Part:BBa_K3464002

Reverse Primer for the detection of the rs4149056

The rs4149056 is located on the gene SLCO1B1 on chromosome 12 (Chromosome 12:21178615 forward strand). The alleles for this single nucleotide variant are T/C with the T as the ancestral allele (wild type). The primer is designed to amplify a fragment of SLCO1B1 gene (Part:BBa_K3464003) along with its forward primers (Part:BBa_K3464000) (Part:BBa_K3464001). The Reverse primer (Part:BBa_K3464002) is placed further downstream on the gene and does not contain the targeted variant in its sequence.

Usage and Biology

In our project, to detect the two alleles of the rs4149056, was used the allele-specific Polymerase Chain Reaction (AS-PCR). The principle of the AS-PCR is based on the modification of the primers for different alleles: the 3’ end of the primers is modified so as the one set of the primers (Part:BBa_K3464000) (Part:BBa_K3464001) is designed to amplify the normal allele and the other one the alternative. The mismatch single base is introduced at the 3’ end of the primer and allows each primer to amplify one single allele. The expected product length from the Forward Primers along with the Reverse primer when there was no mismatch is 535bp.

The mismatch of the last base of the primer is the key factor in achieving the amplification. If the mismatch near the 3’ end is weak the chance of amplification is higher, whereas, a strong mismatch near the 3’ end in the non-complementary allele terminates the amplification of the gene fragment, since it’s not complementary to the target DNA sequence.

Figure 1. AS-PCR products
Sequence & Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]