BLAST使用方法PPT教學(xué)課件

1、第2頁/共39頁第1頁/共39頁SequenceSequence第3頁/共39頁第2頁/共39頁BlastBlast第4頁/共39頁第3頁/共39頁第5頁/共39頁第4頁/共39頁DNA sequenceDNA sequence第6頁/共39頁第5頁/共39頁The The result of result of blastblast第7頁/共39頁第6頁/共39頁StatisticStatisticWhat are you interested?What are you interested?第8頁/共39頁第7頁/共39頁Full-length cDNA of the geneFull-l

2、ength cDNA of the geneFirst-step:First-step:Design of the special primersDesign of the special primersPrimer premier-Primer premier-Design-DesignDNAStartDNAStart-Evaluation-Evaluation第9頁/共39頁第8頁/共39頁DNA sequenceDNA sequence第10頁/共39頁第9頁/共39頁第11頁/共39頁第10頁/共39頁DNAStar-EditSeqDNAStar-EditSeqDNA Sequence

3、DNA Sequence第12頁/共39頁第11頁/共39頁第13頁/共39頁第12頁/共39頁DNAStar-PrimerSelectDNAStar-PrimerSelect第14頁/共39頁第13頁/共39頁第15頁/共39頁第14頁/共39頁第16頁/共39頁第15頁/共39頁%GC%GCTmTmDimerDimer3 3- -第17頁/共39頁第16頁/共39頁Second-step:Second-step:PCR amplicationPCR amplication第18頁/共39頁第17頁/共39頁On the other hand, we can obtain gene acco

4、rding to On the other hand, we can obtain gene according to homologous analysishomologous analysis ! ! First-step: First-step: Search for homologous sequences in NCBI. Search for homologous sequences in NCBI. 第19頁/共39頁第18頁/共39頁第20頁/共39頁第19頁/共39頁GoGoComplete cdsComplete cds第21頁/共39頁第20頁/共39頁Save as S

5、ave as “EditSeq DNA EditSeq DNA Sequence FileSequence File”第22頁/共39頁第21頁/共39頁Then, DNAStar-MegAlignThen, DNAStar-MegAlign第23頁/共39頁第22頁/共39頁Degeneration Primers:Degeneration Primers: Nest PCR Nest PCR %GC %GC Tm Tm Dimer Dimer 3 3- -第24頁/共39頁第23頁/共39頁Third, PCR amplification:Third, PCR amplification:

6、 Template Template Nest-PCR Nest-PCR 第25頁/共39頁第24頁/共39頁二二. The characterization of gene expression. The characterization of gene expressionNorthern blottingNorthern blottingSemi-quantitative RT-PCRSemi-quantitative RT-PCRReal-time PCRReal-time PCRIn-situ hydribationIn-situ hydribationWestern blottin

7、g Western blotting 第26頁/共39頁第25頁/共39頁Real-time PCRReal-time PCRInternal reference gene Interested geneInternal reference gene Interested geneSpecial primers:Special primers:第27頁/共39頁第26頁/共39頁第28頁/共39頁第27頁/共39頁第29頁/共39頁第28頁/共39頁第30頁/共39頁第29頁/共39頁三三. The function of gene. The function of geneExpressio

8、n in vitroExpression in vitroCell transfectionCell transfectionRNAi (RNA interference)RNAi (RNA interference)Yeast two-hybrid Yeast two-hybrid 第31頁/共39頁第30頁/共39頁Prokaryotic expressionProkaryotic expression ORF of interested geneORF of interested geneVectorVectorSelection of restriction enzymeSelecti

9、on of restriction enzymePrimers containing the sequence of restriction enzymesPrimers containing the sequence of restriction enzymesCleaved by restriction enzymesCleaved by restriction enzymesLigation and transferLigation and transfer第32頁/共39頁第31頁/共39頁Search ORF-DNAStar EditSeqDNAStar EditSeqForward

10、 PrimerForward PrimerReverse PrimerReverse Primer第33頁/共39頁第32頁/共39頁第34頁/共39頁第33頁/共39頁第35頁/共39頁第34頁/共39頁PrimersPrimersFP: 5-CATGCCATGGACGAAGACC.-3 NcoIRP:5-GGGTTCGAACTGCTTCTT-3 HindIII第36頁/共39頁第35頁/共39頁Digested by two enzymesDigested by two enzymesLigation and transfer to Ligation and transfer to E. coliE. coli DH5 DH5SequenceSequenceTransfer to Transfer to E. coliE. coli BL21 (DE3) BL