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Beginning genotyping experiments?

Use PrimeTime® Mini LNA Probes to test your preliminary assays

When beginning a genotyping experiment, scientists typically first test a small sample subset to refine and optimize their LNA probe designs. PrimeTime® Mini LNA Probes provide enough material to perform 100 reactions, ideal for preliminary experiments, without the cost and waste of a larger scale yield. Once the researcher is ready to perform large scale screening, the 250 nmole scale product will provide them with 8000 reactions at a lower cost per reaction.

Figure 1 demonstrates the use of PrimeTime Mini LNA Probes la­beled with FAM and HEX dyes. For this experiment, the probes were designed with each targeting one of two alleles (Allele 1: FAM, Allele 2: HEX) in the fat mass and obesity (FTO) gene (rs3751812). Amplification reactions (10 μL), including Coriell gDNA (10 ng) of known genotype (homozygous variant 1, homozygous variant 2, heterozygous) and 250 nM probe, were performed in triplicate. An allelic discrimination plot was prepared using RFU at cycle 40 (Figure 1). Blue squares and orange circles represent DNA samples determined to be T (male) or G (female) homozygous DNA samples respectively, while green triangles represent DNA from heterozygous females. Open diamonds represent no template controls.

 


Figure 1. SNP assays using PrimeTime® Mini LNA Probes accurately identify 2 alleles in the FTO gene (rs3751812). SNP assays (10 μL) containing Coriell gDNA (10 ng) of known genotype (homozygous variant 1, homozygous variant 2, heterozygous), 250 nM PrimeTime Mini LNA Probes labeled with FAM (Allele 1) and HEX (Allele 2) dyes, 500 nM primers, and Immolase DNA Polymerase were run in triplicate on a BioRad CFX384™ Real-Time PCR Detection System. Allelic discrimination plots were prepared using RFU at cycle 40 and demonstrate that these probes were able to accurately discriminate between homozygous and heterozygous genotypes for all samples tested.

PrimeTime Mini LNA Probes are provided at 0.5 nmoles normalized yield, and are available with FAM/IBFQ* or Hex/IBFQ dye/quencher combinations. LNA probes are also available at a 250 nmole scale. Order online.

* IBFQ = Iowa Black Fluorescent Quencher

Additional reading

Developing onsite genotyping of Antarctic penguins—Research profile: This article highlights the increasing need for mobile qPCR testing using an example from conservation biology. PrimeTime® Custom qPCR Assays have been designed to distinguish genetic variants of Adélie penguins.

Discriminating highly similar transcripts using rhPCR—Research profile: Read about the uses of RNase H-dependent PCR, a technology developed to increase PCR specificity and eliminate unwanted interactions between primer sets (e.g., primer-dimers, etc.). An example is provided in which it is used to distinguishing highly similar alternatively spliced sequences. This technology can also be useful in genotyping applications, in highly multiplexed qPCR assays, library construction for NGS, and for rare allele detection, where the added specificity provided by the blocked-cleavable primers enables detection of a rare mutant allele in a background of large amounts of wild-type DNA.

Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

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