PCR is amplifying non-specific fragment

[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

 

 

 

[hypervalent_iodine]

It might be easier for your question to be answered with more specific knowledge of your primer sequence, the thermocycling conditions you used, etc.

 

In any case, here's a few points I picked up in my time working in a genetics lab (I should note a disclaimer here that I am actually a chemist and my time in said lab was both brief and a few years ago, so some of my points may need checking):

 

• If your annealing temperature is too low, you will get your primers binding quite strongly at other sites of the template, resulting in false product or no product at all. Too high will mean that the primers are unable to bind properly, if at all, which results in no product. If your equipment allows it, it may be worth-while doing multiple PCR's simultaneously with a annealing temperature gradient from about 40^oCon the cool end of the heating block to 60^oC on the other end. If you run a gel of the amplified cDNA, you should be able to work out the approximate optimal temperature and furthermore, if the temperature is affecting where in the template you're amplifying (assuming there is sufficient size difference).
• Is it possible to excise the fragment you're after with a restriction enzyme at all? Even if you can get it within a few hundred bp flanking either side, this may help you get more specific binding of your primers to where you want.
• If you have too many A-T pairings in your primer (more than 50% is probably too much), the primer may not form strong enough bonds with your target, resulting in mismatch. This also affects the annealing temperatures you should use; as a general rule of thumb, G-C pairs (being the more strongly bonded base pair due to their one extra hydrogen bond) will be able to withstand higher annealing temperatures than will A-T pairs.
• How big is your target fragment? Too large a fragment will probably mean the extension phase isn't working properly, which may be why you aren't seeing it. If it's too small, there's a chance you're missing detecting it when you're extracting it from the gel.
• Avoid using A-T pairs on the 3' end of your primer. A-T pairs on this end result in something known as 'breathing' and may give you mismatches. Similarly, runs of three or more G's or C's on the 3' end may also promote mismatches at other G or C rich areas of the DNA sequence.
• Your primers should be about 18 or so bp long. If they're too short, there is an increased likelihood of mismatch.

To summarise, I would test to see if the annealing temperature is affecting specificity in your samples and then have a look at your primer design. It sounds like you will have to change one or both of your primers.

[biochemistry3096]

It might be easier for your question to be answered with more specific knowledge of your primer sequence, the thermocycling conditions you used, etc.

 

In any case, here's a few points I picked up in my time working in a genetics lab (I should note a disclaimer here that I am actually a chemist and my time in said lab was both brief and a few years ago, so some of my points may need checking):

 

• If your annealing temperature is too low, you will get your primers binding quite strongly at other sites of the template, resulting in false product or no product at all. Too high will mean that the primers are unable to bind properly, if at all, which results in no product. If your equipment allows it, it may be worth-while doing multiple PCR's simultaneously with a annealing temperature gradient from about 40^oCon the cool end of the heating block to 60^oC on the other end. If you run a gel of the amplified cDNA, you should be able to work out the approximate optimal temperature and furthermore, if the temperature is affecting where in the template you're amplifying (assuming there is sufficient size difference).
• Is it possible to excise the fragment you're after with a restriction enzyme at all? Even if you can get it within a few hundred bp flanking either side, this may help you get more specific binding of your primers to where you want.
• If you have too many A-T pairings in your primer (more than 50% is probably too much), the primer may not form strong enough bonds with your target, resulting in mismatch. This also affects the annealing temperatures you should use; as a general rule of thumb, G-C pairs (being the more strongly bonded base pair due to their one extra hydrogen bond) will be able to withstand higher annealing temperatures than will A-T pairs.
• How big is your target fragment? Too large a fragment will probably mean the extension phase isn't working properly, which may be why you aren't seeing it. If it's too small, there's a chance you're missing detecting it when you're extracting it from the gel.
• Avoid using A-T pairs on the 3' end of your primer. A-T pairs on this end result in something known as 'breathing' and may give you mismatches. Similarly, runs of three or more G's or C's on the 3' end may also promote mismatches at other G or C rich areas of the DNA sequence.
• Your primers should be about 18 or so bp long. If they're too short, there is an increased likelihood of mismatch.

To summarise, I would test to see if the annealing temperature is affecting specificity in your samples and then have a look at your primer design. It sounds like you will have to change one or both of your primers.

 

 

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.....

[hypervalent_iodine]

That is strange. Did you notice any other bands in the gel at all? I suppose you'll just have to change primers and see what happens.

[Pendragon04]

I had the same experience a while back. I knew that my primers were specific for my gene of interest but when I sent the products off for sequencing, I got totally different hits.

 

If you're not getting the right amplicon size when you conduct PCR, then chances are you're not getting your gene of interest. Possibly because the DNA template you're using might have some mutations in that gene or the primers are not that specific. Or like what the others said, you need to further optimize your conditions.

 

Also, another advice is you need to open the sequence chromatogram file and check whether or not the automatic base calling was correct. If your PCR products weren't purified or if it's too small, the polymerase from the sequencing reaction might not be able to compliment the correct base or there could be some degradation. Anomalies like this often happen. So for endpoint PCR, ideally you'd have to send products more than a hundred base pairs long to account for end degradation. Also, make sure that the ones you send do not contain nonspecific products as this could interfere with your results. You can do gel extraction or there are spin columns available to separate the PCR mix from the products and deactivate the polymerase and other enzymes you used in amplification.

[CharonY]

What, precisely, is your template? A cDNA library or a single cDNA template, for instance? Or purified RT-PCR products? A quick check (prior to sequencing) to see whether the amplificate is correct is doing a quick restriction analysis, btw.

[Greippi]

You might need an even higher temperature than you've been using. I recently had the same problem and had to go up to 72 degrees before it worked.

[biochemistry3096]

the cDNA template was derived by extracting RNA was reverse transcribing it to make cDNA.

 

Are there any other suggestions for amplifying the wrong fragment during PCR apart from the ones suggested above ? I have checked my primers specificity by conducing a blast search which yeilded my desired amplicon as the only hit, so i dont understand why i amplified the wrong cDNA fragment???

[CharonY]

There are a lot of sources for mispriming events. Was the RT-PCR conducted with random primers or the same for the re-amplification (big source here). Was the cDNA cloned and checked for accuracy or immediately amplified? Also note that short sequences (most primers are around 20 bp) are not well suited for BLAST searches, make sure that you have enabled short and near exact matches (or something similar, forgot the precise option). Other common strategies involve concatenation of both primers with Ns in between.

[CTS]

Usually before sending off something for sequencing, I load the 1-2 ul of the sample onto the gel. If and only if I get one single band, I send it for sequencing. Could you please post a picture over here so that we can all help?