Hi Friends,
I have been having a nightmare genotyping these transgenic mice. What I have are heterozygous mice with a few knock-in point mutations.
Originally, we used the same WT reverse primer with either a WT forward or mutant forward primer to amplify WT and mutant allele, respectively. The WT primers always worked very well, but the mutant PCR was horrible (non-specific results, smeared bands, low amplification).
I have tried EVERYTHING to get it to work better (increase annealing temp, temp-gradient PCR, HPLC-grade primers, less/more genomic DNA, less or more primer concentration, different brands of reagents, different thermocyclers, and the list goes on....) I even tried the band-stab method, to stab my band of interest from one gel, so I could make a new PCR reaction to get a single band and even that turned out smeared!
So, I instead made another WT primer upstream of my domain of interest, so I could get a clean PCR product and send all the samples for sequencing. Surely I would be able to tell between WT and Het from the chromatogram.
All my positive control (original heterozygous mice that were gifted to our lab) came back with WT sequences... The chromatogram should have had double peaks, where there were point mutations.
My only idea is that the PCR is preferentially amplifying the WT allele. So my question, how can I make regular PCR allele specific?
Please advise ! :)
In the meantime, I'm outsourcing my genotyping to a company.
Thank you!!
Note: I am not introducing mutations or doing real time PCR... Just regular genotyping by PCR and running samples on agarose gel, purifying the samples, and sending them for sequencing.
Hi Sarah,
I think if you design a primer with the 3' nucelotide specific for your allele of interest only (perhaps one of your point mutations?), you should only get amplification in the presence of this allele (5'-3' extension is only possible if the 3' nucleotide of the primer aligns perfectly with the DNA template). It's the same theory behind site-specific primer (SSP) PCR. This technique allows the simultaneous amplification of alleles to give different sized amplicons due to the primer design, as (I hope!) is shown in the attached figure (Lane 1 and 2 = WT homo; Lane 3 and 4 = hets). Hope this helps.
Hi Sarah
you can design several primers that will amplify the both alleles.
However, after the PCR amplification you can clone the PCR product into plasmids (like pGEM) and then select 5-10 colonies for sequencing. this will help to distinguish between the alleles since only one PCR product will be cloned each time.
Good luck.
Hi Sarah,
I think if you design a primer with the 3' nucelotide specific for your allele of interest only (perhaps one of your point mutations?), you should only get amplification in the presence of this allele (5'-3' extension is only possible if the 3' nucleotide of the primer aligns perfectly with the DNA template). It's the same theory behind site-specific primer (SSP) PCR. This technique allows the simultaneous amplification of alleles to give different sized amplicons due to the primer design, as (I hope!) is shown in the attached figure (Lane 1 and 2 = WT homo; Lane 3 and 4 = hets). Hope this helps.
I had this issue with a point mutation line at one point. Three more possible solutions:
The idea of the high-resolution melting analysis is a good one as well, although I haven't had much luck with it yet in my lab...
Note: I am not introducing mutations or doing real time PCR... Just regular genotyping by PCR and running samples on agarose gel, purifying the samples, and sending them for sequencing.
I think that the better than you can do is use reverse and direct primer that amplifies both amplicons (heterozigous and homozigous). Then you can sequence it and if you confirm the mutation you may try make HRM.
Hi Sarah, I am using SNP genotyping by using High Resolution melting. It is a very sensitive technique and can pick up differences of even 1 nucleotide. You would need some optimization before but once optimized, the technique is easy and high throughput. You can run up to 96 samples in one go. However, the price per sample is relatively high as compared to gel electrophoresis technique and you would need access to real time PCR equipment. If you need a protocol, you can send me a message.
To make a PCR allele specific you should design a primer on one of the variant nucleotides, with the variant on the extreme 3'end. To increase the allele specificity you add more mismatches into the primer at -2 for instance, also the primer lenght should be short, the more effect these mismatches will have on the specificity.
Just an update: I tried to outsource my mice to two different genotyping companies (Transnetyx and Genewiz), and neither could genotype them based on the paper the mice were from. Although the allele specific PCR did not work, they tried neomycin PCR and all of the samples came up positive... So, at this point I am going to TA clone my gene to see if the point mutations are actually there or not... Thank you everyone :)
Dear all,
I have 2 questions regarding Moran's answer in this post:
"after the PCR amplification you can clone the PCR product into plasmids (like pGEM) and then select 5-10 colonies for sequencing. this will help to distinguish between the alleles since only one PCR product will be cloned each time"
1) Why will a colony have only one PCR product? Isnt it possible/likely that some bact cells were transformed with more than 1 plasmid?
2) Does anyone have a good protocol for this? or do you just use the manufacturers protocol (i.e pGEM)?
Just fyi: before PCR my samples are a pool of 2 (max 3) similar DNA sequences (CRISPR modified alleles) from a monoclonal line and should in theory be present in nearly equimolar concentrations. I just want to sequence the alleles present in my sample. Any advice is very welcome. Thanks in advance.
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