PCR, followed by sequencing... (View forum version)



nightingale

Posted 17 August 2009 - 10:39 PM

Dear all,
a question that is on my mind :) ...

in the application in which :
do PCR,
gel extraction for the band,
sequencing

i already know the base sequence for my gene/mutated one from the PCR
so,,
why sequencing ( as if once again ?? ) ???

is it because of the amplification error ???
if this is the case, i can choose a high fidelity enzyme
which,i think, is cheeper than using a whole new device to know the sequence ...

my apologies, if my question seems silly ...

thanks for ur time :)

gfischer

Posted 18 August 2009 - 06:15 AM

There are a couple of reasons why you might do this. Some really paranoid people sequence their product to be extra sure they have the right product. IMHO, this is overkill if you've BLASTed your primers. Also, if you clone the product into a plasmid before sequencing, you can use the plasmid as a standard for qPCR.

fishdoc

Posted 18 August 2009 - 06:39 AM

Dear all,
a question that is on my mind :) ...

in the application in which :
do PCR,
gel extraction for the band,
sequencing

i already know the base sequence for my gene/mutated one from the PCR
so,,
why sequencing ( as if once again ?? ) ???

is it because of the amplification error ???
if this is the case, i can choose a high fidelity enzyme
which,i think, is cheeper than using a whole new device to know the sequence ...

my apologies, if my question seems silly ...

thanks for ur time :)




I've been using Phusion High Fidelity polymerase of late for cloning, and have gotten many many point mutations in my sequences, and the point mutations vary by individual colony, so it's not a mistake in the original sequence. I don't know if it's my polymerase or bad E. coli cells, but I now suspect the polymerase.

In the past (and in some instances currently) Phusion has given me no problems like this, so it may just be a bad lot of polymerase.

In either case, PCR is not always perfect, even if you have a high fidelity polymerase. You can have a bad lot of the polymerase, or it could have been left out of the freezer for too long by another lab member, and the enzyme could be affected.

Furthermore, if you're making a mutant, you better be sure it's what you think it is. A bad PCR product could knock a downstream gene out of frame (if it's a bacterial gene in an operon). If you're doing allelic exchange (homologous recombination), if you're sequence flanking your mutation is mutated, it could directly affect a upstream or downstream flanking gene, if that mutation is transferred.

To me, there's too much at risk to NOT sequence.

nightingale

Posted 19 August 2009 - 02:42 AM

thanx alot for ur explanation ;)