Essential Bioin as long as matics Resources as long as Designing PCR Primers in addition to Oligos as long as Various Applications Workshop Outline Primer3 Primer3Plus PrimerZ

Essential Bioin as long as matics Resources as long as Designing PCR Primers in addition to Oligos as long as Various Applications Workshop Outline Primer3 Primer3Plus PrimerZ www.phwiki.com

Essential Bioin as long as matics Resources as long as Designing PCR Primers in addition to Oligos as long as Various Applications Workshop Outline Primer3 Primer3Plus PrimerZ

Doyle, Alicia, Contributor has reference to this Academic Journal, PHwiki organized this Journal Essential Bioin as long as matics Resources as long as Designing PCR Primers in addition to Oligos as long as Various Applications Please complete the workshop sign-in as long as m. Essential Bioin as long as matics Resources as long as Designing PCR Primers in addition to Oligos as long as Various Applications Yi-Bu Chen, Ph.D. Bioin as long as matics Specialist Norris Medical Library University of Southern Cali as long as nia 323-442-3309 yibuchen@belen.hsc.usc.edu Workshop Outline The General Rules as long as PCR Primer Design Resources as long as General Purpose PCR Primer Design Resources as long as Real-Time q-PCR Primer Design Resources as long as Site-Directed Mutagenesis PCR Primer Design Resources as long as PCR Primers/Oligos Quality Analysis Resources as long as Multiplex PCR Primer Design Resources as long as Microarray Probes Design Resources as long as SNPs in addition to Genotyping PCR Applications Resources as long as Degenerate PCR Primer Design Resources Methylation PCR Primer Design

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The Polymerase Chain Reaction (PCR) revolutionized life sciences as it provides a sensitive, reliable, efficient, in addition to convenient means of amplifying relatively large quantities of DNA Invented in 1983 by Kary Mullis, who won a Nobel Prize 1993 The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus, discovered in hot springs. The primary materials used in PCR: – DNA nucleotides: the building blocks as long as the new DNA – Template DNA: the DNA sequence that you want to amplify – Primers: single-str in addition to ed short DNA (16-50 nucleotides long) that are complementary to a short region on either end of the template DNA – DNA polymerase: a heat stable enzyme that catalyzes the synthesis of new DNA PCR: the technology that changed the world we knew Primers dictate the successfulness of a PCR Specificity Proper annealing to the template Be as long as e you design your own primers – Don’t reinvent the wheels!

Be as long as e you start designing primers – Find in addition to use the right resources! What are the primers as long as General purpose amplification SNPs detection/validation Methylation study Real-time PCR Microarray probes Degenerate PCR Multiplex PCR What do you have to begin with Single DNA/protein sequence Multiple DNA/protein sequence files GenBank ID/Gene ID/Gene Symbol/rsSNP ID After you have your primers designed – Consider a second opinion! Most likely your primers can be designed by several different software Different software may vary significantly in: Concepts in addition to overall approaches Designing criteria in addition to default settings Comprehensiveness Usability Accessibility in addition to speed Consider a second opinion when You are new to such design task/application You don’t have a lot of confidence in the initial result General rules as long as primer design – Primer in addition to amplicon length Primer length determines the specificity in addition to significantly affect its annealing to the template Too short – low specificity, resulting in non-specific amplification Too long – decrease the template-binding efficiency at normal annealing temperature due to the higher probability of as long as ming secondary structures such as hairpins. Optimal primer length 18-24 bp as long as general applications 30-35 bp as long as multiplex PCR Optimal amplicon size 300-1000 bp as long as general application, avoid > 3 kb 50-150 bp as long as real-time PCR, avoid > 400 bp

General rules as long as primer design – Melting temperature (Tm) Tm is the temperature at which 50% of the DNA duplex dissociates to become single str in addition to ed Determined by primer length, base composition in addition to concentration. Also affected by the salt concentration of the PCR reaction mix Working approximation: Tm=2(A+T)+4(G+C) (suitable only as long as 18mer or shorter). Optimal melting temperature 52°C- 60°C Tm above 65°C should be generally avoided because of the potential as long as secondary annealing. Higher Tm (75°C- 80°C) is recommended as long as amplifying high GC content targets. Primer pair Tm mismatch Significant primer pair Tm mismatch can lead to poor amplification Desirable Tm difference < 5°C between the primer pair General rules as long as primer design - Specificity in addition to cross homology Specificity Determined primarily by primer length as well as sequence The adequacy of primer specificity is dependent on the nature of the template used in the PCR reaction. Cross homology Cross homology may become a problem when PCR template is genomic DNA or consists of mixed gene fragments. Primers containing highly repetitive sequence are prone to generate non-specific amplicons when amplifying genomic DNA. Avoid non-specific amplification BLASTing PCR primers against NCBI non-redundant sequence database is a common way to avoid designing primers that may amplify non-targeted homologous regions. Primers spanning intron-exon boundaries to avoid non-specific amplification of gDNA due to cDNA contamination. Primers spanning exon-exon boundaries to avoid non-specific amplification cDNA due to gDNA contamination. General rules as long as primer design - GC content; repeats in addition to runs Primer G/C content Optimal G/C content: 45-55% Common G/C content range: 40-60% Runs (single base stretches) Long runs increases mis-priming (non-specific annealing) potential The maximum acceptable number of runs is 4 bp Repeats (consecutive di-nucleotide) Repeats increases mis-priming potential The maximum acceptable number of repeats is 4 di-nucleotide General rules as long as primer design - Primer secondary structures Hairpins Formed via intra-molecular interactions Negatively affect primer-template binding, leading to poor or no amplification Acceptable G (free energy required to break the structure): >-2 kcal/mol as long as 3’end hairpin; >-3 kcal/mol as long as internal hairpin; Self-Dimer (homodimer) Formed by inter-molecular interactions between the two same primers Acceptable G: >-5 kcal/mol as long as 3’end self-dimer; >-6 kcal/mol as long as internal self-dimer; Cross-Dimer (heterodimer) Formed by inter-molecular interactions between the sense in addition to antisense primers Acceptable G: >-5 kcal/mol as long as 3’end cross-dimer; >-6 kcal/mol as long as internal cross-dimer; General rules as long as primer design – GC clamp in addition to max 3’ end stability GC clamp Refers to the presence of G or C within the last 4 bases from the 3’ end of primers Essential as long as preventing mis-priming in addition to enhancing specific primer-template binding Avoid >3 G’s or C’s near the 3’ end Max 3’end stability Refers to the maximum G of the 5 bases from the 3’end of primers. While higher 3’end stability improves priming efficiency, too higher stability could negatively affect specificity because of 3’-terminal partial hybridization induced non-specific extension. Avoid G < -9. General rules as long as primer design - Annealing temperatures in addition to other considerations Ta (Annealing temperature) vs. Tm Ta is determined by the Tm of both primers in addition to amplicons: optimal Ta=0.3 x Tm(primer)+0.7 x Tm(product)-25 General rule: Ta is 5°C lower than Tm Higher Ta enhances specific amplification but may lower yields Crucial in detecting polymorphisms Primer location on template Dictated by the purpose of the experiment For detection purpose, section towards 3’ end may be preferred. When using composite primers Initial calculations in addition to considerations should emphasize on the template-specific part of the primers Consider nested PCR http://www.hsls.pitt.edu/guides/genetics/obrc http://www.usc.edu/hsc/nml/lib-services/bioin as long as matics/index.html http://search.hsls.pitt.edu/vivisimo/cgi-bin/query-metainput- as long as m=molbio-simple&query=pcr+primer&v%3Asources=OBRC&v%3Aproject=molbio Resources as long as General Purpose PCR Primer Design Primer3 Primer3Plus PrimerZ PerlPrimer Vector NTI Advantage 10 General Purpose PCR Primer Design Tool– Primer3 Web Site: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3-www.cgi More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1043858198/info General Purpose PCR Primer Design Tool– Primer3Plus Web Site: http://www.bioin as long as matics.nl/primer3plus More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1191263055/info General Purpose PCR Primer Design Tool– PrimerZ Web Site: http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1190992855/info General Purpose PCR Primer Design Tool – PerlPrimer Web Site: http://perlprimer.source as long as ge.net/index.html PerlPrimer screenshots: http://perlprimer.source as long as ge.net/screenshots.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1167845497/info General Purpose PCR Primer Design Tool– Vector NTI Advance 10 Web Site as long as NML Workshop: http://www.usc.edu/hsc/nml/lib-services/bioin as long as matics/vector-nti-advance-10-workshop.html More Info On Vector NTI Advance 10: http://www.usc.edu/hsc/nml/lib-services/bioin as long as matics/vector-nti-advance-10.html Primer Design Resources as long as Real-time PCR NCBI Probe Database RTPrimerDB Primer Bank qPrimerDepot PCR-QPPD PerlPrimer Doyle, Alicia Ventura County Star Contributor www.phwiki.com

Public PCR Primers/Oligo Probes Repository – The NCBI Probe Database Web Site: Database Overview: http://www.ncbi.nlm.nih.gov/genome/probe/doc/Overview.shtml Database Query Tips: http://www.ncbi.nlm.nih.gov/genome/probe/doc/QueryTips.shtml http://www.ncbi.nlm.nih.gov/sites/entrezdb=probe Resources as long as real time PCR– RTPrimerDB Web Site: http://medgen.ugent.be/rtprimerdb/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1099597360/info

Resources as long as real time PCR– Primer Bank Web Site: http://pga.mgh.harvard.edu/primerbank/ More Info: http://www.ncbi.nlm.nih.gov/sites/entrezDb=pubmed&Cmd=ShowDetailView&TermToSearch=14654707 Resources as long as real time PCR– qPrimerDepot Web Site as long as Human Genes: http://primerdepot.nci.nih.gov/ Web Site as long as Mouse Genes: http://mouseprimerdepot.nci.nih.gov/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1174922412/info Resources as long as real time PCR– QPPD Web Site: http://web.ncifcrf.gov/rtp/GEL/primerdb/default.asp More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1152117830/info

Primer Design Tools as long as Methylation PCR– PerlPrimer Web Site: http://perlprimer.source as long as ge.net/index.html PerlPrimer screenshots: http://perlprimer.source as long as ge.net/screenshots.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr-oligos/URL1167845497/info Please evaluate this workshop to help me improving future presentations: http://www.zoomerang.com/survey.zgip=WEB2277FTDR3AJ Have questions or comments about this workshop Please contact: Yi-Bu Chen, Ph.D. Bioin as long as matics Specialist Norris Medical Library University of Southern Cali as long as nia 323-442-3309 yibuchen@belen.hsc.usc.edu Useful web sites as long as design degenerate PCR primers http://boneslab.bio.ntnu.no/degpcrshortguide.htm http://info.med.yale.edu/mbb/koelle/protocols/protocol-degenerate-PCR.html http://www.mcb.uct.ac.za//pcroptim.htm Degenerate http://www.protocol-online.org/prot/Molecular-Biology/PCR/Degenerate-PCR/ http://cgat.ukm.my/protease/degpcr.html

Doyle, Alicia Contributor

Doyle, Alicia is from United States and they belong to Ventura County Star and they are from  Camarillo, United States got related to this Particular Journal. and Doyle, Alicia deal with the subjects like Local News; Regional News

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