I am getting PCR product sequenced for one gene. Is it necessary to to do sequencing with both forward and reverse primer? What is the relevance and benefit of doing this?
it depends on many things. Size. You can sequence about 900 bases so amplimers longer than 900 will need both ends. Short sequences need both ends very often because you cannot sequence under the primer or for the nearest 40 bases from the 3'end of the primer so the ends of short sequences can only be sequenced from the other end. Sometimes conformational problems like loops and repeats will sequence well on one strand but not on the other. If your primers are 40 or more bases from the sequence of interest then it is often not worth sequencing both strands if the signal is strong and you get all of your target.example for a short exon primers are often 60 bases away from the splice sites so you get all of the exon from either direction
Thanks Dr. Paul,
My primer has 20 bases forward and 21 bases reverse.
I am trying to sequence PCR product showing amplification ~560bp. After sequencing I am getting ~485 - 509 bp sequences, in this case is it necessary to confirm it by reverse primer ? I am not getting las 20-30 bases all the time . Is it common for all the time in Sanger sequencing? or this is problem of DNA Analyser ?
This sounds very good sequencing. You cannot get any meaningful sequence from under the primer because if the pcr has worked then the primer sequences are in every amplimer even if there was a . under the primer. Not getting sequence close to each primer is a consequence of the sequencing kit chemistry. The ratio of ntps to dideoxy ntps and the amount od manganese are designed to give 900 bases of sequencing and to achieve this good sequencing close to the primers has been sacrificed
If you make up your own sequencing mix you can do this but it is much better
use a commercial kit and move the primers further out if you want this sequence.
You may get a slightly longer sequence read if you load more sequenced sample if the original signal strength is weak at high base numbers but it is often better to have good sequence at both ends by doing both sequences. It depends what you want to do with the sequence. If all of the sequence is important and you are cloning and doing expression then you must be certain of the sequence and you must sequence both directions but if you are comparing your sequence with another sequence and you have good signal strength and low background over all of the compared sequence then on direction should be fine and is half the price although most people will think that their time is more valuable than the cost of another sequence
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