1. DNA sequencing Direct determination of nucleotide sequence
(previously identified by back translation
of amino acid sequences)
Basic principles
Chemical sequencing / Maxam-Gilbert method
Enzymatic sequencing / Sanger method

2. Maxam-Gilbert method Older technique / Not frequently used
Base-specific chemical reaction
Different sets of chemical reactions
Dimethyl sulfate selectively react to purine
Hydrazine selectively react to pyrimidine

3. DMS reaction

4. Hydrazine reaction

5. Maxam-Gilbert method End labeling of DNA sequence
Chemical modification and removal of specific bases
Piperidine to cleave phosphodiester bond
Reactions controlled to get 1 break per molecule
Subsets of labeled DNA with different lengths

6. Maxam-Gilbert method Size fractionation side by side
Polyacrylamide gel electrophoresis
Denatured by 7 M Urea at high temp
Sequence read directly from bottom up

7. Maxam-Gilbert method

8. Maxam-Gilbert method G DMS + Piperidine
G/A DMS + Formic acid + Piperidine
C/T Hydrazine + Piperidine
C Hydrazine M NaCl + Piperidine

9. Maxam-Gilbert method

10. Maxam-Gilbert method

11. Sanger method Mostly used Based on the ability of DNA polymerase
Incorporation of dideoxynucleotide
terminates DNA synthesis

12. Ribose structure DNA synthesis requires 3’ OH
For formation of phosphodiester bond

13. Sanger method Strand labeling/primer annealing step
Labeled short DNA primer
Internal label by dNTP*
End label by ddNTP*
radioactive or fluorescent label

14. Sanger method Chain termination step
Four separate reactions (1 or 4 tubes)
Klenow fragment of DNA polymerase
Each reaction: 4 dNTPs + 1 ddNTP
Size fractionation by PAGE

15. Dideoxy chain termination
2’3’ dideoxynucleoside triphosphate

16. Sanger method

17. Sanger method

18. Sanger method

19. Sanger method

20. Sequencing enzymes Klenow / AMV RT read more than 80% of target
low rate of substrate incorporation
Taq polymerase / modified T7 DNA polymerase
high rate of incorporation
consistent band intensity
low background with high degree of accuracy

21. Sequencing enzymes Klenow DNA template / Good for AT-rich region
10-12 dNTP per second
AMV RT
DNA/RNA template / Good for GC-rich region
4 dNTP per second

22. Sequencing enzymes Taq polymerase reaction carried out at high temp
increase stringency with primer
decrease secondary structure

23. Sequence search Public Databases EST / expressed sequence tag GENBANK
TIGR
EMBL
NCBI