Essential Bioinformatics Resources for Designing PCR Primers and Oligos for Various Applications

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Essential Bioinformatics Resources for Designing
PCR Primers and Oligos for Various Applications
Please complete the workshop sign-in form.
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Essential Bioinformatics Resources for Designing
PCR Primers and Oligos for Various Applications
Yi-Bu Chen, Ph.D. Bioinformatics Specialist
Norris Medical Library University of Southern
California 323-442-3309 yibuchen_at_belen.hsc.usc.e
du
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Workshop Outline

1. The General Rules for PCR Primer Design
2. Resources for General Purpose PCR Primer Design
3. Resources for Real-Time q-PCR Primer Design
4. Resources for Site-Directed Mutagenesis PCR
   Primer Design
5. Resources for PCR Primers/Oligos Quality Analysis
6. Resources for Multiplex PCR Primer Design
7. Resources for Microarray Probes Design
8. Resources for SNPs and Genotyping PCR
   Applications
9. Resources for Degenerate PCR Primer Design
10. Resources Methylation PCR Primer Design

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PCR the technology that changed the world we knew

• The Polymerase Chain Reaction (PCR)
  revolutionized life sciences as it provides a
  sensitive, reliable, efficient, and 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 for the
  new DNA
• - Template DNA the DNA sequence that you want
  to amplify
• - Primers single-stranded 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

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Primers dictate the successfulness of a PCR
Specificity?
Proper annealing to the template?
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Before you design your own primers Dont
reinvent the wheels!
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Before you start designing primers Find and
use the right resources!

• What are the primers for?
• 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?

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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 and overall approaches
• Designing criteria and default settings
• Comprehensiveness
• Usability
• Accessibility and speed
• Consider a second opinion when
• You are new to such design task/application
• You dont have a lot of confidence in the initial
  result

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General rules for primer design-- Primer and
amplicon length

• Primer length determines the specificity and
  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 forming secondary
  structures such as hairpins.
• Optimal primer length
• 18-24 bp for general applications
• 30-35 bp for multiplex PCR
• Optimal amplicon size
• 300-1000 bp for general application, avoid gt 3 kb
• 50-150 bp for real-time PCR, avoid gt 400 bp

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General rules for primer design-- Melting
temperature (Tm)

• Tm is the temperature at which 50 of the DNA
  duplex dissociates to become single stranded
• Determined by primer length, base composition and
  concentration.
• Also affected by the salt concentration of the
  PCR reaction mix
• Working approximation Tm2(AT)4(GC) (suitable
  only for 18mer or shorter).
• Optimal melting temperature
• 52C-- 60C
• Tm above 65C should be generally avoided because
  of the potential for secondary annealing.
• Higher Tm (75C-- 80C) is recommended for
  amplifying high GC content targets.
• Primer pair Tm mismatch
• Significant primer pair Tm mismatch can lead to
  poor amplification
• Desirable Tm difference lt 5C between the primer
  pair

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General rules for primer design-- Specificity
and 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.

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General rules for primer design-- GC content
repeats and 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

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General rules for 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) gt-2 kcal/mol for 3end hairpin gt-3
  kcal/mol for internal hairpin
• Self-Dimer (homodimer)
• Formed by inter-molecular interactions between
  the two same primers
• Acceptable ?G gt-5 kcal/mol for 3end self-dimer
  gt-6 kcal/mol for internal self-dimer
• Cross-Dimer (heterodimer)
• Formed by inter-molecular interactions between
  the sense and antisense primers
• Acceptable ?G gt-5 kcal/mol for 3end
  cross-dimer gt-6 kcal/mol for internal
  cross-dimer

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General rules for primer design-- GC clamp and
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 for preventing mis-priming and
  enhancing specific primer-template binding
• Avoid gt3 Gs or Cs near the 3 end
• Max 3end stability
• Refers to the maximum ?G of the 5 bases from the
  3end of primers.
• While higher 3end stability improves priming
  efficiency, too higher stability could negatively
  affect specificity because of 3-terminal partial
  hybridization induced non-specific extension.
• Avoid ?G lt -9.

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General rules for primer design-- Annealing
temperatures and other considerations

• Ta (Annealing temperature) vs. Tm
• Ta is determined by the Tm of both primers and
  amplicons
• optimal Ta0.3 x Tm(primer)0.7 x Tm(product)-25
• General rule Ta is 5C 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 and considerations should
  emphasize on the template-specific part of the
  primers
• Consider nested PCR

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http//www.hsls.pitt.edu/guides/genetics/obrc
http//www.usc.edu/hsc/nml/lib-services/bioinforma
tics/index.html
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http//search.hsls.pitt.edu/vivisimo/cgi-bin/query
-meta?input-formmolbio-simplequerypcrprimerv
3AsourcesOBRCv3Aprojectmolbio
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Resources for General Purpose PCR Primer Design

• Primer3
• Primer3Plus
• PrimerZ
• PerlPrimer
• Vector NTI Advantage 10

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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.e
du/guides/genetics/obrc/dna/pcr_oligos/URL10438581
98/info
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General Purpose PCR Primer Design Tool
Primer3Plus
Web Site http//www.bioinformatics.nl/primer3plus
More Info http//www.hsls.pitt.edu/guides/genet
ics/obrc/dna/pcr_oligos/URL1191263055/info
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General Purpose PCR Primer Design Tool PrimerZ
Web Site http//genepipe.ngc.sinica.edu.tw/primer
z/beginDesign.do More Info http//www.hsls.pitt.
edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992
855/info
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General Purpose PCR Primer Design Tool
PerlPrimer
Web Site http//perlprimer.sourceforge.net/index.
html PerlPrimer screenshots http//perlprimer.so
urceforge.net/screenshots.html More Info
http//www.hsls.pitt.edu/guides/genetics/obrc/dna
/pcr_oligos/URL1167845497/info
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General Purpose PCR Primer Design Tool Vector
NTI Advance 10
Web Site for NML Workshop http//www.usc.edu/hsc
/nml/lib-services/bioinformatics/vector_nti_advanc
e_10_workshop.html More Info On Vector NTI
Advance 10 http//www.usc.edu/hsc/nml/lib-servic
es/bioinformatics/vector_nti_advance_10.html
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Primer Design Resources for Real-time PCR

• NCBI Probe Database
• RTPrimerDB
• Primer Bank
• qPrimerDepot
• PCR-QPPD
• PerlPrimer

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Public PCR Primers/Oligo Probes Repository The
NCBI Probe Database
Web Site Database Overview
http//www.ncbi.nlm.nih.gov/genome/probe/doc/Over
view.shtml Database Query Tips
http//www.ncbi.nlm.nih.gov/genome/probe/doc/Quer
yTips.shtml
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http//www.ncbi.nlm.nih.gov/sites/entrez?dbprobe
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Resources for real time PCR RTPrimerDB
Web Site http//medgen.ugent.be/rtprimerdb/
More Info http//www.hsls.pitt.edu/guides/geneti
cs/obrc/dna/pcr_oligos/URL1099597360/info
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Resources for real time PCR Primer Bank
Web Site http//pga.mgh.harvard.edu/primerbank/ M
ore Info http//www.ncbi.nlm.nih.gov/sites/entrez
?DbpubmedCmdShowDetailViewTermToSearch1465470
7
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Resources for real time PCR qPrimerDepot
Web Site for Human Genes http//primerdepot.nci.n
ih.gov/ Web Site for Mouse Genes http//mousepr
imerdepot.nci.nih.gov/ More Info
http//www.hsls.pitt.edu/guides/genetics/obrc/dna
/pcr_oligos/URL1174922412/info
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Resources for 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
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Resources for real time PCR PerlPrimer
Web Site http//perlprimer.sourceforge.net/index.
html PerlPrimer screenshots http//perlprimer.so
urceforge.net/screenshots.html More Info
http//www.hsls.pitt.edu/guides/genetics/obrc/dna
/pcr_oligos/URL1167845497/info
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Resources for Site-Directed Mutagenesis PCR
PrimerX
Web Site http//www.bioinformatics.org/primerx/
More Info http//www.hsls.pitt.edu/guides/gene
tics/obrc/dna/pcr_oligos/URL1175091818/info
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Resources for PCR Primer or Oligo Analysis

• AutoDimer
• IDT OligoAnalyzer 3.0
• PUNS
• NCBI BLAST
• UCSC In-Silico PCR

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Resources for PCR Primer or Oligo Analysis
AutoDimer
Web Site http//www.cstl.nist.gov/div831/strbase/
AutoDimerHomepage/AutoDimerProgramHomepage.htm
More Info http//www.hsls.pitt.edu/guides/gene
tics/obrc/dna/pcr_oligos/URL1154964478/info
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Resources for PCR Primer or Oligo AnalysisIDT
OligoAnalyzer 3.0
Web Site http//www.idtdna.com/analyzer/Applicati
ons/OligoAnalyzer/ Online Instruction
http//www.idtdna.com/Analyzer/Applications/Instr
uctions/Default.aspx?AnalyzerInstructionstrue
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Resources for PCR Primer Specificity Analysis
PUNS
Web Site http//okeylabimac.med.utoronto.ca/PUNS/
More Info http//www.hsls.pitt.edu/guides/gene
tics/obrc/dna/pcr_oligos/URL1175092441/info
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Resources for PCR Primer Specificity Analysis
NCBI BLAST
http//www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGEN
ucleotidesPROGRAMblastnMEGABLASTonBLAST_PROGR
AMSmegaBlastPAGE_TYPEBlastSearchSHOW_DEFAULTS
on
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Resources for PCR Primer Mapping UCSC In-Silico
PCR
http//genome.ucsc.edu/cgi-bin/hgPcr?dbmm9
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Resources for PCR Primer Mapping/Amplicon Size
SMS Tool
http//www.bioinformatics.org/sms2/pcr_products.ht
ml
http//www.bioinformatics.org/sms2/index.html
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Please evaluate this workshop to help me
improving future presentations http//www.zoomera
ng.com/survey.zgi?pWEB2277FTDR3AJ Have
questions or comments about this workshop?
Please contact Yi-Bu Chen, Ph.D. Bioinformatics
Specialist Norris Medical Library University of
Southern California 323-442-3309 yibuchen_at_belen.h
sc.usc.edu
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Primer Design Tools for Multiplex PCR

• MultiPLX
• PrimerStation

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Primer Design Tools for Multiplex PCR MultiPLX
Web Site http//bioinfo.ebc.ee/multiplx/ More
Info http//www.ncbi.nlm.nih.gov/sites/entrez?cmd
RetrievedbpubmeddoptAbstractPluslist_uids15
598831
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Primer Design Tools for Multiplex PCR
PrimerStation
Web Site http//ps.cb.k.u-tokyo.ac.jp/index.html
More Info http//www.hsls.pitt.edu/guides/geneti
cs/obrc/dna/pcr_oligos/URL1154793164/info
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Resources for Microarray Probe Design

• NCBI Probe Database
• OligoWiz 2.0
• ROSO
• YODA

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Resources for Microarray Probe Design OligoWiz
2.0
Web Site http//www.cbs.dtu.dk/services/OligoWiz2
/ More Info http//www.hsls.pitt.edu/guides/gen
etics/obrc/gene_expression/microarray_design_probe
s/URL1118767631/info
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Resources for Microarray Probe Design ROSO
Web Site http//pbil.univ-lyon1.fr/roso/Home.php
More Info http//www.ncbi.nlm.nih.gov/sites/ent
rez?DbpubmedCmdShowDetailViewTermToSearch1473
4320
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Resources for Microarray Probe Design YODA
Web Site http//pathport.vbi.vt.edu/YODA/ More
Info http//bioinformatics.oxfordjournals.org/cg
i/content/full/21/8/1365
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PCR Primer Design Resources for SNPs and
Genotyping Purposes

• NCBI Probe Database
• PrimerZ
• MuPlex
• SNPBox

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Public PCR Primers/Oligo Probes Repository The
NCBI Probe Database
Web Site Database Overview
http//www.ncbi.nlm.nih.gov/genome/probe/doc/Over
view.shtml Database Query Tips
http//www.ncbi.nlm.nih.gov/genome/probe/doc/Quer
yTips.shtml
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PCR Primer Design Resources for SNPs and
Genotyping Purposes PrimerZ
Web Site http//genepipe.ngc.sinica.edu.tw/primer
z/beginDesign.do More Info http//www.hsls.pitt.
edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992
855/info
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PCR Primer Design Tools for SNPs and Genotyping
Purposes MuPlex
Web Site http//genomics14.bu.edu8080/MuPlex/MuP
lex.html More Info http//www.hsls.pitt.edu/guid
es/genetics/obrc/dna/pcr_oligos/URL1135006767/info

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PCR Primer Design Tools for SNPs and Genotyping
Purposes SNPBox
Web Site http//www.snpbox.org/ More Info
http//www.hsls.pitt.edu/guides/genetics/obrc/dna/
pcr_oligos/URL1097782408/info
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Primer Design Tools for Degenerate PCR

• Primaclade
• GeneFisher2
• CODEHOP

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Primer Design Tools for Degenerate PCR Primaclade
Web Site http//www.umsl.edu/services/kellogg/pri
maclade.html More Info http//www.hsls.pitt.edu/
guides/genetics/obrc/dna/pcr_oligos/URL1167846864/
info
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Primer Design Tools for Degenerate PCR
GeneFisher2
Web Site http//bibiserv.techfak.uni-bielefeld.de
/genefisher2/ More Info http//www.ncbi.nlm.nih.
gov/sites/entrez?DbpubmedCmdShowDetailViewTerm
ToSearch8877506
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Primer Design Tools for Degenerate PCR CODEHOP
Web Site http//blocks.fhcrc.org/codehop.html
More Info http//www.hsls.pitt.edu/guides/geneti
cs/obrc/dna/pcr_oligos/URL1118954832/info
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Primer Design Resources for Methylation PCR

• MethPrimer
• methBLAST and methPrimerDB
• BiSearch
• PerlPrimer

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Primer Design Resources for Methylation PCR
MethPrimer
Web Site http//www.urogene.org/methprimer/
More Info http//www.hsls.pitt.edu/guides/geneti
cs/obrc/dna/pcr_oligos/URL1167846108/info
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Primer Design Tools for Methylation PCR
methBLAST and methPrimerDB
methPrimerDB Web Site http//medgen.ugent.be/meth
primerdb/ methBLAST Web Site http//medgen.ugent
.be/methBLAST/ More Info http//www.biomedcentr
al.com/1471-2105/7/496
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Primer Design Tools for Methylation PCR
BiSearch
Web Site http//bisearch.enzim.hu/ More Info
http//www.ncbi.nlm.nih.gov/sites/entrez?Dbpubme
dCmdShowDetailViewTermToSearch15653630
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Primer Design Tools for Methylation PCR
PerlPrimer
Web Site http//perlprimer.sourceforge.net/index.
html PerlPrimer screenshots http//perlprimer.so
urceforge.net/screenshots.html More Info
http//www.hsls.pitt.edu/guides/genetics/obrc/dna
/pcr_oligos/URL1167845497/info
62
Please evaluate this workshop to help me
improving future presentations http//www.zoomera
ng.com/survey.zgi?pWEB2277FTDR3AJ Have
questions or comments about this workshop?
Please contact Yi-Bu Chen, Ph.D. Bioinformatics
Specialist Norris Medical Library University of
Southern California 323-442-3309 yibuchen_at_belen.h
sc.usc.edu
63
Useful web sites for design degenerate PCR primers
http//boneslab.bio.ntnu.no/degpcrshortguide.htm
http//info.med.yale.edu/mbb/koelle/protocols/prot
ocol_degenerate_PCR.html
http//www.mcb.uct.ac.za//pcroptim.htmDegenerate
http//www.protocol-online.org/prot/Molecular_Biol
ogy/PCR/Degenerate_PCR/
http//cgat.ukm.my/protease/degpcr.html