TL;DR: I think I had a PCR/Sequencing artifact fool me into thinking I had a homozygous deletion mutant, where it may actually be heterozygous (or vice versa). I want to know how to avoid this.
I'm doing CRISPR in RPE-1 cells and the method that I'm using for analyzing mutants is PCR followed by sequencing. There was one clonal colony that I sequenced with this method, and I got a clean sequencing result, suggesting 1 allele. However, when I repeated the genomic DNA extraction a month later, PCR amplified, and sequenced the gene, I found a clear sign of heterozygosity. One of these alleles is wild-type, while the other has an indel. I'm assuming this was either a PCR or sequencing artifact where I got selective amplification of one allele vs. another. But this concerns me because I don't know whether I need to re-check all of my mutant lines again.
Are there any ways of checking for heterozygosity that don't depend on PCR amplification, so I can avoid artifacts like this in the future? I know of Surveyor and T7Endo, but these both rely on PCR amplification followed by duplex formation to check for heterozygosity.
There is often an amplification bias that will tend to make more copies of a smaller product with Indel mutations.
Here's what you do:
sequence the PCR product from the Indel allele.
Determine the exact location of the deletion.
Design a pair of primers where one primer is located inside the deletion (primer A) region and the other is outside (primer B).
Set up two separate PCR reactions - one with your original primers you designed for the wild type allele (primer 1 & primer 2); a second PCR reaction with your new primers (primer A and primer B).
You might be able to design a "deletion spanning" primer that will only anneal to the allele that has the deletion. Then you will have a positive result in your homozygous mutant cells. Use a second primer that is outside the deletion
You will get these results:
DNA primer 1& 2 primer A & B. deletion spanning
Homozygous wildtype large band band no band
heterozygous small band* band band
homozygous mutant small band no band band
*might see both large & small bands in heterozygous
You should check all of your mutant lines again. Good luck!
There is often an amplification bias that will tend to make more copies of a smaller product with Indel mutations.
Here's what you do:
sequence the PCR product from the Indel allele.
Determine the exact location of the deletion.
Design a pair of primers where one primer is located inside the deletion (primer A) region and the other is outside (primer B).
Set up two separate PCR reactions - one with your original primers you designed for the wild type allele (primer 1 & primer 2); a second PCR reaction with your new primers (primer A and primer B).
You might be able to design a "deletion spanning" primer that will only anneal to the allele that has the deletion. Then you will have a positive result in your homozygous mutant cells. Use a second primer that is outside the deletion
You will get these results:
DNA primer 1& 2 primer A & B. deletion spanning
Homozygous wildtype large band band no band
heterozygous small band* band band
homozygous mutant small band no band band
*might see both large & small bands in heterozygous
You should check all of your mutant lines again. Good luck!
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