SNP Detection Using LNA Oligonucleotides
Discriminate closely related sequences with sensitivity and specificity
LNA probes are superior to DNA probes for SNP detection
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.
- 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.