Primers for PCR - Forward and Reverse Primer Sequences (Dec/23/2010 )
I am confused by the sequnces of primers which are used in PCR making DNA fingerprints.
For example, I often come across the term 'forward and reverse primers', where one sequnce is complementary to the other sequance, such as:
GATCTTAGCTTTAAAGCCC
CTAGAATCGAAATTTCGGG
Furthermore, I also come across websites which provide sequences for specific loci (STR's), but these sequnces are not complementary, for example:
CAA ACC CGA CTA CCA GCA AC
GAG CCA TGT TCA TGC CAC TG
What is the difference and which would you use as PCR primers if you were targeting specific loci?
The first set of primers are unrelated. It would be highly accidental if they were used to amplify a pcr product. I suspect you are misreading the sequences, or failing to note which are the 5' or 3' ends. When written without further annotation, the 5' end of the sequence is (by convention) on the left, and 3' on the right. But if you write the reverse complement strand below a sequence, then that string will have the 3' end at the left, and the 5' end on the right.
The much more common case is the second one, where the primers bear no apparent relationship to one another -- they bind to different places on the target DNA strand, surrounding the pcr product location. The "forward" primer by convention will bind to the sense strand at the left, while the "reverse" primer will bind to the anti-sense strand, on the right. This means that when you look at the sense strand, you must search for the reverse complement of the reverse primer to find its binding location.
phage434 on Fri Dec 24 02:39:56 2010 said:
The "forward" primer by convention will bind to the sense strand at the left, while the "reverse" primer will bind to the anti-sense strand, on the right.
Hey, just a question, Wouldn't the forward primer bind to the anti-sense strand and make the sense strand and the other way around for the reverse primer.... or is it just me
Just as you said. Right.
The second pair is the one you need to amplify D18S51
5'-CAA ACC CGA CTA CCA GCA AC-3'
5'-GAG CCA TGT TCA TGC CAC TG-3'
See:http://www.cstl.nist.gov/biotech/strbase/str_D18S51.htm