DNA Sequencing

1. DNA Sequencing

2. DNA sequencing …ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…

3. DNA sequencing • Determination of nucleotide sequence • Two similar methods: 1. Maxam and Gilbert method 2. Sanger method • They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end • This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined

4. Maxam and Gilbert Method • The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate • It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal • The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base 1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine • The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified

5. Frederick Sanger • Discovered DNA sequencing by chain termination method • Nobel Prize 1 (1958) • Complete amino acid sequence of insulin • Nobel Prize 2 (1980) • For DNA sequencing

6. Sanger Method • DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides • Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system • Incorporation of dideoxynucleotides into growing strand terminates synthesis • Synthesized strand sizes are determined for each dideoxynucleotide rxn by using gel or capillary electrophoresis

7. Dideoxynucleotide 5’ BASE CH2 PPP O O 3’ no hydroxyl group at 3’ end prevents strand extension

8. Dideoxy nucleotides • Incorporation of a dideoxynucleotide to growing DNA strand terminates its further extension • Are added in small proportion • dATP ddATP • dGTP ddGTP • dCTP ddCTP • dTTP ddTTP

9. Chain Termination

10. Target ddC ddA Template-Primer ddG ddT Labeled Terminators ddA A ddC AC ddG ACG ddT Chain Terminator Basics TGCA Extend dN : ddN 100 : 1 Ladder n, n+1...

11. 3’ CCGTAC 5’ primer 5’ 3’ dNTP ddTTP ddCTP ddGTP ddATP GGCA GGCAT GGC G GG GGCATG ATCG

12. All Possible Terminations

13. Polyacrylamide Gel Electrophoresis Separates fragments based on size

14. Electrophoresis

15. DNA Sequencing – vectors DNA Shake DNA fragments Known location (restriction site) Vector Circular genome (bacterium, plasmid) + =

16. Different types of vectors

17. DNA Sequencing – gel electrophoresis • Start at primer (restriction site) • Grow DNA chain • Include dideoxynucleoside (modified a, c, g, t) • Stops reaction at all possible points • Separate products with length, using gel electrophoresis

18. Template • ssDNA vectors • M13 • pUC • PCR • dsDNA (+/- PCR)

19. Primers • Universal primers • cheap, reliable, easy, fast, parallel • BULK sequencing • Custom primers • expensive, slow, one-at-a-time • ADAPTABLE

20. Extension Chemistry • Polymerase • Sequenase • Thermostable (Cycle Sequencing) • Terminators • Dye labels (“Big Dye”) • spectrally different, high fluorescence • ddA,C,G,T with primer labels

21. Separation • Gel Electrophoresis • Capillary Electrophoresis • suited to automation • rapid (2 hrs vs 12 hrs) • re-usable • simple temperature control • 96 well format

22. Sequencing Strategies • Ordered • Divide and Conquer • Random Sequence • Brute Force

23. DNA Sequencing 5.17)

24. Sequencing of DNA by the Sanger method