Enhanced Diagnostic Tools
For regular DNA primers: Design primers that are specific to the 3’ end nucleotide of the SNP and the 3rd nucleotide from the 3’ end is mismatched with the template DNA.
Design primers that are specific to the 2nd nucleotide from the 3’ end of the SNP and the 3rd nucleotide from the 3’ end is mismatched with the template DNA.
To avoid the “pseudopositive problem”: Figure 2 illustrates the concepts in the rational design of biological active peptides and peptidomimetics.
Design primers that are specific to the 3’ nucleotide of the SNP and the other two nucleotides located downstream from the location of the SNP are mismatched with the template DNA.
For BNA based primers: Add one BNA/LNA monomer per primer at the 3’ end where the SNP is located.
To address the “pseudopositive problem” and to minimize the “pseudopositive” signal the primers were designed by Yaku et al. in 2008 with a 3’ nucleotide matching the template plus 2 mismatch nucleotides.
Additionally, Yaku et al. showed that selecting a primer having a 3’ terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence, can be used as an allele-specific primer to eliminate the pseudopositive problem.
Using primers for the human ABO genes, the researchers demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.
However, the literature citing experimental data indicates that the proper design of primer DNA sequences is important for the efficient detection of SNP by PCR. Allele-specific primers are usually designed to complement template DNA and contain a nucleotide specific to the SNP at the 3’ end.
The SNP-specific nucleotide forms a base pairing or mismatch pairing depending on the base pair identity of the SNP. Only a proper base pairing at the end of the primer/template duplex will produce a PCR product. Less PCR products are produced for terminal mismatch pairings due to decreased DNA polymerase binding and inefficiencies in the incorporation of 2’-deoxyribonucleoside triphosphates. However, undesired primer extension with mismatched DNA primers can occur during the PCR reaction under unsuitable reaction conditions when the proper amplification cycle, reaction time, temperature, and 2’-deoxyribonucleoside triphosphate concentrations are not optimized. This is called the “pseudopositive problem” that can also arise due to specific DNA primer sequences. Primer extension reactions are often observed when a single mismatch is formed at the 3’ end with the primer. When SNP typing is used to distinguish homozygotes and heterozygotes, this problem becomes more serious since the pseudopositive signal should be less than 1% of those obtained with matched primers. Therefore, allele-specific primers have been designed so that the 3’ end nucleotide was specific to the SNP and the 3rd nucleotide from the 3’ end was mismatched with the template DNA (Kambaraet al., 2004). Another research group developed primers so that the 2nd nucleotide from the 3’ end was specific to the SNP and the 3rd nucleotide from the 3’ end was mismatched with the template DNA (Aono et al., 2000). Methods using locked nucleic acids (LNA), phosphorothioate-modified primer, and dideoxynucleotide-terminated primer have been reported.
Taq Polymerase ->
X1 = A, T, or C; X2X3 = CA, CT, CC, AA, AT, AC, TA, TT, OR TC
<- Taq Polymerase
1 TAGGAAGGAT GTCCTCGTGG TGACCCCTTG GCTGGCTCCC
41 ATTGTCTGGG AGGGCACATT CAACATCGAC ATCCTCAACG
81 AGCAGTTCAG GCTCCAGAAC ACCACCATTG GGTTAACTGT
121 GTTTGCCATC AAGAA
1 TAGGAAGGAT GTCCTCGTGG TACCCCTTGG CTGGCTCCCA
41 TTGTCTGGGA GGGCACATTC AACATCGACA TCCTCAACGA
81 GCAGTTCAGG CTCCAGAACA CCACCATTGG GTTAACTGTG
121 TTTGCCATCA AGAA
The selected target for human blood genotyping was the 22nd base pair in exon 6 of the ABO. This base pair is a G/C base pair in the A and B alleles and an A/T base pair in the O allele due to deletion of the G/C base pair found in the A and B alleles.
The 22nd, base pair in exon 6 is the homo G/C base pair for blood type AB and the homo A/T base pair for blood type O.
A two-step PCR was carried out to detect allelic difference in the 22nd base pair of exon 6 of the ABO gene.
(Source: Yaku et al., 2008)
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