Why do I always get these same 4 point mutations ? Help !!! - (Jul/29/2010 )

I have been doing this construction for a long time, but every time I do it I get exactly the same 4 point mutations. The construction is like this: 2k PCR fragment + 11.9 gel extracted digestion product. The mutations are on the 2k PCR part. I sequenced PCR, it was fine but I sequence the colony I got, there are always same 4 point mutations. I don't know what to do now. Where would those mutations come from if not from PCR ? Would there be anything wrong with the competent cell, I use XL-1. Please help me !!!

Ablat on Thu Jul 29 21:19:44 2010 said:

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.

Are you repeating the PCR, and each time your clones have the same point mutations, or did you just do the PCR once, and are repeatedly cloning from that same pool of DNA?