sequencing

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srujana
srujana's picture
sequencing

I have designed primers that bind to conserved regions,so that variable regions inbetween the conserved regions get amplified.
My main aim is to detect the microbial diversity in a given sample by amplifying the variable regions of different microbes.
I used to amplify variable regions and get a 340 bp product and with this product i used to clone it by topo vector and later on used to sequence each single clone by sanger sequencing and the sequences were very fine.
But when i gave the 340 bp PCR product directly for sanger sequencing there was presence of muliple peaks.so iam stuck here.Why my sequencing is not working.what is the problem? i  give exosap treatment before sequencing.some one please help me out what went wrong in my experiment or is it not possible to get sequences of variable region amplicon?
please help me out with your valuable suggestion

srujana

hcheung
hcheung's picture
Hi Srujana,

Hi Srujana,

What sequencing primer did you use for sequencing the clones in the top vector?
What sequencing primer are you using to sequence the 340 bp PCR product directly?

DougB
DougB's picture
Hi Srujana,

Hi Srujana,

There could be a number of possibilities for double sequence in the PCR products. Since this has been your stopping point in the experiment, can I assume that the products have not been cloned into a vector? I know you said that you have been successul with Sanger sequencing on previous samples. However, you did not say these problematic products were cloned and sequenced.

Starting with the more obvious cause, you might be amplifying multiple products. It is also important to note that multiple products of similar length may not separate by agarose gel electrophoresis and therefore cannot be seen.

Another possibility is that the exosap may not be working. Do you have any control sample that will allow you to test the exosap. In this case both PCR primers would be present in the product. and amplify in the Sanger reaction.

It is also possible that the conserved region is not actually conserved.

I have also seen cases where one of the PCR primers (or both) may have multiple sequences. It would only require that some of the primer in the mix misses one base. Amplification of multiple products would produce double sequence where one sequence was different than the second sequence by one base. This would depend on whether this pair of primers had been used before.

This is a couple of possibilities. I will let you know if I can think of others.

DougB

srujana
srujana's picture
hello DougB,

hello DougB,
I have sorted out the problem.
As there were multiple variable regions that were getting amplified.i have got multiple peaks in sequencing.
Iam going to clone the pcr product and give it for sequencing.
Thank you
srujana

srujana
srujana's picture
hi hcheung,

hi hcheung,
i havent cloned the pcr product.i will be doing it know to sort out my problem.
thank you
srujana