NEW YORK (GenomeWeb) – The human genome has an estimated 300,000 known SNPs that lie in regions of high GC content, according to researchers at The Hospital for Sick Children and the University of Toronto. Probe-based PCR assays often fail in these regions, but new research from the SickKids-Toronto team has now shown that adding a pre-amplification step to TaqMan SNP genotyping protocols allows better discrimination.
In a study described this month in BioTechniques, the researchers developed the pre-amplification method for AmpliTaq assays from Thermo Fisher's Life Technologies business, testing it on three SNP variants in regions whose GC content ranged from about 70 to 95 percent.
The modified protocol allowed separation of genotype clusters that, without pre-amplification, appeared as clumps of data from which a genotype call could not be made with confidence. Importantly, adding the pre-PCR step did not appear to bias results toward one allele or the other.
"It’s simple," said Tara Paton, manager of the Genetic Analysis and Sequencing facilities of the Centre for Applied Genomics at the Hospital for Sick Children and an author on the study. "Just one step before [PCR], and you can get better results," she said.
SNPs in GC-rich regions like CpG islands or first exons are notoriously hard to detect, and there may be a paucity of population data on them, Paton said. This could change as whole-genome sequencing "does a better job of covering these regions," she said, but traditional approaches, like microarray studies, may have historically yielded less information for SNPs in GC-rich regions.
"When people are looking at their next-generation data deciding if they want to prioritize a variant based on how common it is, they may be misled into thinking their SNP is rare when in fact it's just not very well studied," noted Paton.
For small numbers of samples, researchers might currently be getting around this issue using sequencing. However, Paton explained that if a researcher wanted to look at a SNP in many hundreds of samples, "sequencing would be cost-prohibitive, not to mention laborious and time consuming."
The Centre at SickKids is a sort of core facility providing research and technical support for scientists from all over the globe, Paton said. "People come to us with the different research projects they want to do, and because of that, we work on a variety of projects and also encounter a variety of situations where we have to be a little creative," she said.
Her group had noticed a pattern in the assay failures over the past ten years — in those failing in the design pipeline as well as formulated assays in the lab — observing that these tended to be assays for SNPs in GC-rich regions.
However, the group wanted to keep using the Life Tech Primer Express software and the TaqMan MGB probes if possible, Paton said.
In standard assays from the company, primers and probes come in pre-mixed formulations, but designing and ordering each of these separately was the key that allowed the group to add the pre-amplification step using the primers alone.
The group then used the resulting PCR product, not genomic DNA, as the input DNA for real-time PCR analysis.
The method may also be more broadly applicable to any allelic discrimination that is probe-based, including microarrays, Paton noted.
Paton said her group did not collaborate with Thermo Fisher on this study, but she did notify the company of the publication. Life Tech, meanwhile, also markets three other products specific for GC-rich regions.
One caveat of the method described in BioTechniques is that researchers still need to identify PCR primers "that are in the 60 to 62 degree [Celsius] melting temperature range in order to be able to amplify," Paton said, and SNP detection may remain challenging if the surrounding region is such that PCR primers can't be placed.
A number of other researchers have recently published protocols using pre-amplification to solve problems with PCR, as reported in GenomeWeb. A team in Australia, for example, included this step to get around problems related to small amounts of DNA run on multiplex STR kits for forensic analyses. The method has also been used to overcome problems inherent in employing formalin-fixed, paraffin-embedded tissue samples for lung cancer diagnosis.
To Paton's knowledge, her group is the first to publish this type of pre-amplification method for SNPs in regions of high GC content. She said she hopes that formalizing the method in the literature will help other researchers.
"There are probably a lot of people who don't think to do this, or just give up on that variant and move on to something else that's a little easier, so maybe our method will make [SNPs in GC-rich regions] a bit more accessible to people," said Paton.
"The more information we can get about the SNPs in these regions, the better the community will be in terms of knowing how important these might be," she added.