Hi, I sent my PCR product sample for sequencing. The sequencing company will purify the sample. The expected sequencing region is around 800 bp. However, the sequencing can read only 200 bp. The PCR product is a single band at 800 bp. The primer is around 23–30 bp with a GC > 60. What is wrong with my sequencing result?
One possibility is that you have and indel where the sequence breaks up. Please attach an original .abi sequence file of the sample and things will be a lot simpler. Insertions or deletions mean that there are 2 sequences after the indel and it is usually possible to separate the 2 sequences to define the change. It will be useful to sequence from the other end (reverse primer) as well
We hope you have checked the sequence length provided by the company. Some companies sequence only up to a particular length. For an example there are companies which will sequence your product up to 400 bp or even 900 bp. Check the instruments available with the company before sending the samples. Certain instruments can sequence only short lengths. But most of the instruments can be calibrated to sequence longer reads. Let them know you need to sequence the full length.
Or, the other possibility is that the company scientist may have used excess template (PCR product) for sequencing reaction. If that has happened, then the read length of the sequence will be dramatically reduced.
Or, if the Primer concentration is too high, then the sequence length will again be shortened. This happens when the ddNTPs will be utilized to synthesize new strands.
So, the scientists who processed your sample may have all the details. Ask him/her to reduce the pcr product template and primer concentrations. It might work. Hope this information helps. I assumed that you have used Sanger sequencing method.
Let us know what has happened. Good Luck!
If the region of interest is 800 bp, and the sequence reads out to 200 bp, does the sequence stop abruptly at 200 bp, or do you get a double sequence after 200 bp? If the sequence stops abruptly, you may have a region of secondary structure in the template inhibiting the formation of extension products. You may want to try using GTP mix or design a primer close to the region where the sequence stops. Are you sequencing from the other/reverse direction?
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication