• Primer3 program at the Whitehead Institute is the most reliable and versatile tool currently available.
Primers for a COL3A1 variant
• The human COL3A1 gene has a variant at amino acid 531 of the triple helix.
• Ala or Thr encoded in exon 31 of the gene.
• AluI restriction enzyme site present in the Ala allele but absent in the Thr allele.
• PCR amplify the region and genotype by digestion of PCR products with AluI.
Running Primer3
• Paste the DNA sequence into Primer3 with the “target” enclosed in square brackets.
• Select a mispriming library — only human and rodent available at present.
• Select option for a 1-base 3´ “GC Clamp”.
• Select PCR product size range (>600 bp).
• Click the “Pick Primers” button.
• Marvel at the ease and simplicity.
The COL3A1 Ala/Thr PCR
• The PCR primers amplify from the start of exon 31 to just beyond exon 33 — 656 bp.
• Ala alleles are digested by AluI, producing fragments of 82 & 574 bp.
Will Other Genes Amplify Too?
• The primers have been designed on the
basis of the DNA sequence of a single gene.
• Might the primers also amplify other
segments whose sequence we have not
taken into account?
• Need to consider the sequence of the entire genome to answer this.
Virtual PCR Results
• Virtual PCR searches entire genome looking for potential primer sites within 10,000 bases of one another.
• If found, it performs a virtual PCR reaction.
• Primers for Ala/Thr polymorphism in human COL3A1.
Applications of PCR
• Mutation testing, e.g. cystic fibrosis.
• Diagnosis or screening of acquired diseases,e.g. AIDS.
• Genetic profiling in forensic, legal and biodiversity applications.
• Site-directed mutagenesis of genes.
• Quantitation of mRNA in cells or tissues.
Multiplex PCR
• PCR reactions can be devised in which several targets are amplified simultaneously
—often used in diagnostic applications.
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