分子生物学复习1.docVIP

Genomics sequence project Technique support for genome sequence project: Sanger chain-terminator method PCR Automated DNA sequencing machine Bioinformatic software and facility Two strategies in genome sequence project: Clone by Clone (逐步克隆法) Whole Genome Shot-gun (全基因组鸟枪（霰弹）法) Item strategy Whole genome shot-gun Clone by clone Genetic background No requirement Require physical map Speed quick slow Expense low high The capacity of computer Require high capacity low capacity Apply to Without huge repeated sequence With huge repeated sequence Representative species Drosophila, Arabidopsis, rice Human, C. elegans Clone by Clone Construct physical map →Construct BAC library →Assemble BAC contigs according to STS marker →Assembling shotgun sequence →Assembling whole genome sequence and finishing Physical map: A genetic map based on physical characteristics of the DNA, such as restriction sites rather than on locations of genes. STS (Sequence Tagged Site): a short stretch of DNA that can be identified by amplifying it using PCR with defined primers. Each STS marker has its specific location on chromosome. Physical map = STS map Construct BAC library NotI、SacI are enzymes used to cut genomic genes into 200kb DNA. And they will be inserted into vectors. After growing in E. coil, the genes can be detected by screening on antibiotic medium. Each colony has one insertion. Assemble clone contig Screen the BAC colons by “STS-PCR pooling” assemble clone contigs, and fill interclone gaps by BAC end sequencing walking or fingerprinting walking. Assemble BAC on chromosome according to STS The density of STS markers on chromosome is not enough, many regions would not be covered by clones, Walking by End Sequence or Walking by Fingerprinting can help to select new clones cover the empty regions. Walking by End Sequence（末端序列步行法 ） Sequence the clone locates on the end of contig, the specific new sequence can be regarded as a new STS marker to screen ne