自学基因组和dna测序原理.ppt

The Sanger method requires Cycle sequencing Sequenase (T7) polymerase has been a widely used enzyme for DNA sequencing with radioactive nucleotides because of its high processivity and low error rate. However using this type of enzyme for fluorescent DNA sequencing is not optimal because the amount of fluorescent DNA produced is low and thus a large amount of template is required to produce good fluorescent signals. The enzyme currently in general use for automated fluorescent DNA sequencing is a variant of Taq DNA polymerase. The thermostability of Taq polymerase allows the sequencing reactions to be carried out like PCR reactions, and the reactions are called cycle sequencing reactions. The advantage of using the thermostable Taq polymerase over the use of Sequenase is that multiple rounds of sequencing can be performed without the need to add fresh enzyme. Inside the sequencer A Sequence print-out from a control sample DNA template is denatured to single strands. DNA primer (with 3’ end near sequence of interest) is annealed to the template DNA and extended with DNA polymerase. Four reactions are set up, each containing: DNA template Primer annealed to template DNA DNA polymerase dNTPS (dATP, dTTP, dCTP, and dGTP) Next, a different radio-labeled dideoxynucleotide (ddATP, ddTTP, ddCTP, or ddGTP) is added to each of the four reaction tubes at 1/100th the concentration of normal dNTPs. ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with normal dNTPS, and produce no phosphodiester bond. Manual Dideoxy DNA sequencing -How it works: Whenever the radio-labeled ddNTPs are incorporated in the chain, DNA synthesis terminates. Each of the four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions. Extension products in each of the four reaction mixutes also end with a different radio-labeled ddNTP (depending on the base). Next, each reaction mixture is electrophoresed in a separate lane (4 lanes) at