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Nested BSP Primer Design

Nested PCR BSP Methylation

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6 replies to this topic

#1 epibet

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Posted 14 April 2014 - 12:17 AM

Hello eveyone

I am trying to design BSP primers for a region with a high CG content, it is almost impossible to avoid CGs in regular sized (18-22) primers. I have designed 2 primers outside the region of my interest, both primers are CG free but they are 16bp long and there are A and T runs. For the nested primers, I could not excluded CGs (one CG) in forward but reverse is ok but again the problem is forward primer 17bp, reverse primer 16bp long. I was wondering has anyone tried to use BSP primers that short and what do you think whether will it be specific since I am using nested primers.

 

I also tried to blast my primers but methblast is under maintenance, so I found a web site http://bisearch.enzim.hu/ for blast. But I was expecting my 1st round of PCR to produce a 922bp product, the result gave a lot of smaller products and not located in the region of my interest. Has anyone used bisearch. 

 

I am giving the primers below, it is really important for me I could appreciate any feedback, thank you.

 

1st round F (5-3) :GTGTAGGTTTATGTGG

1st round R(5-3)  :AATAAATCAAAAACCC

 

2nd round F (5-3): GGAGAGCGTTTTTTGAG

2nd round R (5-3): AAAACCCAACCTCTCC



#2 phage434

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Posted 14 April 2014 - 03:55 PM

You will be much better off using a longer primer with CG's. The C's may or may not be converted, but you can make primers which will work with either  by specifying a Y in that primer location. With the very high AT content of primers for converted DNA and a short primer, you would have a very difficult, perhaps impossible, time getting the PCR to work. i would aim for a 22-26 bp primer. Good binding on the 3' end is most important.



#3 epibet

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Posted 16 April 2014 - 03:15 AM

Thank you very much for the reply. We are stil trying to decide which primer to choose since degenerate primers tend to have different efficiencies in different annealing temperatures. Longer primers with YG dinucleoitedes can cause a bias due to this difference. 



#4 pcrman

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Posted 17 April 2014 - 10:28 AM

In general, BSP primers should be longer than those for regular PCR, definitely not shorter.  You may not need nested pairs, just re-amplify using the same pair. 



#5 epibet

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Posted 18 April 2014 - 05:09 AM

Thank you very much for the reply. We decided to use longer primers with degenerate bases.

Can I ask one more question about using same primers for the 2nd round of PCR? I have designed BSP primers containing degenerate bases but the PCR did not work, then I decided to design one forward primer(new) outside of my region to perform a semi-nested PCR. So in the 1st round of PCR I used this new forward primer and previously designed reverse primer. I performed the 2nd round of PCR with previous primers, so I have used the same reverse primers for both PCRs. I have cloned and sequenced it. Methylation pattern in th primers were different from each other. Forward primer (contains two CGs) have both methylated and unmethylated CGs in the clones however reverse primer (containing 1 CG) has always methylated CG in the clones.

I thought if a primer creates a bias in the BSP PCR using it in the 2nd round of PCR can improve this bias and make it prominent. What do you think about this? Have you ever experienced anything like that when you perform 2nd round of PCR with the same primers? Thanks again :)



#6 phage434

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Posted 18 April 2014 - 05:56 AM

The methylation pattern within the primer sequence is poorly determined. The primers may bind with mismatches, and the amplified PCR product will have the priimer sequence, not the template sequence. I would ignore the sequence reads within the primer sequence.



#7 epibet

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Posted 24 April 2014 - 05:54 AM

Thank you very much, this was very helpful rolleyes.gif







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