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引物设计和Primer-BLAST的应用

Primer-BLAST 2. 3. 4. Polymerase Chain Reaction 1971 Khorana 1985 KaryMullis PCR1986 5 Mullis The Cold Spring Harbor Laboratory CSHL PCR 1. DNA PCR PCR Inverse PCR IPCR PCR anchored PCR RACE Rapid Amplification of cDNA Ends PCR ligation-mediated PCR LM-PCR 2. PCR nested PCR 3. PCR PCR real-time quantitative PCR RQ-PCR G+C 40%-60% 4 18-27 3bp 3bp 3 Tm Tm 5 C Tm 10 C3 3 G C 3 NNGC NNCG GC rich AT rich 3 T/C A/G Primer Premier 6 P6 Beacon Designer 8 DB Oligo 6 Vector NTI Suit Primer Express PCR *Omiga* serpine1 NCBI Reference Sequence: Primer Premier

附3：微滴式数字PCR（Droplet Digital PCR，ddPCR） ddPCR系统在传统的PCR扩增 前对样品进行微滴化处理，即将 含有核酸分子的反应体系分成数 万个纳升级的微滴，其中每个微 滴或不含待检核酸靶分子，或者 含有一个至数个待检核酸靶分子。 经PCR扩增后，对每个微滴的荧

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Transcription of 引物设计和Primer-BLAST的应用

1 Primer-BLAST 2. 3. 4. Polymerase Chain Reaction 1971 Khorana 1985 KaryMullis PCR1986 5 Mullis The Cold Spring Harbor Laboratory CSHL PCR 1. DNA PCR PCR Inverse PCR IPCR PCR anchored PCR RACE Rapid Amplification of cDNA Ends PCR ligation-mediated PCR LM-PCR 2. PCR nested PCR 3. PCR PCR real-time quantitative PCR RQ-PCR G+C 40%-60% 4 18-27 3bp 3bp 3 Tm Tm 5 C Tm 10 C3 3 G C 3 NNGC NNCG GC rich AT rich 3 T/C A/G Primer Premier 6 P6 Beacon Designer 8 DB Oligo 6 Vector NTI Suit Primer Express PCR *Omiga* serpine1 NCBI Reference Sequence.

2 Primer Premier 6(PP6) Tm 57 C+2 CLength 18+27 bpAmplicon Length 80 200 bp(G+C)%=40% 60%Beacon Designer 8(BD8) Tm 66 C+2 CLength 18+27 bpAmplicon Length 80 200 bp(G+C)%=40% 60% Primer 6 BD8 Beacon Designer 8 BD BD 8 mRNA 1 2 3 4 BD 8 1 2 3 4 Tm GC G DNA 3 G 9 5 G DNA 1 2 3 4 BD 8 Tm GC% 1 2 3 4 BD 8 FP AGTGAAGATGGAGTGGAGGTAGRP TCTGGCTGGCTGAAGTCTATC AA GCGC Integrated DNA Technologies Web Primer PrimerDesign NCBI Primer.

3 Primer-Blast Primer-BLAST Primer3 NCBI Blast Primer-BLAST Blast Primer-BLAST Primer-Blast Primer-Blast PCR Tm The Tm calculation is controlled by Table of thermodynamic parametersand Salt correction formula(under advanced parameters). The default Table of thermodynamic parameters is "SantaLucia1998" and the default Salt correction formula is "SantaLucia1998" as recommended by primer3 program. PCR Tm GC GC clamp GC 5 30-40bp GC PCR 3 3 GC Primer-Blast Primer-Blast PrimerParameters specificitycheck Tm Primer-Blast 1 Primer-Blast Primer-Blast 2 Tm GC 3 refseq_rna genomedatabase nr probeBase 2016 probeBaseis a curated database of rRNA-targetedoligonucleotide probes and rRNA

4 RRNA probeBase2016 SearchSearchprobeBasefor target organisms, probe names, primers, target sites, references, rRNA sequence(s) against probeBase and find all published probes targeting your sequence(s). ProxyMatchpartial rRNAsequence(s) against full-length sequences inSILVAand find published probes potentially targeting your sequence(s).ListsView lists of probes(according to probe categories), primers, microarrays, references, etc. SubmissionSubmit new or missing probes to probeBase. LinksWebsites relevant to ribosomal RNA rRNA rRNA rRNA probeBase 1 16S rRNA , probeBase 16S rRNA 2 FISH , ARB probeBase Loy, Frank Maixner, Michael Wagner and Matthias Horn* 2006 probeBase an online resource for rRNA-targeted oligonucleotide probes: new features 2007 ,Nucleic Acids Research, Vol.

5 35, D800 D804 Database ,A., Horn,M. and Wagner,M. (2003) probeBase: an online resourceforrRNA-targeted oligonucleotide probes. Nucleic Acids Res.,31, 514 Ye1*, George Coulouris1 , Irena Zaretskaya1 , IoanaCutcutache2 , Steve Rozen2 and Thomas L Madden1 Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction, Ye et al. BMC Bioinformatics 2012, 13:1344. , , , , .16S rRNA . , 2015, 35(9):2769-2788 1 PCR Inverse PCR, IPCR PCR DNA DNA 3 DNA DNA DNA PCR 2 PCR real-time quantitative PCR.

6 RQ-PCR PCR SYBR SYBR DNA SYBR PCR PCR PCR PCR Taq 5 -3 DNA PCR 3 PCR droplet digital PCR ddPCR ddPCR PCR PCR 1 0 THANKS

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