Dna sequencing

sequencing techniques

1. DNA SEQUENCING AUTOMATED SEQUENCING AND RESEQUENCING

2. DNA SEQUENCING The information content of DNA is encoded in the form of four bases (A,G,C and T) DNA sequencing  is the process of determining the precise order of nucleotides within a DNA  molecule DNA fragments can be analyzed to determine the nucleotide sequence of DNA and to determine the distribution and location of restriction sites.

3. METHODS METHODS OF DNA SEQUENCING: Conventional DNA sequencing methods Chemical degradation method. Chain termination method. (B) Cycle sequencing. (C) Automated DNA sequencing. (D) Pyrosequencing.

4. AUTOMATED DNA SEQUENCING It is possible to automate DNA sequencing by replacing radioactive labels(used in sanger’s method) with fluorescent labels It is desirable to acquire sequence data in real time by detecting the DNA bands within the gel during the electrophoretic separation The process involves fluorescent tags that are specific to each of the four dideoxynucleotides

5. METHOD The fluorescent tags are attached to the chain terminating nucleotides. Each of the four dideoxynucleotides carries a spectrally different fluorophore The tag is incorporated into the DNA molecule by the DNA polymerase and accomplishes two operations in one step: It terminates synthesis and I t attaches the fluorophore to the end of the molecule . Fluorescent primers can be used with non-labelled dideoxynucleotides By using four different fluorescent dyes it is possible to electrophorese all four chain terminating reactions together in one lane of the sequencing gel

6. METHOD ( ctd) The DNA bands are detected by their fluorescence as they electrophorese past a detector If the detector is made to scan horizontally across the base of a gel slab, many separate sequences can be scanned, one sequence per lane This method is called as four dye system An alternative to this method is to start with a single fluorescent labelled p rimer which is used in all four sequencing reactions The resulting labelled strands are separated in four different lanes in the electrophoresis ( one dye system)

7. Automated sequencer

8. CAPILLARY ARRAY ELECTROPHORESIS The method is undertaken in high-purity fused silica capillaries with an internal diameter of 50µm Silica capillaries are very flexible and are easily incorporated into automated instruments The product of the sequencing reaction is applied to the top of a capillary gel The labelled DNA fragments migrate through the capillary and emerge at the end in a vertical stream where they are detected

9. Capillary array electrophoresis

10. ADVANTAGES OF AUTOMATED SEQUENCING Manual sequencing generates excellent data but often results in poor autoradiography that makes sequence reading difficult or impossible hence automated sequencing is widely used Skilled DNA sequencers ignore bad sequencing tracks but many laboratory procedures do not It is desirable to sequence a piece of DNA several times and on both strands, to eliminate errors caused by technical problems

11. RESEQUENCING Resequencing projects are when an organism’s genome is sequenced and assembly is done using the reference genome as a template The key reason for carrying out resequencing is to compare differences between genomes from the same species.

12. RESEQUENCING Sequencing by hybridization is now performed using microarrays (gene chips) Two methods are particularly useful for sequencing short stretches of DNA – pyrosequencing and massively parallel signature sequencing (MPSS) Pyrosequencing most closely resembles sanger sequencing in that it involves DNA synthesis whereas MPSS has similarities to sequencing by hybridization

13. PYROSEQUENCING Pyrosequencing is a DNA sequencing method that involves determining which of the four bases is incorporated at each step in the copying of a DNA template As DNA polymerase moves along a single stranded template, each of the four nucleoside triphosphates is fed sequentially and then removed If one of the four bases is incorporated then pyrophosphate is released and this is detected in an enzyme cascade that emits light

14. Principle of pyrosequencing (oligonucleotide) n + nucleotide ---------> (oligonucleotide) n+1 + PPi PPi + APS ------------------> ATP + sulfate ATP + luciferin + O 2 ---------------------> AMP + PPi + oxyluciferin + LIGHT A polymerase catalyses the incorporation of nucleotides into a nucleic acid chain. As each nucleotide is incorporated a pyrophosphate(PPi) molecule is released and incorporated into ATP by ATP sulfurlyase. On addition of luciferin and the enzyme luciferase, this ATP is degraded to AMP with the production of light

15. VARIANTS OF PYROSEQUENCING There are two variants of pyrosequencing- solid phase pyrosequencing, liquid phase pyrosequencing In solid phase pyrosequencing, the DNA to be sequenced is immobilised and a washing step is used to remove excess substrate after each nucleotide addition In liquid phase pyrosequencing, a nucleotide degrading enzyme (apyrase) is introduced to make a four-enzyme system. Addition of this enzyme has eliminated the need for a solid support and intermediate washing thereby enabling the pyrosequencing reaction to be performed in a single tube

16. Solid phase pyrosequencing

17. Liquid phase pyrosequencing

18. Sequencing DNA by hybridization using microarrays Two different experimental configurations have been developed for the hybridization reaction Either the target sequence is immobilised and the oligonucleotides labelled or the oligonucleotides maybe immobilised and the target sequence is labelled The method is routinely performed and it involves labelling the sequence being examined and hybridizing it with immobilised oligomers

19. Sequencing DNA by hybridization using microarrays

20. Microchips Microchips, although have not been used for de novo sequencing of a genome, they have been used for resequencing genomes as exemplified by human mitochondrial DNA For this purpose, a 4L tiled array is set up in which L corresponds to the length of the sequence to be analysed The sequence is probed with a series of oligomers of length P which exactly match the target sequence expect for one position which is systemically substituted with each of the four bases A, T, G, C

21. Microchips

22. Microchips Microchips are particularly used for detecting mutations and polymorphisms, particularly SNPs A variety of microchips are now commercially available for the purpose of sequencing

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