投影片 1

各位參予本次課程的學員大家好 : 很謝謝您的認真參與讓本次課程順利落幕! 課程中最後介紹的線上工具 :primerx 的應用方向描述有誤, 在此向大家致歉, 並且更正如下 : PrimerX 是設計 mutagenic PCR primers 的線上工具 主要功能是讓使用者可以輸入一段原始序列, 並且指定變異點 PrimerX 便會自動設計出一對 primer 利用這對 primer 去進行 PCR 實驗, 便可以產生一段包含變異點的新序列 最後非常感謝所有在課後提供寶貴建言的學員, 期許這次的課程讓大家都能互相有所進益 敬祝研安

Online Tools for Primer Design and Analysis 林依蓉 20090630 Institute of Biomedical Sciences, Academia Sinica. Phone: +886-2-2782-5258 ext 4323 Email: rong@ibms.sinica.edu.tw

Primer Design Primer Length: 18-22 mer Tm v.s. Ta: Ta (annealing temperature) Tm (melting temperature): Tm should be within 55~65,Tm=2(A+T)+4(G+C) keep the Ta of the primers as close as possible(<5 ) Primer GC content within 45~60% 3' End Stability : It is the maximum ΔG value of the five bases from the 3' end. An unstable 3' end (less negative ΔG) will result in less false priming. Avoid Cross homology : Blast In-Silico PCR

loop or dimer Cross Dimer

Online Tools for Primer Design and Analysis Primer3Plus: http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi GENEPIPE/ PRIMERZ: http://genepipe.ngc.sinica.edu.tw/ NCBI/ Primer-BLAST: http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi ExonPrimer http://ihg2.helmholtz-muenchen.de/ihg/exonprimer.html

PRIMER3

Primer3

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi

APOE :exon3

APOE: exon3 with 200bp franking region

Primer3 plus: input page

Primer3 plus: General Setting

Primer3 plus: result page

Exercise APOE : Exon4 TOMM40 NM_001128916.1 Exon10

Primer Z A Primer Design Tool for Gene and SNP Bioinformatics Team Biomedical IT Core

Primer Z: Introduction PrimerZ is a high throughput web-based system for designing primers of the genes or SNPs. The users can get all primer data of a gene in one page. 16

Originally it took much time and a lot of steps to design the primers of a gene. 17

The workflow of PrimerZ Z 5 upstream 1440bps EXON intron 240bps EXON intron 240bps >360bps? EXON Assignment There will be 1440 base pairs Design primers The 240 bp of flanking retrieved in 5' upstream 5 upstream intron intron which will for Z the gene If there is an exon EXON longer sequence than 360 in EXON bps, each this exon will be be used for designing the primers of program selected 240bps will divide 240bpsthis exon into many part the promoters. so that every part of this exon won't be longer ACE than Maximum Exon Length. 240bps 240bps 5 upstream EXON 360 240bps 360 360 240bps 360 240bps 240bps Exon Flanking sequences Length primer intron EXON Z intron 60 60 EXON EXON primer Excluded Regions EXON Z EXON 18

Primer Z: EXON Z EXON 19

Primer Z home page Design Parameters (I) Users can select an option to design primer for a gene, a SNP, or SNPs by uploading file. Select one sepcies for primer design Enter gene name or Ensembl ID PrimerZ will divide the exon into many part so that every part of this exon won't be longer than Maximum Exon Length. The flanking intron sequence length of target exon. A list of product size ranges, for example 150-250 100-300 301-400 Primer oligos may not overlap any region called Excluded Region. 20

Primer Z: Advance Options

Primer Z: Gene result page (I) Primer s Parameters Graphical index of exons, users can click on it and the page is directly go to selected exon primer section. All primer data of a gene designed by Primer Z will be shown in one page. 22

Gene result page (II) 23

Designing primers for SNPs rs2772 Enter SNP rs ID or Affy_probID Specify the length of sequence that surrounding by the target SNP. 24

Designing primers for SNPs 25

Designing primers for SNPs 26

Error Messages (I) Source Web Server response is too slow or the connection of server and website is lost. 27

Error Messages (II) 28

Error Messages (III) The input Gene name has been found many records in Ensemble database. 29

Exercise http://genepipe.ngc.sinica.edu.tw/ Gene: Brca1 PVRL2 SNPs rs11542036 rs11542029 rs11542031 rs11542038 rs71693603 rs11542040 rs11542033 rs11542037 rs429358

NCBI Primer-BLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi

NCBI Primer-BLAST

NCBI Primer-BLAST

NCBI Primer-BLAST

NCBI Primer-BLAST: result page

NCBI Primer-BLAST : primer reports

Exercise NCBI Primer-BLAST NCBI Reference Sequence: NM_001042724.1 NCBI Reference Sequence: NM_002856.2 NCBI Reference Sequence: NM_012116.3 NCBI Reference Sequence: NM_001130852.1

ExonPrimer http://ihg2.helmholtz-muenchen.de/ihg/exonprimer.html

ExonPrimer

ExonPrimer

注意 : 更正謬誤 PrimerX http://www.bioinformatics.org/primerx/ PrimerX 是設計 mutagenic PCR primers 的線上工具 主要功能是讓使用者可以輸入一段原始序列, 並且指定變異點 PrimerX 便會自動設計出一對 primer 利用這對 primer 去進行 PCR 實驗, 便可以產生一段包含變異點的新序列

PrimerX 注意 : 更正謬誤 輸入一段原始序列 指定變異點

PrimerX 注意 : 更正謬誤 驗證序列資料

PrimerX 利用這對 primer 去進行 PCR 實驗, 便可以產生一段包含變異點的新序列 注意 : 更正謬誤

AutoPrime

Thanks for your attention! Institute of Biomedical Sciences, Academia Sinica. Phone: +886-2-2782-5258 ext 4323 Email: rong@ibms.sinica.edu.tw