DNA Sequencing

1. DNA Sequencing

2. DNA sequencing • Determination of nucleotide sequence • the determination of the precise sequence of nucleotides in a sample of DNA • 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

3. Maxam-Gilbert • Walter Gilbert • Harvard physicist • Knew James Watson • Became intrigued with the biological side • Became a biophysicist • Allan Maxam

4. The Maxam-Gilbert Technique • Principle - Chemical Degradation of Purines • Purines (A, G) damaged by dimethylsulfate • Methylation of base • Heat releases base • Alkali cleaves G • Dilute acid cleave A>G

5. Maxam-Gilbert Technique • Principle Chemical Degradation of Pyrimidines • Pyrimidines (C, T) are damaged by hydrazine • Piperidine cleaves the backbone • 2 M NaCl inhibits the reaction with T

6. Maxam and Gilbert Method • Chemical degradation of purified fragments (chemical degradation) • 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

7. Maxam-Gilbert sequencing - modifications

8. Maxam-Gilbert sequencing- summary

9. Advantages/disadvantagesMaxam-Gilbert sequencing • Requires lots of purified DNA, and many intermediate purification steps • Relatively short readings • Automation not available (sequencers) • Remaining use for ‘footprinting’ (partial protection against DNA modification when proteins bind to specific regions, and that produce ‘holes’ in the sequence ladder) In contrast, the Sanger sequencing methodology requires little if any DNA purification, no restriction digests, and no labeling of the DNA sequencing template

10. Sanger Method • Fred Sanger, 1958 • Was originally a protein chemist • Made his first mark in sequencing proteins • Made his second mark in sequencing RNA • 1980 dideoxy sequencing

11. Original Sanger Method • Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA • Requires DNA polymerase I • Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: beta-galactosidase screening, polylinker) • Uses 32P-deoxynucleoside triphosphates

12. Sanger Method • in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their integration • 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 di-deoxynucleotides into growing strand terminates synthesis • Synthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary electrophoresis • Enzymatic methods

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

14. The principles • Partial copies of DNA fragments made with DNA polymerase • Collection of DNA fragments that terminate with A,C,G or T using ddNTP • Separate by gel electrophoresis • Read DNA sequence

15. 3’ CCGTAC 5’ primer 5’ 3’ dNTP ddTTP ddCTP ddGTP ddATP GGCA GGCAT GGC G GG GGCATG ATCG

16. DideoxyChain Terminator • Template • Primer • Extension Chemistry • polymerase • termination • labeling • Separation • Detection

17. 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...

19. Electrophoresis

20. Sanger Method Sequencing Gel

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

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

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

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

25. 1 lane Sample Output

27. Sequencing of DNA by the Sanger method

28. Sanger Method Enzymatic Requires DNA synthesis Termination of chain elongation Maxam Gilbert Method Chemical Requires DNA Requires long stretches of DNA Breaks DNA at different nucleotides Comparison