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RExPrimerPongsakorn Wangkumhang, M.Sc.
Biostatistics and Informatics Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand
Motivations• de facto Primer3 program has limitations• Graphical user interface for visualizing gene
structure is crucial for the resequencing primer design
• Existing primer designing tools do not consider▫ SNP-in-Primer
mis-matching problem▫ Pseudogene form
mis-priming problem▫ Structural variation (CNV)
mismatching (deletion) and mis-priming (duplication)
Generic steps for primer design
•Identify target sequence▫Genome sequence, intron/exon boundaries
•Design primers▫Use de facto Primer3
•Check for Specificity▫Use BLAST, primerBLAST or UCSC In-Silico
PCR
What are the tools out there?
• PrimerZ [Tsai et al, 2007 ]• EasyExonPrimer [Wu and Munroe, 2006 ]• MutScreener [Yao et al, 2006 ]• VariantSEQr [Applied Biosystem, 2005]• ELXR [Schageman et al, 2004]• SNPbox [Weckx , 2004]• ExonPrimer (ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html)• Genomic Primer (www-fgg.eur.nl/kgen/primer/Genomic_Primers.html)
Software FunctionalityPrimerZ
MutScreener
EasyExonPrimer
VariantSEQr
ELXRSNPbox
ExonPrimer
Genomic Primer
Sequence information retrievals
and annotations + + + - + + - -Promoter
resequencing ++ ++ - ++ - ++ - -Continuous genomic DNA resequencing - ++ - - - ++ - -
Combining two short exons as one
template - + - - - - ++ -Overlapping primers
for large exons ++ ++ ++ ++++ - ++ -
Redesign + - - - - - - -++ : Very good + : Available with limitation- : Not available
Problems to be solved!!•Mis-matching (No amplification due to
variation in primer)▫SNP-in-primer▫Insertion/deletion (indel) polymorphisms▫Copy number variation (CNV)
•Mis-priming (Non-target homology seq. binding) ▫Structural complexity of the genome ▫Pseudogene forms▫Chromosome segmental duplications▫Repetitive elements (e.g. SINES, LINES,
satellite sequences, etc.)
Why RExPrimer (features must have)•DNA Resequencing/SNP genotyping
primer design•Integrated to Human genome database,
variation databases•Intuitive and comprehensive visualization
for avoiding unwanted regions at first•Re-designable to escape previous
unwanted regions
RExPrimer Workflow
Case Study : CYP2D6 Resequencing
• Cytochrome P450 2D6 gene• Important drug metabolizing enzyme• Ethnic-specific resequencing/genotyping CYP2D6 is
crucial for medical treatments• Highly polymorphic i.e. SNPs, CNVs and Pseudogenes• Pseudogenes, CYP2D7P and CYP2D8P, share 98%
homology with CYP2D6
Case Study: CYP2D6 Resequencing
SNP Genotyping
DNA Resequencing
SNP Genotyping
DNA ResequencingCYP2D6
Case Study: CYP2D6 Resequencing
Gene location of CYP2D6 and its pseudogenes
Case Study: CYP2D6 Resequencing
Gene location of CYP2D6 and its pseudogenesChromosome mapping of CYP2D6 and its pseudogenes
Case Study: CYP2D6 Resequencing
Gene location of CYP2D6 and its pseudogenesChromosome mapping of CYP2D6 and its pseudogenes
Multiple sequence alignment of CYP2D6 and its pseudogenes
CYP2D6 gene
Case Study: CYP2D6 Resequencing
pseudogenes
Case Study: CYP2D6 Resequencing
Example Scenario: CYP2D6 Resequencing
CYP2D6 gene
pseudogenes
CNVs
2191
8000..2191
9500
Example Scenario: CYP2D6 Resequencing
219126902191292
0
Example Scenario: CYP2D6 Resequencing
CYP2D6 gene
pseudogenes
CNVs
RExPrimer Results
Experimental Validation
Name Sequence 5'→ 3'
CYP2D6_1F GGCCTACCCTGGGTAAGGGCCTGGAGCAGGA
CYP2D6_1R CTCAGCCTCAACGTACCCCTGTCTCAAATGCG
CYP2D6_2F GAGACTCCTCGGTCTCTCG
CYP2D6_2R TAATGCCTTCATGGCCACGCG
CYP2D6_3F AGGCCTTCCTGGCAGAGATGAAG
CYP2D6_3R CCCCTGCACTGTTTCCCAGA
CYP2D6_4F CCAGCCACCATGGTGTCTTTG
CYP2D6_4R GCCTCAACGTACCCCTGTCTC
CYP2D6_5F GTACCCCTGTCTCAAATGCG
CYP2D6_5R GTAAGGGCCTGGAGCAGGA
Purpose
whole gene amplification
exon 1-2 amplification
exon 9 amplification
exon 3-5 PCR amplification
exon 6-8 amplification
Amplification Results
Whole gene amplicon of CYP2D6 gene amplificationfrom 5 diff samples
4.6 kb
468 bp
624 bp 860 bp 1180 bp
1 2 3 4 5
Exon 1-2
Exon 3-5
Exon 6-8
Exon 9
100 bp M
1
2 Nested PCR yieldsexon 1-9 amplicon
Conclusions
RExPrimer is successful at :• designing resequencing primers; specific to the
target gene • Guiding/assisting users to make the PCR
experimental design• All-in-one graphic user-interface of
gene structure view alignment true/pseudogenes SNP from several ethnics CNVs
• www4a.biotec.or.th/rexprimer
Future Work
Currently adding more organisms to the RExPrimer platform
Bovine Porcine Mouse Canine Chicken Horse Chimpanzee
Acknowledgements
• Jittima Piriyapongsa• Chumpol Ngamphiw• Anunchai Assawamakin• Payiarat Suwannasri• Uttapong Ruangrit• Sissades Tongsima• Philip J. Shaw • BIOTEC• Siriraj Hospital, Mahidol
University
Thank you for your attention
Questions?