SNP Detection

SNP Detection Using LNA Oligonucleotides

The high specificity and sensitivity of LNA oligonucleotides makes them ideal for discriminating between closely related sequences. Single nucleotide polymorphisms (SNPs) can be detected via allele-specific PCR, using either primers or probes. Several techniques are available for detecting SNPs, including hyperchromicity, intercalating dyes, colorimetric or fluorescent dye detection and fluorescence polarization melting curve analysis.

Capture probes can be used in several different ways to detect SNPs. They can be used in real-time PCR applications, but also in the detection of SNP from PCR amplicons using ELISA-like assays or microarray analysis.
One of the most important features of a SNP detection probe is its ability to discriminate between its target and a highly related mismatch sequence. For this discrimination to be successful, there needs to be a clear difference in the melting temperatures of the duplexes (i.e., a high ΔTm value). The high specificity of LNA probes make them ideal for this application.

The high affinity of LNA probes for their targets means that probes as short as 12 nucleotides in length can routinely be used for SNP detection. Significant differences in ΔTm for the duplexes can be observed when such probes are hybridized to perfectly matched targets compared to targets containing single mismatches. In fact, the ΔTm is often around 20°C for single mismatches. This remarkable level of discrimination is not possible with DNA probes and makes LNA probes highly suitable for SNP detection in LightTyper assays.
The improved specificity and sensitivity of LNA oligonucleotides over traditional DNA oligonucleotides make them a very good choice for use in allele-specific primers. In a comparison between LNA primers and DNA primers, LNA nucleotides in the 3’ position of the primers dramatically improve the discriminatory power of the primer. Another strategy for LNA primer design involves incorporating the LNA nucleotide one position away from the 3’ end. These oligonucleotides also offer improved mismatch discrimination compared to DNA oligonucleotides.

Design your own LNA-enhanced oligonucleotides for SNP detection using the design guidelines below. When designing allele-specific primers or probes for SNP detection, vary the length and LNA positioning to obtain comparable melting temperatures (Tm) for the alleles, while keeping the difference in melting temperatures (ΔTm) between the perfect match and mismatch binding as high as possible.

Use the guidelines below to design your own probes, primers and clamps.

SNP probe design:

  • Capture probes should be approximately 12 nucleotides in length.
  • 2–3 LNA bases should be positioned directly at the SNP site.
  • The position of the mismatch in the capture probe is flexible, but the SNP should ideally be positioned centrally.
  • A Tm of approximately 65°C is recommended.
  • No LNA bases should be positioned in palindrome sequences (GC base pairs are more critical than AT base pairs).

Allele-specific primer design:

  • Follow general practices for the design of PCR primers.
  • If possible, avoid placing an LNA nucleotide at the extreme 3’ end of the primer, as this can result in low PCR efficiency. Instead, try positioning the LNA one position away from the 3’ end.
  • LNA nucleotides should be positioned at the position of the polymorphism and/or immediately 5’ of the polymorphism.

Clamp design:

  • LNA-enhanced clamps are oligonucleotides that compete with probes and primers for binding. They are very similar to the primers or probes they compete with, but are designed to perfectly match undesired PCR products or templates that should not be amplified.
  • The clamps are designed not to be extended during the PCR reaction. This can be achieved by introduction of 3’ modifications, such as inversed T or phosphorylation.