Hi there
I isolated bacteria from serum sample did the extraction of DNA for bacteria, followed by PCR. PCR band was of 100bp I sent it for sequencing. The results were approx 43 bp forward primer and 57 bp reverse primer. I reverse complemented the reverse primer seq and did the BLAST of both forward and reverse.
I got BLAST matches but for both there is no single match. The error message shown in BLAST search is, “No significant similarity found.”
I did following one by one;
1. I did the blast separately and also joined the two seq and did again
2. I did un check the low complexity region
3. I did the Mega-blast
but the result is same.
My primers were highly specific primers. I am suspecting following things;
1. Primer got attach to non-specific site for some reason and amplified junk.
2. During gel purification the sequence was denatured
3. It is actually some human intron sequence and not present on NCBI
4. And last but very least suspected it is some new sequence.
Help me I am lost and worried I have to submit my results so please help me.
Many Thanks
With short PCR products you may need to clone them before sequencing to get reliable results. Check e.g. discussion here: https://www.researchgate.net/post/How_can_I_best_carry_out_sequencing_small_PCR_amplicon_70_bp_by_Sanger_sequencing or here: https://www.researchgate.net/post/How_to_sequence_short_DNA_fragement_less_than_100_bp
If BLAST doesn't give you significant hits, you may try to increase the e-value limit and fiddle with the word length (lower word length takes longer to calculate but finds more hits). At certain e-value though, even if you get hits, you're gonna get hits that may simply represent random noise, especially if your query is not a proper sequence of some biological material, but say some artefact of PCR or sequencing.
With short PCR products you may need to clone them before sequencing to get reliable results. Check e.g. discussion here: https://www.researchgate.net/post/How_can_I_best_carry_out_sequencing_small_PCR_amplicon_70_bp_by_Sanger_sequencing or here: https://www.researchgate.net/post/How_to_sequence_short_DNA_fragement_less_than_100_bp
If BLAST doesn't give you significant hits, you may try to increase the e-value limit and fiddle with the word length (lower word length takes longer to calculate but finds more hits). At certain e-value though, even if you get hits, you're gonna get hits that may simply represent random noise, especially if your query is not a proper sequence of some biological material, but say some artefact of PCR or sequencing.
thank you everyone for your advise. I have started with a new primer set for a large PCR product.
Your desired fragment is 100 bp, it's too small for direct sequencing based on the specific primer. Generally in sequencing process 1st 25 to 50 bp was not giving the required resolution hence there are mismatched and misreading may come. Hence you may go for cloning by using any cloning method like T/A or other else and Target the sequencing primer from the vector sequence by flanking additional minimum 50 bp of your target sequence. I think you will solve your problem.
- At 100bp it should definitely be possible to amplify a specific fragment
- If that were not true then it would not be possible to selectively and specifically amplify by PCR with primers approx to 20bp
- If as you say you have checked the specificity of your primers by BLAST then the most likely explanation is that you have amplified something non specific due to low stringent PCR conditions
- I very much doubt this is a BLAST stringeny issue; rather a PCR stringeny issue
- Try increasing the temp of your PCR by 1-2C and in any case make sure the annealing temp in your PCR is about TM-2C
- As mentioned cloning might not be a bad idea as well; if only to ensure you a single clone and by cloning making large quantities of your fragment for clean unambiguous sequencing
- Remember if you do clone to send off 6 minipreps derived from 6 picked colonies for sequencing. Hopefully among those 6 1-2 will yield specific unambiguous sequence
- Furthermore sequence around 100bp per kb of plasmid plus insert: that will maximise your S:N and render BLAST comparisons specific
Clone your PCR product to a TA cloning vector and sequence the vector using vector primers.
Hi, What size where you expecting when you did the PCR? Was it 100bp?. If it wasn't then you should repeat. Your fragment could be primer dimer. As it has been described, a primer dimer is made up of primer molecules that have bound to each other because of complementary bases in the primers. The DNA polymerase may amplify the primer dimer, which may lead to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification.
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