
SNP Detection
SNP Detection Using LNA Oligonucleotides
Discriminate closely related sequences with sensitivity and specificity
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.
LNA probes are superior to DNA probes for SNP detection
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.
Allele-specific LNA primers improve SNP detection
Coverage and design guidelines
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.