SNP Pol DNA Polymerase

SNP Pol DNA polymerase for easy, reliable and rapid allele-specific discrimination, eg. CRISPR / Cas9 point mutations, for detecting incorrect CRISPR / Cas9 products, or validating sequencing results. The SNP Pol DNA polymerase distinguishes highly specific, whether a mismatch of the primer-template-complex is present or not. The mismatch (point mutation) must be at the 3 'end of the primer. Thus, mutant alleles can be distinguished exactly from wild-type alleles - without sequencing, since the polymerase simply does not amplify in the case of a mismatch.

Just place your primer on the supposed point mutation (Important: The point mutation must be at the 3 'end) and the polymerase will detect a mismatch in this region with almost 100% accuracy: If the template base complementary to the 3' end of the primer shows the mutation and the primer does not, no amplification takes place - 100% certainty within a short time!
The SNP Pol DNA polymerase can therefore be easily, time and cost-effectively used for the screening of point mutations.

The variant SNP PolTaq DNA polymerase > has 5'-3 'nuclease activity and can therefore be used for specific hydrolysi probes such as Taqman® probes or Molecular beacons.

For more information on our SNP DNA polymerases and applications, read our blog now.

Description

SNP Pol DNA Polymerase is a highly selective DNA polymerase for the detection of Single Nucleotide Polymorphisms. It was developed specifically for allele-specific discrimination, where a very high discrimination rate is required e.g., in allele-specific PCR (ASA; AS-PCR), allele-specific primer extension (AS-PEX), SNP analysis, genotyping or in methylation-specific PCRs (MSP). Many other DNA polymerases tolerate mismatched primer-template complexes and are therefor not suitable. The SNP Pol DNA polymerase, on the other hand, distinguishes these specifically (high discrimination) and supplies only PCR products with perfectly matching primer pairs! The Genaxxon SNP Pol and SNP PolTaq DNA polymerases differ up to 100% by means of allele-specific PCR between the two alleles and, after a simple qPCR, give a clear result as to which allele is present. Thus, the principly great potential of the CRISPR/Cas9 technology can be used for human medicine and plant biotechnology especially together with the SNP PolTaq or SNP Pol DNA polymerase.

Allele-specific PCR can be used to quantify the mutation rate in a pool or background of wild-type sequences. The verification of mutation frequencies determined by NGS can also be verified by means of allele-specific PCR and SNP Pol DNA polymerase. The SNP PolTaq DNA polymerase is very suitable for the analysis of liquid biopsy samples. With SNP PolTaq, the presence and frequency of cancer mutations can be analyzed and quantified very well.

Picture below: Application note SNP Pol DNA Polymerase

We recommend short amplicon length (about 60-200 bp) for best results, but also longer amplicon lengths are possible. The addition of additional Magnesium (+0.5 - 1.5mM) might be needed in case of longer amplicons >500 bp.

Trouble shooting:
No bands after PCR of 30 cycles!
Optimisation procedure for missing or weak bands.

Annealing temperature is too low! 

Attention: The SNP Pol is an aptamer inhibited DNA polymerase. The aptamer oligo used reversibly inhibits the polymerase at temperatures <55°C! Therefore, the primers should ideally have an annealing temperature of >57°C. 

- realtime PCR approach
- Check the dNTP concentration. This should be between 200 and 300µM (in the PCR).
- Increase the number of cycles (at least 35, preferably equal to 40) 

Test optimisation - number of cycles
In endpoint detection, a cycle count of 30 is not always sufficient to generate a clearly visible band. For example, with less than 200 DNA copies as a starting value. The test should therefore be run using real-time PCR, or five parallel PCRs should be used, one of which is taken from the PCR device after each of five different cycles (example below). 

Endpoint detection:
Set up five identical PCR reactions in parallel using the SNP Pol DNA polymerase. Remove one of the PCR tubes from the cycler at each of 20, 25, 30, 35 and 40 cycles and apply all five reactions to an agarose gel at the end. This makes it easy to recognise which is the optimum number of cycles in the PCR for the given application (starting material, target, primer). 

SNP Pol PCR with cell lysates
Animal cells and E.coli can be used directly for PCR with the SNP Pol DNA polymerase! No separate lysis or proteinase K digestion is necessary. 

PCR procedure:

1. 50 - 500 cells are required per PCR batch!
2. prepare PCR mix with primers and buffer and place on ice!
3. add the picked colony directly to 10-20µL water (no separate lysis and no proteinase K digestion), vortex briefly (5 seconds), then add this cell suspension directly to the PCR reaction mixture, e.g. 10µL cell suspension for a PCR preparation with a total volume of 20µL. An initial denaturation time of 2-3 minutes is sufficient is sufficient, can be extended to 5 minutes if necessary.
Genomic DNA per reaction max. 100 copies is relatively low, but should still work. The remainder of this cell suspension can also be frozen away in order to carry out further tests. 

Optional:
4. if possible: do real-time PCR!

The SNP Pol DNA polymerase (M3009 > or M3061 >) can be used together with non-specific fluorescent dyes (eg Genaxxon's Green DNA Dye > or SybrGreen®) in real-time PCR. When working with specific PCR probes, only the SNP PolTaq DNA polymerase can be used, since only these have a 5'-3 'exonuclease activity.

With our high quality dNTPs as Set (M3015.4100) > or Mix (M3016.1010) > or our DNALadders > and our favourable standard agarose (M3044) > we can offer additional products for your PCR.

Application areas for SNP Pol DNA and SNP Pol DNA polymerase
- Monitoring, verification and detection of point mutations
- Identification of correct or wrong CRISPR/Cas9 products
- Verification/validation of sequencing results
- Quantification of mutations (e.g. NGS results)
- SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR
- Methylation specific PCRs (MSP) after bisulfite treated DNA (CpG methylation sides)
- HLA genotyping
- micro sequencing
- realtime PCR with hydrolysis probes
- realtime multiplex PCRs

- DamID-seq data in C. elegans.

As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans.

Sharma R, Ritler D, Meister P.
Tools for DNA adenine methyltransferase identification analysis of nuclear organization during C. elegans development. Genesis. 2016 Feb 4. doi: 10.1002/dvg.22925.

- Minisequencing SNP genotyping with SNPase DNA Polymerase can be carried out by the procedure described in:

Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson S, Syvänen AC. 
Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA. Nucleic Acids Res. 2003;31:e129.

- Allel specific mismatch selectivity by the HiDi DNA polymerase

Drum M, Kranaster R, Ewald C, Blasczyk R, Marx A (2014) Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification. PLoS ONE 9(5): e96640. doi:10.1371/journal.pone.0096640