I want to determine the level of crossing of one rapeseed variety ("A") with another variety ("B") that has a known SNP in the target gene. If it comes to a crossing, then in DNA isolated from seeds we will have two forms of the gene (mutated-SNP and not mutated). The quantification can be calculated with respect to a gene that is not mutated or another reference gene that SNP occurs in a known number of copies. The number of copies of the gene with the known SNP relative to the reference gene will determine the degree of crossing.
Probably a good method is pyrosequencing to determine levels of each but this is quite expensive.and probably not what you meant
With appropriate control samples you could try ARMS pcr where only one of the alleles amplifies in each reaction or DCAPS (ACRS) where you introduce a second change in the primer which makes a restriction site .This second method could be used with dye labelled oligo and capillary electrophoresis ( sequencing apparatus) to quantify the amplification
Thank you Paul Rutland, but how can I quantify the number of SNP, is it possible by this method?
For the analysis of c-kit mutation D816V Mutation we use a MAMA-Taqman PCR for the c-kit mutated allel and a Taqman-PCR for our house-keeping gene. Quantification can be done by real-time PCR with the delta-delta Ct method after having determined the PCR efficiency of both PCRs. However, this only gives relative quantification results, the ratio mutated allel/housekeeping; using plasmids (mutated allel and housekeeping gene) as standards, copy numbers could be established. An alternative to the delta-delta Ct method is the use of digitial PCR, e.g. digital droplet PCR, which allows absolute quantification by using Poisson distribution.
Hope could be of help.
Joanna, which range of mutant DNA copies do you respect?
Hi..
It will be better if you go for ICE-COLD PCR-based approach. In this context, I was looking for some references and found the following research article. Please go through it. Hope it will be helpful.
http://www.sciencedirect.com/science/article/pii/S0009898116304983
If it is intended for high-throughput genotyping, maybe you should consider competitive allele-specific Taqman PCR (cast-PCR). This consists in a set of 3 primers and a Taqman probe. Two of these are forward primers, one should match the mutation (allele) at the 3'-end and provide the 3-OH for amplification. The other forward primer should match the wild-type allele, but its 3'-end should be modified to 3-H (dideoxynucletoide). In this way, the wild-type allele will not be amplified and you will only get signal of the mutation allele. You should run a parallel reaction targeted to another exon, in order to get the reference for copy-number or %.
As far as I know the number of SNP mutants is very low, that's why we need specific reaction which amplify only the mutant DNA.
So you can try to block your wildtype with LNA probe (like this https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448309/) or eliminate wildtype with DSN (https://www.ncbi.nlm.nih.gov/pubmed/27431322).
@ Joanna Chojak-Koźniewska,
When you said that you are measuring 'the level of crossing', did you mean the transmission efficiency of this SNP-allele to the next generation? I cannot understand the concept of 'the level of crossing' in your question.
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