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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