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How to genotype low-penetrance SNPs in non-coding, distal regulatory regions

rhAmp SNP Genotyping System helps link data from genome-wide association study to HIV susceptibility genes

In an approach that combines genome-wide association study (GWAS) data with genome wide functional data, a senior scientist at Rotterdam’s University Medical Center is genotyping low penetrance single nucleotide polymorphisms (SNPs) in distal non-coding DNA to identify HIV susceptibility genes and their regulatory regions. Read why Dr Palstra and colleagues chose the rhAmp SNP Genotyping System to pursue variants present at very low frequency.

Dr Robert-Jan Palstra, Senior Scientist at Erasmus University Medical Center, Rotterdam, the Netherlands, had limited experience with genotyping methods but wanted to identify a link between variation in the human genome and HIV susceptibility. He chose the rhAmp SNP Genotyping System as an easy-to-use way to enter this technology.

“Our research had relied on RFLP analysis. The rhAmp SNP Genotyping System is easy to use, and it saves me a lot of time.”
– Dr Robert-Jan Palstra

SNP analysis: A common problem in GWAS

In his early academic career, Dr Palstra studied how gene regulatory elements located far from the genes they regulate (i.e., distal regulatory elements) are brought into juxtaposition with those genes via chromatin loops. Working with Prof. Manfred Kayser, Head of the Genetic Identification Department at Erasmus University Medical Center, the researchers used iPLEX® assays (Sequenom) and restriction fragment length polymorphism (RFLP) to identify individuals that carried a variant located in a non-coding region that correlated with eye, skin, and hair color.

In general, GWAS studies suffer from several limitations; SNP variants associated with specific traits are:

  1. Often located in non-coding regions, so how they influence a trait is obscure
  2. Low-penetrance SNPs, thus requiring large cohorts to provide statistically significant results, which then only show a statistical association, and not a mechanism of action

An innovative, PCR-based SNP genotyping system

In an approach combining GWAS data with genome-wide functional data to assign function to specific gene regulation, Dr Palstra and colleagues pursue variants present at very low frequency. Their current model is HIV susceptibility genes.

Palstra and colleagues genotyped large numbers of primary CD4+ cell samples isolated from donors. They looked at up to 8 variant assays per sample across 80–100 samples and found the rhAmp SNP Genotyping System very convenient for this purpose. While their experiments are ongoing, they hope to correlate expression of specific variants with regulation of gene expression in their samples, and then extend their findings to other samples and variants.

Palstra notes,

“When genotyping, it is, of course, important to optimize the amount of input DNA. We have been successful using low input amounts (4 ng). Once this factor was determined, we have been very satisfied with the rhAmp SNP Genotyping System’s consistent performance. We have also found the custom double-stranded DNA fragments, gBlocks Gene Fragments, to be useful in these studies. We use them to clone the regulatory regions we study, to modify the variants, and to move these sequences into luciferase expressing vectors.”

Accurate and affordable genotyping with superior performance

SNPs and their associated phenotypes provide crucial information for elucidating gene function and how gene variations influence disease. The rhAmp SNP Genotyping System is a novel, cost-effective genotyping solution that is easy to use and provides superior allelic discrimination. This dual-enzyme system offers:

  • The ability to interrogate SNPs in difficult sequence regions with amplicon lengths as short as 40 bp
  • An extensive predesigned assay collection, a custom design tool, optimized reagent mixes, and optional synthetic control templates
  • Compatibility with any commonly available qPCR instrument for SNP detection

Learn more about the rhAmp SNP Genotyping System and assay design tool.

Published Apr 29, 2019