Aptamer-Inhibited DNA Polymerases for Precise SNP Analysis

October 28, 2024 N. Tröndle
Science SNP, Annealing, Annealingtemperatur, Aptamer

Optimizing SNP Analysis with Aptamer-Based Inhibited DNA Polymerases: Key Tips for Successful PCR

Genaxxon offers specially aptamer-inhibited DNA polymerases that are ideal for SNP analysis. These highly selective enzymes are specifically modified for precise PCR tests and mutation detection—perfect when you are looking to “find the needle in the haystack.”

What Are Aptamers?

Aptamers are short, single-stranded oligonucleotides designed to recognize and bind to specific target molecules, such as proteins or small molecules (https://de.wikipedia.org/wiki/Aptamer). Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process, aptamers are optimized for their specificity and affinity.

In PCR applications, aptamers offer several advantages:

Low molecular weight
Easy modifiability
Excellent stability

Aptamer-Based Inhibition of DNA Polymerases

At Genaxxon, oligo-aptamers are used to specifically inhibit the activity of SNP DNA polymerases. These aptamers bind to the active site of the polymerase at low temperatures, blocking its activity. Only when the temperature exceeds 54°C do the aptamers detach, allowing the reaction to proceed.

Since aptamers do not denature at higher temperatures, they re-bind when the temperature drops below 54°C. This means that for successful SNP analysis using our SNP DNA polymerases, the annealing temperature must consistently be above 55°C to ensure efficient amplification. This must be considered during primer design.

Optimizing the Annealing Temperature

If the annealing temperature is below 55°C, amplification may be significantly reduced or completely inhibited. In the case of AT-rich sequences, longer primers should be used to ensure stronger binding.

Another method to increase the annealing temperature is to use Locked Nucleic Acids (LNA). These modified nucleic acids were independently described in 1997 by Jesper Wengel (1) and Takeshi Imanishi (2) and have since become a staple in hybridization-based applications. Incorporating individual LNA bases increases the melting or annealing temperature of a primer by approximately 2-3°C per LNA base (3, 4). For SNP analysis, LNA bases should preferably be positioned in the middle of the primer, as placing them near the ends is less effective.

Conclusion

Aptamer-based inhibitors provide significant advantages in SNP analysis, particularly in terms of precise polymerase activity control and high specificity. Optimal PCR performance, however, requires a sufficiently high annealing temperature. If necessary, LNA oligonucleotides can be used to further enhance efficiency and achieve the best possible results.

Literatur

1. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, Olsen CE, Wengel J; Biochemistry (2006), 45 (23), S. 7447–7455; doi:10.1021/bi060307w.

2. Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering. Obika S, Nanbu D, Hari Y, Morio KI, In Y, Ishida T, Imanishi T; Tetrahedron Letters (1997), 38 (50), S. 8735–8738; doi:10.1016/S0040-4039(97)10322-7.

3. Structures, dynamics, and stabilities of fully modified locked nucleic acid (β-D-LNA and α-L-LNA) duplexes in comparison to pure DNA and RNA duplexes. Suresh G, Priyakumar UD; J Phys Chem B. (2013), 117(18):5556-64. doi: 10.1021/jp4016068.

4. Biological Activity and Biotechnological Aspects of Locked Nucleid Acids. Lundin KE, Højland T, Hansen BR, Persson R, Bramsen JB, Kjems J, Koch T, Wengel J, Smith CI; Adv Genet. (2013), 82:47-107. doi: 10.1016/B978-0-12-407676-1.00002-0