SNP Detection Using LNA Oligonucleotides

Discriminate closely related sequences with sensitivity and specificity
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LNA-enhanced oligonucleotides offer the specificity and sensitivity needed to discriminate between closely related sequences. Use them for successful detection of single nucleotide polymorphisms (SNP).
Discrimination of closely related sequences
LNA probes are superior to DNA probes for SNP detection
Allele-specific LNA primers improve SNP detection
Coverage and design guidelines
Discrimination of closely related sequences
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.

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LNA probes are superior to DNA probes for SNP detection
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.

In the figure LNA probes are superior to DNA probes when it comes to detecting SNPs, DNA- and LNA-based capture probes are used to detect a SNP in the ApoB gene. The DNA-based system performs poorly because of the small ΔTm of the probe targeting allele 2. Most likely, the low discrimination is caused by the relatively high stability of a G-T mismatch. When using LNA, the ΔTm of the probes were enhanced by 38% and 300%, respectively.

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 (see figure LNA probes are highly sensitive for detection of 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 (see figure SNPs can be conveniently detected using LNA probes in LightTyper assays).

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Allele-specific LNA primers improve SNP detection
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, Latorra and colleagues found that LNA nucleotides in the 3’ position of the primers dramatically improve the discriminatory power of the primer (see figure SNP detection using LNA oligonucleotides).

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 (see figure Effects of single nucleotide LNA substitutions on melting temperatures). Di Guisto and colleagues have found that such primers perform better in proofreading allele specific extension (PRASE) PCR experiments.

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Coverage and design guidelines
Design your own LNA-enhanced oligonucleotides for SNP detection using the design guidelines below. Alternatively, let us design the allele-specific primer or probe for you. Contact us for more information.

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.

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