Hello everyone.
I'm trying to make CRISPR knock-in cell line. Maternal cell line is HCT116 which is known as diploid cancer cell.
I want to knock-in fluorescence protein into C-term of my target gene on chromosome 1 as homozygous.
First, I tried knock-in and successfully selected the positive cells with FACS.
Most of sorted cells were heterozygous.
Second, I tried knock-in again the fluorescence protein with T2A-HygroR into these heterozygous cells. I incubated these cells with hygromycin containing medium for more than 10 days. I picked survived monoclones and tried genotyping with primers on both side of knocked-in site(expected band size : WT - 1kb, FP embedded - 2kb, FP-T2A-HygroR embedded - 3kb).
I supposed there should be two band of 2kb and 3kb if one allele contains FP and the other allele contains FP-T2A-HygroR, but there were three bands of 1kb, 2kb and 3kb.
How can it happened? Please let me get any advice.
Thank you for your reading.
This is most likely a heteroduplex -- nothing to worry about. These form as an artifact in PCR when amplifying long heterozygous insertions.
Imagine during PCR at the melting step, all product molecules become single-stranded. Then, during the annealing step, you form three kinds of double-stranded products: Hygro paired with hygro, FP paired with FP, and Hygro paired with FP. These could be the 3 bands you're seeing.
It's best to confirm with a single molecule sequencing method, like nanopore.
@John Schloendorn
Thank you for your kind answer.
According to your answer, It seems there should be longer PCR product produced by heteroduplex artifact, rather than 1kb which was same size with negative control. Is it possible that the size of heteroduplex size is short like this?
I seem to have seen it, but it's unusual. You can't know for sure what you've got unless you sequence.
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