In PCR-based genotyping, can the DNA extraction method interfere with the mobility of the final PCR amplicon?
DNA samples obtained with an alkaline lysis protocol from mice tails produces amplicon bands with affected mobility (slower, heavier, bigger) in comparison to samples which DNA was obtained with a proteinase K protocol. Any suggestion of what might be happening?
Dear Joao
i routinely carry out PCR based gritting from alkaline lysis preps and have performed similar genotyping on prot K/SDS fully column purified or phenol chloroforn purified preps
i have to confess in all my years of performing PCR based genotyping from both types of preps I have not whitnessed what you are describing. Generally speaking the only difference in the past I have noticed is that on occasions some difficult target sequences amplify more efficiently and give a more definitive answer when using prot K digested and cleaned up genomic DNA as compared with crude alkaline lysates
That said it is possible for proteins to bind to DNA and retard mobility: this is the basis of so called gel retardation assays
Is this difference confined to one target region and/or one set of primers ? I'm unable to offer constructive advice other than suggest trying alternative primers and/or amplifying an alternative target fragment for genotyping if at all possible ?
Yes, it is true, the DNA quality is influence to the mobility of the final PCR amplicons.
The presence of polysaccharides - the main problem, they interfere with DNA mobility during electrophoresis.
You may also notice the difference in the mobility of DNA samples in the gel at different concentrations of the loading buffer based on Ficoll 400.
yes,
if you are interested kindly read my article "Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA of Terminalia arjuna"
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