51
Views
3
CrossRef citations to date
0
Altmetric
TECHNICAL NOTE

Genotyping of the reduced folate carrier‐1 c.80G>A polymorphism by pyrosequencing technology: Importance of PCR and pre‐PCR optimization

, &
Pages 166-170 | Received 27 May 2007, Accepted 10 Jul 2007, Published online: 08 Jul 2009
 

Abstract

When developing a genotyping assay by Pyrosequencing technology for the RFC1 (SLC19A1) c.80G>A polymorphism (rs1051266), unequal peak heights in the pyrograms were observed, probably due to unequal amplification of the mutated and wild‐type alleles. This rarely occurring problem could potentially render assignment of heterozygous genotypes uncertain. When the PCR conditions were studied, it was found that substitution of the dGTP nucleotide in the master mix by dGTP and dITP in proportion 1:1 largely overcame this problem. Heat denaturation of the DNA at 95°C before PCR also counteracted the problem. A combination of these two modifications of the standard pyrosequencing PCR protocol gave the best results. We conclude that, with these modifications, the RFC1 c.80G>A SNP can be reliably assayed by pyrosequencing.

Acknowledgement

Financial support was obtained from Nyckelfonden, Örebro and Forskningskommittén, Örebro county council.

Reprints and Corporate Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

To request a reprint or corporate permissions for this article, please click on the relevant link below:

Academic Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

Obtain permissions instantly via Rightslink by clicking on the button below:

If you are unable to obtain permissions via Rightslink, please complete and submit this Permissions form. For more information, please visit our Permissions help page.