By PCR I got a ~500 bp amplicon from a microorganism. I did gel purification and sent for Sanger sequencing with the PCR primers from both ends. Now the sequencing result from the forward primer is around 1000 bp and the result from the reverse primer is around 500 bp. The reverse complimentary sequence from the reverse primer sequencing can be aligned with the first 500 bp of the forward primer sequencing.
Blast the first 500 bp and the last 500 bp got the same results. They are both the microorganism I PCRed from.
So, does it mean the amplicon purified from the gel contained the template microorganism DNA?
yes you have the right amplicon and species. I think the most likely reason for the longer sequence is that a weak background signal is being read as a proper sequence from the forward primer after the first 500 bases. Can you post the 2 sequences in the .ABI file format then it will be easier to explain the extra bases
1. You said that the first-half (500bp) sequence (amplified with forward primer) matches what you want. What about the second-half of the sequence? Can it be clearly read? If so, did you blast it? What does it belong to? the same organism?
2. If the amplicon purified from the gel contaminated with the template microorganism DNA, as you questioned, how come the reverse-primer only amplified 500-bp product. It could be longer than 500 bp.
3. If problem remains, you can quickly clone the PCR product into a TA-cloning vector for sequencing. Then you should have a single species of DNA template for sequencing.
How qood is the quality of your sequence after the 500 bp of correct sequence? Does the signal significantly drop and is of poor quality? This may be due to signals emitted from the neighboring capillary; you simply need to trim your sequence to the correct size, there is nothing wrong with your amplicon.
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