment. These points were all portion of a late completed DEIS program in the school. Working together as a staff incolved: discoursing school development precedences at staff meetings, professional development on whole-school issues, jointly prioritizing specific actions to better acquisition. Capable sections were seen as organizing a cardinal portion of distributed leading. However, in the school they are considered to be a forum for sharing resources and are merely now being used for capable planning. The responses from the unfastened inquiry on distributed leading emphasised community and coaction instead than hierarchy. The 2nd unfastened inquiry on the questionnaires asked instructors to call the factors that back up them in their work. The cardinal issue emerging was the demand for support and aid from both co-workers and direction, peculiarly in covering with student behavioural or disciplinary issues. They besides referred to the accessibility, handiness and openness of the principal as being an of import factor in enabling them to make their occupation good. Other factors stated were encouragement from direction, being trusted and treated as a professional, good administration and planning and being allowed to seek out new thoughts without intervention. The positions of distributed leading expressed by respondents in the questionnaires were reinforced in the focal point group treatment, peculiarly by the post-holders. However, different positions on the nature of decision-making were expressed by the post-holders ‘ in the focal point group ; foremost, if leading is distributed so that should intend doing determinations together but on the other manus, â€Å" sometimes it is of import for a principal to do a determination. There might be a determination that the squad ca n't hold on and it is a atrocious determination and the principal has to do the determination. † The participants in the focal point group agreed that it was of import that everybody has a voice. The thought of holding a voice was extended farther by a non post-holder, who stated that â€Å" if, at a staff meeting, people are listened to, so you are traveling to acquire the message that this is a good topographic point to portion enterprise and portion thoughts. † Participants besides agreed that instructors are function theoretical accounts for the pupils and that their behavior and interactions with co-workers, every bit good as with pupils, have a major influence on pupils. ‘We ‘re function theoretical accounts for pupils in what we do, in how we interact and speak with each other ‘ . The focal point group treatment allowed for a grade of interaction, dissension and argument about issues and constructs that was non possible in reacting to a questionnaire. Leadership and direction were debated. While there was a general consensus that leading involved everybody in the school, there was some argument about the function of postholders. The consensus among the group was that all instructors, non merely post-holders, can be empowered to take. The principal saw distributed leading as widening beyond schoolroom leading to whole-school issues. ‘Now about every member of staff will either hold authorization delegated to them for a peculiar country or will take it on their ain back to organize something. Whether you are a coach or whatever it is, there is much more involvement in school life now than there was in the yesteryear ‘ . ‘I would see leading as leading wherever it expresses itself throughout the school, whether it is in direction or whether it is running the school musical or whatever it might be that it is the capacity of the individual to convey people with you to accomplish a peculiar undertaking ‘ . Concepts mentioned by both the principal and deputy principal included authorization, giving independency, engagement in decision-making, recognizing expertness, taking by illustration, deputation and making an environment where people are non afraid to take hazards and are encouraged to take enterprise. There is strong overlap between the positions expressed by the principal and deputy chief and those expressed by both post-holders and non post-holders. There was really strong understanding among all participants in the focal point group that leading is a construct that can use to all instructors, whether they hold a place or station of duty or non. The participants all agreed that distributed leading is about authorising people, leting them to take enterprise and be involved in decision-making. They besides agreed that it is about the ambiance in the school that encourages instructors to take leading functions in specific facets of the school, e.g. extra-curricular activities and particular maps that occur in the school from clip to clip. There was understanding that distributed leading incorporates the thought of instructors working together in squads and join forcesing in planning and supplying larning chances for pupils. This applies at both capable section degree and at whole school degree, for illustration holding a squad attack to policy development. There was understanding that if all instructors took leading duty beyond their schoolroom, e.g. for pupils ‘ behavior in the corridor, it would be a really good school. But a note of cautiousness was sounded about some instructors taking on excessively much power and the demand for the principal to â€Å" direct † came through strongly â€Å" he directs us to do certain we ‘re all talking with one voice to pupils and parents † . All were in understanding that the principal and deputy play a peculiar leading function, whether in pull offing staff or directing patterns, so that there will be a shared vision in the school. The post-holders themselves discussed the readying and preparation they received when appointed to their stations of duty. There was strong understanding that they had no formal preparation for their station. They watched other post-holders making similar occupations, particularly twelvemonth caputs. They all agreed that non merely did you watch them but you consulted with them and asked their advice and sentiment. Some stations, nevertheless, are new and their officeholders hence have no ‘predecessor ‘ or co-workers to confer with with. These stations require â€Å" an atrocious batch of enterprise † . Some clip was given to discoursing the system in topographic point for communicating between post-holders and the principal or deputy chief. The participants in the group see meetings, whether formal or informal, with the principal or deputy as being a signifier of support to them in their function. The agreements for formal meetings varied significantly between schools and besides between the two degrees of postholders, i.e. adjunct principals are more likely to hold formal meetings with the principal and deputy than particular responsibilities instructors. The participants agreed that the particular responsibilities instructors were non seen as a squad because they ne'er meet. Generally all Assistant Principals held twelvemonth caput places, while the particular responsibilities maps were more varied which may travel some manner in explicating why meetings ne'er took topographic point. The treatment led to a argument about remaining after school for meetings. In a neighbouring school this is the norm one time a month. Post holders stated they would non be willing to make this, as stations were supposed to be carried out during the school twenty-four hours. However, a non station holder mentioned that because post-holders get an extra allowance they should be willing to remain on after school to transport out responsibilities related to their station. Cipher responded to this statement. There was a important grade of similarity in participants understanding of distributed leading. It is something that must pervade the whole school and is apparent through the prevalent civilization and atmosphere. The principal and deputy chief drama a really of import function in puting this ambiance and they do this in both formal and informal ways. The general ‘approachability ‘ of both chief and deputy plays a cardinal function – demoing a echt involvement in and concern for the work of each person teacher helps to put the tone for how people approach their work. But distributed leading goes beyond that to supplying chances for instructors to exert leading. This may be through actions like chairing a meeting, taking a new enterprise or taking an extra-curricular activity. This chance to exert leading must be facilitated from the top, i.e. the principal or deputy. There was besides understanding that constructions were an of import component of distributed leading as they allow for leading to be exercised by a assortment of people. Structures included capable sections and squads set up to turn to a assortment of school development issues from clip to clip. To be considered a ‘structure ‘ , they must hold clip to run into and peculiar ends to accomplish. Different people may play different functions from clip to clip within these constructions and in that manner they allow for instructors ‘ voices to be heard, therefore including them in the overall decision-making of the school. They besides provide chances for instructors to exert their influence, whether they hold a formal place of leading or non. In the responses to the questionnaires 93 % of respondents agreed that when instructors work together pupil acquisition is enhanced. The focal point group besides agreed that the pupils benefit when everybody works together. ‘I believe they [ pupils ] pick up on an ambiance where everybody is working together, and where more cognition is transferred between sections, staff is more cognizant of how pupils are making. They pick up on those sorts of things that are in the ethos of the school ‘ . Capable sections are now playing a more of import function than in the yesteryear. The capable section meetings offer the chance to be after lessons together, to synchronize learning across a twelvemonth group and to discourse learning methods for peculiar elements of the course of study. There was understanding that many capable sections are in the early phases of development, and that holding formal capable meetings is indispensable for this development to go on. But capable section meetings are non the lone structures that enable instructors to work together. An illustration was given of a meeting, set up by a principal, to reexamine the advancement of a peculiar pupil. All instructors involved with this pupil were invited to analyze the state of affairs and aid instructors to work together to run into the challenges of back uping this pupil in his acquisition. This meeting required a restructuring of the timetable for the period of the meeting and the proviso of category screen for some instructors but the precedence it was given showed the belief in the power of instructors working together to better the educational experience for this pupil. In the focal point group there were two instructors who had trained and worked in the UK and Australia. They spoke of their experience of working as portion of a squad in their several schools. They both agreed that the constructions were more formal than in Ireland and that these formal constructions non merely enabled instructors to work together but created the outlook that they would. There were formal systems in topographic point for sharing resources, for keeping meetings and for detecting each other ‘s lessons. Both instructors agreed that these systems and constructions were good. There was consensus in the focal point group that when the ambiance is friendly instructors can speak to each other and ask inquiries in an informal scene such as the staffroom. They expressed the support they felt when they work together. It removes the sense of isolation and supports their schoolroom instruction. The quotation mark below is brooding of the consensus among the group. Not merely does it assist instructors but they besides perceived that it has a positive impact on pupils. ‘I will inquire the other instructors inquiries and I think it has truly helped me to loosen up and experience that I am non†¦ a small island on my ain. I can inquire for aid. Thingss like that do pervade out to the pupils every bit good when they see it ‘ . There is besides acknowledgement that newer patterns such as school development planning and Department of Education reviews have encouraged instructors to work together.Decisions and RecommendationsIn the questionnaire in this research, the respondents suggested that pupils and their parents should play a leading function in the school but, in pattern, their voices are frequently non heard in decision-making. A really important position on distributed leading is losing from this study by non including these two component groups. Further research should include both pupils and parents in the data-gathering procedure. Schools are now required to hold both pupil and parents ‘ councils and are expected to include them in policy development in the school..The function of capable sectionsThe function of capable sections has become more outstanding in schools in recent old ages, as a consequence of the school development planning and the whole school rating procedures. In this survey the function of capable sections in heightening pupil acquisition was acknowledged. Capable sections could supply a forum for sharing good thoughts and resources. A more formal attack would better the chances for these sections to act upon schoolroom pattern, for illustration by discoursing teaching method every bit good as course of study proviso. Further probe into the leading of capable sections would add well to the pattern of distributed leading in the school. This is surely an country of untapped potency. If instructors are trained in leading in their capable sections it would better the operation of a section and hence better instruction and acquisition. It would besides take to the betterment of instructors ‘ assurance in their ain leading abilities therefore constructing leading capacity in the school and finally lending to school betterment.Contemplations on my work in this ECAIn this geographic expedition of distributed leading, I had concerns about discoursing distributed leading with research participants before specifying or determining their apprehension of leading in general. For that ground, a questionnaire was given to instructors. This was a really utile exercising in that it produced thoughts about leading and how instructors perceived school leading. It yielded a really wide reading of leading but identified the fact that instructors accept that they play a leading functio n – leading is non the exclusive privilege of the principal and deputy principal. However, in hindsight, more geographic expedition of the difference between leading and direction would hold been helpful. I learned a batch about practician research in one ‘s ain educational administration. While one has the benefit of cognizing the participants, this can besides hold disadvantages. I was witting that participants may non hold been as unfastened and honest with me as they would be with an external research worker and that this may hold some deductions for my findings. In future research, I would prefer to work with staff in a school external to mine. I would interview the principal and deputy chief separate to the focal point group ( in order to guarantee a more unfastened treatment ) and would carry on two separate focal point groups – one for postholders and one for non postholders in order that their positions could be comprehensively compared. A utile result of the research procedure has been the articulation by instructors themselves of their leading function, and a acknowledgment of the influence they exert non merely on the pupils but besides over their co-workers. The research has highlighted certain issues that the school could concentrate on that would heighten instruction and acquisition. If the ambiance is positive, so a civilization of coaction can be developed and the leading function of postholders can be discussed and articulated more clearly. Similarly, more preparation for capable section squad holds the possibility of doing a really positive part to school betterment. The leading of the principal and deputy principal is really of import. First, they create the positive ambiance by paying attending to each person teacher – recognizing that their influence on pupil acquisition is through their instructors. Second, they are in a place to organize constructions and systems that enable instructors to work together and to develop leading accomplishments. Third, professional development is an of import portion of making an ambiance of larning among staff. Having completed the survey, the findings can be used by the school in a figure of ways. The principal will have a study sketching the responses to the questionnaire and a sum-up of findings from the focal point group treatment. The cardinal issues will hold deductions for the principal and deputy principal in that the findings highlight the importance of their leading function in developing a positive acquisition environment but besides in developing single leading accomplishments in instructors and supplying chances for leading to be exercised among co-workers. All of these have the potency to better instruction and acquisition. The studies could besides be used as a footing for treatment among postholders, concentrating on their function in taking acquisition. This research study set out to research what was meant by distributed leading and to see, if practised in a school, would it lend to bettering instruction and acquisition. Through questionnaires and a focal point group treatment the construct of distributed leading was explored and the consequences presented and analysed. A broad runing definition emerged that recognised that all instructors can be leaders, but the extent of their leading maps varies from within the schoolroom to their influence on pupils beyond their single schoolroom, to their leading influence over co-workers. Their apprehension of distributed leading encompassed structural and cultural issues, both of which had the possible to influence instruction and acquisition. This potency is non to the full realised, but with a more knowing focal point on instruction and acquisition and a witting development of leading capacity this state of affairs could alter to the benefit of pupils.