6 replies to this topic

[biochemistry3096]

Hi,

I have recently designed a set of primers to amplify a fragment of a gene present in an cDNA template. I have performed a blast search on my primers and the results have shown a hit with my gene of interest.

However, after conducing the PCR and cloning my fragment into a vector, i sent it off for sequencing. The results were surprising. Sequence analysis showed that the amplified fragment was from a completely unrelated gene.

I was wondering, bearing in mind my designed primers are specific for my fragment of interest, why I amplified a non-specific fragment.

Any suggestions would help.

Thank you,
Biochemistry3096

[pDNA]

it's hard to say why ...but i would go for a nested approach to prevent amplification of non-specific fragments ...thats the way you normally do it for gDNA templates since the probability of mispriming is quite high.

Regards,
p

[Trof]

When you blasted your cloned fragment, did you try to align ends of the fragment, where would the primers bind, for similarity with your primer sequences/specific amplicon?  You can see if there really is the same sequence as your primers, or what mismatches are there and if the optimisation of the reaction conditions would help to amplify more specifically.
There may be many reasons, but first you need to check what exactly did you really amplified.

[biochemistry3096]

Trof, on 26 November 2011 - 08:23 AM, said:

When you blasted your cloned fragment, did you try to align ends of the fragment, where would the primers bind, for similarity with your primer sequences/specific amplicon?  You can see if there really is the same sequence as your primers, or what mismatches are there and if the optimisation of the reaction conditions would help to amplify more specifically.
There may be many reasons, but first you need to check what exactly did you really amplified.

Thank you for your suggestions.
I have designed my primers based on general primer design laws e.g primers 18-22bp, 50-60% AT pairing, annealing temp of 55 degrees, ect ect.
I have used a blast search to see the hits, and my top hit with 100% is the gene i am looking to amplify, however, this did not happen.
I have been using gradient PCR from 50-62 degrees to find the optimal temp, but this has still given me the wrong amplicon. I have also tried altering the primer concentration and Mg concentration but with no success.
I think the next thing would be to design another set of primers, but its just seems strange as to why these primers are not working.....

[pDNA]

http://en.wikipedia...._chain_reaction

Regards,
p

[biochemistry3096]

I was wondering if there are any addtional causes of wrong pcr amplification apart from the ones listed above ?

[Biouday]

Is the PCR product same size what you expected.