Often time I see scientists using both forward and reverse primers for amplification of a particular gene.. Is it really necessary??
Hi there and Happy New Year,
As DNA is double stranded, you need both the forward and reverse primers. Let's say you used only one of the primer, such as forward primer. So during the PCR, it will only bind to the forward strand and amplification of the reverse strand happens. Since, you do not have reverse primer, the forward strand is not being amplified. At the end of the PCR, you'll get a lot of reverse strand but very few/the same amount of forward strand you began with. This also leads to you having single stranded DNA as your PCR product.
If you only use one primer then at each cycle you have 2x the original amount of forward strand so after 10 cycles of one primer pcr you have 20x as much forward strand. With a reverse primer also doubling the total amount of amplified dna after 10 cycles you have 1024 times as much product as you started with. After 20 cycles of 2 primer we have 1,000,000 times as much amplimer. " primers is logarithmically more efficient
Thanks @Andrew Ting and Paul Rutlant.. But are we saying there won't be good result if we use one primer.. Even if the amplification is ultimately for identification like ITS
that is exactly what we ate both saying. There will be no amplification and nothing to detect with only one primer. The only times I have ever known 1 primer to work is when the primer is part of a repeat sequence and two repeats face each other in the genome then one primer can work in both forward and reverse direction but this is not often useful except for a variant of this boxair pcr when large numbers of quite random pcr products can be used for fingerprinting
Well, actually if you want to know how important are the primers, then have a look at what is happening during DNA replication IN VIVO. At the lagging Strand there will be a lot of RNA primers formed by PRIMASE enzyme at the replication fork with only few of them at the leading strand. The idea in both cases is that the DNA polymerase enzyme cannot work on totally single stranded DNA, that is why we use these primers. This will ensure an optimal action of Taq enzyme. In addition, having a pair of primers will determine the size of the target to be amplified.... Best wishes
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