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DNA Sequencing - I SUBJECT FORENSIC SCIENCE Paper No. and Title PAPER No.13: DNA Forensics Module No. and Title MODULE No.21: DNA Sequencing - I Module Tag FSC_P13_M21 FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.21: DNA Sequencing - I TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction 3. First Generation Sequencing Methods 4. Second Generation Sequencing Techniques 5. Third Generation Sequencing – emerging technologies 6. DNA Sequence analysis 7. Summary FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.21: DNA Sequencing - I 1. Learning Outcomes After studying this module, reader shall be able to understand - DNA Sequencing Methods of DNA Sequencing DNA sequence analysis 2. Introduction In 1953, James Watson and Francis Crick discovered double-helix model of DNA, based on crystallized X-ray structures studied by Rosalind Franklin. As per this model, DNA comprises of two strands of nucleotides coiled around each other, allied by hydrogen bonds and moving in opposite directions. Each strand is composed of four complementary nucleotides – adenine (A), cytosine (C), guanine (G) and thymine (T) – with A always paired with T and C always paired with G with 2 & 3 hydrogen bonds respectively. The sequence of the bases (A,T,G,C) along DNA contains the complete set of instructions that make up the genetic inheritance. Defining the arrangement of these nucleotide bases in DNA strand is a primary step in assessing regulatory sequences, coding and non-coding regions. The term DNA sequencing denotes to methods for identifying the sequence of these nucleotides bases in a molecule of DNA. The basis for sequencing proteins was initially placed by the effort of Fred Sanger who by 1955 had accomplished the arrangement of all the amino acids in insulin, a small protein produced by the pancreas. The first technique for identifying DNA order involving a location-specific primer extension scheme was developed by Ray Wu at Cornell University in 1970. Between 1970 and 1973, Wu, R Padmanabhan and co-workers revealed that this technique could be engaged to identify any DNA arrangement by applying synthetic location-specific primers. FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.21: DNA Sequencing - I Frederick Sanger then executed this primer-extension method to advance more rapidly DNA sequencing conducts at the MRC Centre, Cambridge, UK and delivered a method for "DNA sequencing with chain-terminating inhibitors" in 1977. The awareness of DNA sequences of genes and further fragments of the genome of organisms has become crucial for several applied and research fields such as: Diagnostic Biotechnology Forensic Biology Biological Systematics Taxonomy Phylogeny Ecology Genetic studies Progressions in sequencing were assisted by the coexisting expansion of recombinant DNA technology, permitting DNA samples to be separated from sources other than viruses. Development of dye grounded sequencing technique with automated examination, DNA sequencing has become easy to handle and comparatively faster. The speedy sequencing accomplished with contemporary DNA sequencing tools has been influential in the sequencing of the human genome, in the human genome project. Developments in DNA sequencing technologies can be grouped into three stages: 1. First generation sequencing 2. Second generation sequencing or next generation (NGS) 3. Third generation sequencing (TGS) – emerging technologies The Sanger and Gilbert approaches of sequencing DNA are often called "first-generation" sequencing because they were the first to be developed. In the late 1990s, new methods, called second-generation sequencing methods, that were faster and cheaper, began to be developed. The most popular, widely-used second-generation sequencing method was one called Pyrosequencing. Today various newer sequencing methods are available and others are in the progression of being developed. These are often called third generation or next- generation sequencing methods. FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.21: DNA Sequencing - I 3. First Generation Sequencing Methods 3.1 Maxam–Gilbert Sequencing (The Chemical Cleavage) Method During 1976-1977, Allan Maxam and Walter Gilbert established a DNA sequencing technique grounded on chemical alteration of DNA and succeeding cleavage at precise bases. The technique necessitates radioactive tagging towards one end and purification of the DNA fragment to be sequenced. Chemical handling forms breaks at a minor proportion of one or two out of four nucleotide base in individual four reactions (G, A+G, C, C+T). Thus a sequence of marked remains is produced, from the radiolabelled terminal to the first ‘cut’ site in every molecule. The fragments of the four reactions are organized near each other in gel electrophoresis for size by size isolation. To observe the fragments, the gel is exposed to X- ray film for autoradiography, forming a sequence of dark bands each according to a radiolabelled DNA fragment, from which the arrangement may be inferred. It was primary extensively accepted technique for DNA sequencing, however, it is no longer in widespread use because of its methodical complication eliminating the application in standard molecular biology kits, widespread application of dangerous elements, and difficulties with scale-up. Hence, this method has been supplanted by next generation sequencing methods. FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.21: DNA Sequencing - I 3.2 Sanger Sequencing Methods: Frederick Sanger established numerous fast, more effective methods to order DNA (Sanger et al., 1977). Certainly, Sanger's effort in this domain was so ground breaking that he got the Nobel Prize in Chemistry in 1980. The two key methods developed by Sanger are called Chain-termination and Dye- terminator sequencing. 3.2.1 The chain-termination or dideoxy Method: The chain termination technique needs a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labelled nucleotides, and altered nucleotides that halts DNA strand extension. The DNA sample is separated into four distinct sequencing reactions, containing all four of the typical deoxynucleotides (dATP, dGTP, dCTP, dTTP) and the DNA polymerase. To each reaction is added one of the four dideoxynucleotide (ddATP, ddGTP, ddCTP, ddTTP) which are the chain terminating nucleotides, lacking a 3’- OH group needed for the creation of a phosphodiester bond among two nucleotides, thus halting DNA strand elongation and subsequently forming DNA fragments of varying length. The recently produced and labelled DNA fragments are heat denatured, and isolated as per size through gel electrophoresis on a denaturing polyacrylamide-urea gel with all of the four reactions move in one of the four individual lanes (lanes A, T, G,C), the DNA bands are then observed by autoradiography or UV light, and the DNA arrangement can be straight revealed by the X-ray film or gel image. A dark band specifies a DNA portion which is outcome of chain finish after combination of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The comparative location of the various bands amongst the four tracks are then applied to read the DNA sequence. The procedural differences of chain termination arrangement comprise labelling each dideoxynucleotide with fluorescence dye. FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.21: DNA Sequencing - I Dideoxynucleotides are alike to even, or deoxynucleotides, but has only one main difference they do not have hydroxyl group on the 3’ carbon of the sugar circle. In an even nucleotide, the 3’ hydroxyl group perform the function of as “hook," permitting a new nucleotide to be attached to prevailing chain. After the dideoxynucleotide have been attached to the chain, it will have no hydroxyl accessible and no additional nucleotides will be attached. The chain terminates with the dideoxy nucleotide, that is labelled with a specific color of dye according to the base (A, T, C or G) that it transports. 3.2.2. Dye -terminator sequencing Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than 4 reactions as in the tagged- primer method. In this, all of the four dideoxynucleotide chain terminators is marked with fluorescent dyes, all of that has diverse wavelengths of fluorescence and emission. It produces DNA pieces that halt at every nucleotide beside the template strand. The DNA is isolated with capillary electrophoresis as per their size. By the arrangement of fragments, the DNA sequence can be revealed. The short pieces were halted initially, and they processed first from the column, and subsequently different fluorescent labels leaving the column is the sequence of the strand. The DNA sequence revealed is exposed on an electropherogram that is produced by a laser scanner. This technique permits four times more sequencing reactions to be electrophoresed on a gel, because 4 dideoxynucleotide reactions from a single template are in a single lane rather than four separate lanes. FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.21: DNA Sequencing - I Shotgun Sequencing: This technique was originally used by Fred Sanger and his co-workers for sequencing small genomes of viruses and bacteria. The technique is entitled by the analogy with promptly intensifying, quasi-random firing configuration of a shotgun. In shotgun sequencing numerous replicas of the similar chromosome are isolated and fragmented in random locations. All the fragments are sequenced,
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