Why do I always get these same 4 point mutations ? Help !!!
Posted 29 July 2010 - 01:19 PM
Posted 29 July 2010 - 06:22 PM
Just to clarify, these mutations are within the PCR product and are not part of the primer.
How many isolated repeats of this plasmid have you sequenced?
What kind of polymerase are you using?
Sequencing reaction gives the average sequence of the population of molecules being sequenced. Thus it is pointless to sequence a PCR mix, as each molecule is a unique product of the PCR reaction. The 4bp mutations are in mix somewhere
As an example. imagine a PCR reaction that gave rise to 1000 DNA fragments 1000bp long. And each and every one of these DNA fragment had a random mutation. Thus none of these DNA fragment code any functional protein product.
If these 1000 DNA fragments were sequence, we would get a sequence read which was perfect. The reason is at each position there a 999 basepairs which are correct and only one which is wrong. The molecule which contains the mistake changes every time. The DNA sequencing reaction only looks at the average.
IN your single colony, a single DNA fragment has been isolated within the plasmid. SO when the plasmid is sequenced, a population of clones are being sequenced and thus the mutation is uncovered.
Posted 29 July 2010 - 07:04 PM