基因的直接進(jìn)化-天津大學(xué)研究生e-learning平臺(tái)

1、基因直接進(jìn)化研究進(jìn)展 ——DNA Shuffling技術(shù),天津大學(xué),申明華,DNA Shuffling技術(shù),DNA Shuffling,DNA改組DNA洗牌DNA攪亂重排,,1994年，Stemmer等，用DNA Shuffling技術(shù)體外快速進(jìn)化蛋白——有性PCR法1997年 ，F(xiàn)rance Aronld研究組將DNA Shuffling技術(shù)做了改進(jìn)——交錯(cuò)延伸法,What happens after DN

2、A shuffling,Generation of a large library of novel genes (chimeras) Selection for improved/desired bio-functions,crossovers, deletions, insertions, inversions, point mutations,Darwinian EvolutionNatural selection

3、of existing mutations,Directed Evolution Targeted selection of created mutations,What is DNA shuffling?,DNA Shuffling的內(nèi)涵,DNA Shuffling：指DNA分子的體外重組，是基因在分子水平上進(jìn)行有性重組(Sexual Recombination)。通過(guò)改變單個(gè)基因(或基因家族，gene family)原有的核苷

4、酸序列，創(chuàng)造新基因，并賦予表達(dá)產(chǎn)物以新功能。,DNA Shuffling的內(nèi)涵,DNA Shuffling與常規(guī)定向進(jìn)化的比較,How DNA shuffling works - 1,How DNA shuffling is done in the tube Random fragmentation of a pool of related genes; Self-priming polymerase reaction and te

5、mplate switching (causing crossovers); PCR amplification with primers of reassembled products,How DNA shuffling works - 2,Similar mutants generated byerror-prone PCR, random and site-directed mutagenesis,,,Single gene

6、 shufflinglibrary of point mutants,Family gene shufflinglibrary of chimeras,,,Generating chimeras with crossovers of large blocks of sequences,,,,,,,X XXX,X XX X,XX

7、 X,XXX X,XX X,,,,,,X XXX,,,,,,XX X,,,,,,X XX X,XXX X,,,,,,X XXX,,,,,,XX X,,,,,,X

8、 XX X,XXX X,,,,,,,,,XX X,,,,,XX X,,XX XXX X X X X X XX X,Fragment Reassemble Select bestwith DNAseI fra

9、gments recombinants,,Repeat for multiple cycles,,,,How DNA shuffling works - 3,DNA 重組裝的過(guò)程,隨機(jī)引物PCR和重組裝,交錯(cuò)延伸PCR突變法,磷脂酶熱穩(wěn)定－催化活性的分子進(jìn)化（Jae Kwang Song等，2000）,DNA Shuffling技術(shù)的應(yīng)用,枯草桿菌蛋白酶E熱穩(wěn)定性的分子進(jìn)化（Huimin Zhao等，1999）,進(jìn)化