杨荣武分子生物学课件Week3modification.pptVIP

DNA sequencing: methods I. Brief history of sequencing II. Sanger dideoxy method for sequencing III. Sequencing large pieces of DNA VI. The “$1,000 dollar genome” Why sequence DNA? All genes available for an organism to use -- a very important tool for biologists Not just sequence of genes, but also positioning of genes and sequences of regulatory regions New recombinant DNA constructs must be sequenced to verify construction or positions of mutations Etc. Methods of sequencing Sanger dideoxy (primer extension/chain-termination) method: most popular protocol for sequencing, very adaptable, scalable to large sequencing projects Maxam-Gilbert chemical cleavage method: DNA is labelled and then chemically cleaved in a sequence-dependent manner. This method is not easily scaled and is rather tedious Pyrosequencing: measuring chain extension by pyrophosphate monitoring Primers for DNA sequencing Oligonucleotide primers can be synthesized by phosphoramidite chemistry--usually designed manually and then purchased Sequence of the oligo must be complimentary to DNA flanking sequenced region Oligos are usually 15-30 nucleotides in length DNA templates for sequencing: Single stranded DNA isolated from recombinant M13 bacteriophage containing DNA of interest Double-stranded DNA that has been denatured Non-denatured double stranded DNA (cycle sequencing) Reagents for sequencing: DNA polymerases Should be highly processive, and incorporate ddNTPs efficiently Should lack exonuclease activity Thermostability required for “cycle sequencing” Sanger dideoxy sequencing--basic method Sanger dideoxy sequencing: basic method Sanger dideoxy sequencing: basic method How to visualize DNA fragments? Radioactivity Radiolabeled primers (kinase with 32P) Radiolabelled dNTPs (gamma 35S or 32P) Analysis of sequencing products: Polyacrylamide gel electrophoresis--good resolution of fragments differing by a single dNTP Slab gels: as previously described Capillary gels: require only a tiny amo