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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
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
DMS reaction
Hydrazine reaction
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
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
Maxam-Gilbert method
Maxam-Gilbert method
G
G/A
C/T
C
DMS + Piperidine
DMS + Formic acid + Piperidine
Hydrazine + Piperidine
Hydrazine + 1.5 M NaCl + Piperidine
Maxam-Gilbert method
Maxam-Gilbert method
Sanger method
Mostly used
Based on the ability of DNA polymerase
Incorporation of dideoxynucleotide
terminates DNA synthesis
Ribose structure
DNA synthesis requires 3’ OH
For formation of phosphodiester bond
Sanger method
Strand labeling/primer annealing step
Labeled short DNA primer
Internal label by dNTP*
End label by ddNTP*
radioactive or fluorescent label
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
Dideoxy chain termination
2’3’ dideoxynucleoside triphosphate
Sanger method
Sanger method
Sanger method
Sanger method
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
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
Sequencing enzymes
Taq polymerase
reaction carried out at high temp
increase stringency with primer
decrease secondary structure
Sequence search
Public Databases
EST / expressed sequence tag
GENBANK
TIGR
EMBL
NCBI