Hello dear colleagues. Im trying to clone my dna fragment (2000 bp) using TA vector and E.Coli cells.
In PCR step from cdna template i was using high sensitivity polymerase with 5`-3` proofreading activity (HS-Fuzz), which worked fine in other cloning experiments.
Then i purified product from gel > adenilation > ethanol precipitation > ligation in TA vector > picked 20 colonies > PCR screening from external and internal primers for insert > picked 6 samples with right size > propagated them overnight in liquid media > isolated plasmids > restriction analysis > Sanger sequencing of 5 samples, and every sample contains more than 5 mismatches (all different, including insertions of 4 nucleotides). Since the polymerase is verified in other experiments, i tryed another strain of E.Coli (xL-1 Blue), and another PCR product, but nothing changed. What could be a sourse of these mismatches? Maybe revertase or bad transformation?
You could have amplified a cDNA from a related gene, not your gene of interest. Or your reference genome might not be a good reference. Honestly, unless you are working with a highly inbred organism, there is a LOT of genetic variation in most species. Also, the reference genome might not be high quality so you would expect some mismatches.
If your starting material (cDNA) is supposed to be from one individual or cell line (so ~no genetic diversity), I think it's most likely that your PCR step is introducing the errors. I've never heard of your polymerase, but perhaps it doesn't have a very high fidelity, and the reducing number of cycles may help (or try Phusion). Also, poor quality of DNA can give you a higher error rate.
Another thought - did you get high quality sequencing reads from your cloned inserts? Don't mistake crappy Sanger sequencing for mutations.
My cDNA is from high quality RNA from human fibroblasts. My gene has no pseudogenes and has very low expression level, so i use only high sensitivity polymerases (i tried Fussion and Q5 but couldnt get any bands of my fragment). I have no information about my polymerase except that it has 5-3 proofreading activity. But even polymerases without this activity doesnt do so many mistakes (2-3 for every 1000b.p.) and my colleagues worked with it and it was fine. I think maybe reverse transcription is the source of mistakes.
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