分子遗传学理论与技术基础 Chapter VI.DNA Sequencing.ppt

VI. DNA Sequencing technology 1. summary. 1) concept: DNA sequencing is the techni- cal process to check the base sequence of DNA molecular with experiment way. 2) the multiple methods: (1) chemical degradation method(化学裂解 法). Maxmam and Gilbert. In 1977. (2) dideoxymediated chain-termination method(双脱氧末端终止法). 鄂p425. Sanger in the end of 1970s. (3)sequencing by hybridization.(杂交法) in the end of 1980s. (4) automatic sequencing.(that is based on Sanger’s method.) in 1990s.（自动化法) *complete automatic sequencer. capillary electrophoresis and erading picture all automatically. 75~1632 bp x 96(sample) / each time. 2.The principle of dideoxymedia-ted chain-termination method. (1)to join the target DNA fragment and the plasmid vector into recon. after denature, we use the single strand DNA as the template to synthesis complementary DNA chain. the reaction system： target recon primer DNA polymerase dNTP markered ddNTP （见板图） (2) As the ddNTP (dideoxyribonucleoside triphosphate) are joined, so that the extension of the new synthesised single DNA strand are terminated, and we can get the random long DNA fragments that are new synthesised.(请见板图) (3)the high resolution denature polyacryla- mide gel electrophoresis technology can resolve the different long single strand DNA fragments that only are difference in one base long. (4)the radioautograph technology can get the electrophoresis pattern(电泳图谱) for DNA sequences analysis.鄂p427图. 3.Basic require for instruments: High voltage electrophoresis apparatus(2000V). sequencing electrophoresis tank (vertical slab form). eppendorf tube centrifuge(15000rpm). pipetter(0.5~200 ul) and specific tip. vacuum desiccator(抽干机). vacuum filtration manifo