PCR across a highly GC rich tandem repeat region
|
PCR across a highly GC rich tandem repeat region
I am trying to PCR across a tandem repeat region which is highly GC rich
(approx 82%). The repeat region is asymmetrical in that one strand
contains mostly Cs and the opposite strand mostly Gs. The repeat unit
varies in size from 2.5-5Kb with most of the population having one small
and one large allele. All I get when I try the PCR is the smaller
allele and lots of smear. I've tried increasing the annealing temp,
extension temp, increasing the extension time, increasing the
denaturation time and temp. I've tried adding glycerol, DMSO, and
betaine but all to no avail. Does anybody have any tips or tricks I can
try?
Link
In our lab people are amplyfying microsatelites and some of them they only
get by using Expand from Boehringer/now Roche.
Maybe worth a (expensive) try.
Gregor
Link
Try using a polymerase mix i.e. Taq plus a proof-reading pol. Add in
DMSO to 5-10% and Betaine to 1M final. You may also need to raise your
denaturation temp by 1 or 2C, maybe up to even 98C. Unfortunately that
will have a nasty effect on the stability of the Taq component so make
sure you run minimal denaturation times i.e. 5-10 secs max.
Duncan
Link
Hmm - the wonder betaine didn't cut it eh? There was another trick from NAR
where they denatured their DNA with NaOH and then neutralised it before
adding it to the PCR. Worth trying??? As Duncan said, enzyme blend would
also help. Another combination used (can't remember the paper) is TMAC
(60mM) and formamide (2.5%) thay may be useful.
John
Link
I don't know if the reference is in there, but the following
website gives a good list of additives that you can use:
http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html
Link
Last update 27-Jul-2006, Rating Poor of 4 votes.
|
Write your comment
|
how do you oerform this touchdown pcr, i.e. how to step up the temp from 55 t o 65.how to set this.
thanks
rachel
Rating: n/a
Reply
|
|
|
We have a good results for GC-rich templates with Topotaq polymerase from Fidelitysystems (www.fidelitysystems.com)
Rating: n/a
Reply
|
|
|
I did some long and difficult of PCRs, such as 3-5 kb (even cDNA)and 60-80% GC rich. I always try Roche GC rich KIT and use touch town PCR programme, I can work out most of them. If not , I 'll try design new primer for avoid some hard amplified region in primers, then do nest PCR.
The touchdown PCR protocol as follows:
94C 3 min
10 cylces of
94C 20 sec
55-65C 30 sec
68C 3-6mins (for 3-5 kb)
25 cycles of
94C 20 sec
55-65C 30 sec
68C 3-6 mins plus addition 20sec every cycle
68C 7 min
For details , see Roche GC rich kit(cat. no. 12 140 306 001)
Rating: Excellent!
Reply
|
|
|
Hi,
we did lots of difficult of PCRs which didn't work under standard conditions. Therefore, we optimized PCR additives to generate an enhancer, which we now add to all our PCRs. Most of them are improved.
Have a look at
http://www.protocol-online.org/prot/Detailed/4054.html
Bye,
Markus
Rating: n/a
Reply
|
|
Related resource
Protocol for Enhancing PCR of Very Difficult Regions
|
