分子遺傳學(xué)理論與技術(shù)基礎(chǔ) Chapter VIDNA Sequencing

1、VI. DNA Sequencing technology1. 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(化學(xué)裂解 法). Maxmam and Gilbert. In 1977. (2) dideoxymediated chain-termination method(

2、雙脫氧末端終止法). 鄂p425. Sanger in the end of 1970s. (3)sequencing by hybridization.(雜交法) in the end of 1980s. (4) automatic sequencing.(that is based on Sangers method.) in 1990s.（自動(dòng)化法) *complete automatic sequencer. capillary electrophoresis and erading picture all automatically. 751632 bp x 96(sample) /

3、 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 dN

4、TP markered ddNTP （見板圖） (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.(請(qǐng)見板圖) (3)the high resolution denature polyacryla- mide gel e

5、lectrophoresis 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(電泳圖譜) for DNA sequences analysis.鄂p427圖.3.Basic require for instruments: High voltage electrophoresis a

6、pparatus(2000V). sequencing electrophoresis tank (vertical slab form). eppendorf tube centrifuge(15000rpm). pipetter(0.5200 ul) and specific tip. vacuum desiccator(抽干機(jī)). vacuum filtration manifold(真空抽濾裝置). dark room. radioautography apparatus. sequencing reaction reagents box.4.The technology steps

7、of DNAsequencing by handiwork.鄂p427. Preparation of the target DNAs recon. alkali denature (to get single strand DNA for sequencing.) 5ATGCCTTGACAGACGTACGT3 sequencing primers renature(37, 30min.) 5ATGCCTTGACAGACGTACGT3 TGCATGCA5 *Labeling reaction. RT10min. (標(biāo)記反應(yīng)) dNTP and a-32PdNTP sequenase(測(cè)序DNA

8、合成酶) *Termaniting reaction(37 5min.) (終止反應(yīng)). divide into 4 reaction tubes. to add different ddNTP for random joining.ddATP ddGTP ddCTP ddTTP 3ddCTGCATGCA5 3ddCTGTC-5 3ddCGGAACTGTC-5 3ddGTCTGCATGCA5 3ddGAACTGTC-5 3ddGGAACTGTC-5 3ddTCTGCATGCA5 3ddTGTC-5 3ddTACGGAACTGTC-5 3ddACTGTCTGCATGCA5 3ddAACTGTC-

9、53ddACGGAACTGTC-5 hot denature (96 , 2 min) high resolution gel electrophoresis A G C T _ _ _ _ _ _ _ _ _ _ _ _ddC-5 ddG-5 ddG-5 ddA-5 ddA-5 ddC-5 ddT-5 ddG-5 ddT-5 ddCTGCATGCA5 dry *read out the DNA sequences: 5ACGTACGTCTGTCAAGGCAT3 radioautography primer antisense sequences of target DNA fragment

10、to read picture鄂 P16 圖片。6.The principle of completeautomatic sequencer. (1)the preparation of template DNA for sequencing (handiwork). to amplify target DNA fragments with PCR technology. to purify the PCR production. to join the target DNA fragments and vector DNA into recon. to transformate host c

11、ells with the recon, then to clone amplify. to isolate and purify the target recons.(2)Sequencing reaction(handiwork). to hot denature the target recon(96,2). to build the single reaction tube for sequencing reaction. target recons (sequencing template). sequencing primer. buffer(1x) dNTP fluorescei

12、n marked ddNTP mixture. Taq DNA polymerase. PCR reaction for 30 cycle, to get terminal marked DNA fragments that is random long. to purify the production of sequencing reaction.(3) To isolate the sequencing reaction production by capillary electrophoresis. ( to be operated by the automatic sequencer

13、.) * capillary electrophoresis : capillary :20200 um diameter. 10100 cm long. terminal voltage :200400 V / cm. the capillary autowashing; autopouring gel solution; autosampling ; 8 capillary can electrophoresis in same time. there are 96 hole on the DNA sample plate, 9 hole / each line, that are joi

14、ned with the 8 capillary for autosampling. each operation can do sequencing for 96 of DNA samples. Each DNA sample is 180200 bp long best.(4) To read the picture though the examer window. (to operate sequencer) the production of sequencing reaction are random long DNA fragments. 3ddA-5 3ddC-5 3ddT-5 3ddG-5 capillary electr