A fragment of DNA from a genomic prep was amplified by PCR using specific primers. This fragment was PCR purified and sent for sequencing. The sequencing results however indicated that possibly more than one amplicon is present in the sample (mixed sample). What cloning strategy that can be used to resolve this issue so that the nature of the PCR products can be accurately deduced?
I think you question has been well answered by the respondants. Do note, however, that Taq polymerase is quite error-prone. This doesn't matter when you are sequencing the product directly after amplification, because the errors average out. However, cloning isolates and amplifies a single amplicon molecule along with any errors that Taq has put in. Therefore, sequence several 'identical' clones and create a consensus sequence. Alternatively, re-amplify with a high fidelity polymerase.
You may just repeat the PCR with a higher annealing temperature. Concerning cloning: Assumed that you have not used a proof-reading enzyme, I would recommend to use TA-vector. A restriction enzyme analysis of a few (~12) isolated clones may identify diverse inserts. Sequence at least one of them with the expeced size.
I recommend using TA cloning. Are you using Taq based polymerase for your PCR?
Than your amplicons have a-overhangs, and you can go straight to TA cloning. I have used both InsTAclone PCR Cloning Kit from Thermo and TOPO Kits from Invitrogen with good results. Use vector-specific primers for sequencing, so you get a good coverage of your PCR product.
I think you question has been well answered by the respondants. Do note, however, that Taq polymerase is quite error-prone. This doesn't matter when you are sequencing the product directly after amplification, because the errors average out. However, cloning isolates and amplifies a single amplicon molecule along with any errors that Taq has put in. Therefore, sequence several 'identical' clones and create a consensus sequence. Alternatively, re-amplify with a high fidelity polymerase.
You can PCR again and purify the specific fragment by agarose purification (cutting the fragment from the agarose gel), qiagen, promega and zymo have kits for that.
Dear colleagues.
Although ,I think you have received to the best answer until now. However, I think it is better that check your primers first and your sequence after.is it GC rich?If it possible use pfu and pfu buffer instead Taq PCR.
In fact,TA vector could be useful at the next step.and as Andrew said,It may happens consequently when you are sequencing your fragment directly after amplifying.
Best Regards.
I agree with all the above suggestions. Additionally I suggest you to carefully look at your primers once again. We have experience that sometimes primers work fine at higher annealing temperatures during PCR (55-60 degrees annealing temperature) and yield single amplicons. However, during cycle sequencing reaction (annealing at 45-50 degrees centigrade) the same primer may partially anneal to an internal sequence in the template and give a mixed read.
Nevetheless, cloning and sequencing using the vector primers is always a better option.
Best wishes,
SD
Just remember that if you do TA cloning to check on which enzyme you are using to amplify. TA cloning only works if you have the overhangs left by TAQ or some similar enzymes. The error proof amplification enzymes often do not leave overhangs.
A few options come to mind:
1. Your PCR has more then one product (happen a lot) and since you do only PCR purification you do not separate them. You can purify from the gel and sent for sequencing.
2 .More then one product but they are in (approximately( same size so agarose gel does not separate them well enough.
If indeed (2), run the gel, purify, and do blunt cloning into pJET or similar. Pick 5~6 colonies, extract plasmids and sent for sequencing.
Yoram
Hi Nathanael Forde
From your explanation about the question I feel some more information is need to be explained such as amplicon size, presence of multiple copies with some variation sequence etc.
But another clue is there may be possibility that your pcr product is not completely purified from non specific bands provided that prior to purification you might have check pcr product on gel.
Second the size of amplicon is not having detectable difference ...long and high % gel is solution for it.
You can find the chance of contamination if any by doing the blast with two sequences or multiple copies of that
The sequencing error can also misguide while contig assembly so check the quality of sequence first before proceeding most the end of pcr product sequenced are error prone
Good luck....
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