iCODEHOP help: how are the reverse primers supposed to be read? - Degenerate primer design (Sep/27/2010 )
Hi,
I'm trying to design degenerate PCR primers and have hit an unexpected snag using iCODEHOP. I don't understand how the reverse primers generated by the program are supposed to be read. I would assume that in the output from the program they are written 5'-3' (from left to right) - complementary to the reverse strand - as is the convention. However, several confusing bits of information seem to contradict this simple assumption.
First is "iCODEHOP help" 's cryptic description: "the primer's direction; 'forward' primers run 5' to 3' along the selected sequences while 'reverse' primers are designed to hybridize to the anti-sense strand of your target and are shown as if hybridized to their reverse complement beneath the blocks region" - no further explanation is given.
Second, the iCODEHOP primers are, as far as I understand, designed so they have a non-degenerate 5' "clamp" region (CAPITALS) and a degenerate 3' "core" region (lowercase). For example a forward primer: CGGCGGCGGCcayathaayca. In contrast, the output for a reverse primer looks like this: accctygtrcgGATGATGGACGTCATGTTC. Is that 3' - 5' or the conventional 5' - 3'? Is it the true sequence or the complementary sequence?
As I'm ordering degenerate primers the sequence direction and complementarity is naturally important.
Help that sheds light on this mystery is much appreciated!
Cheers,
/Aucklander
Aucklander on Tue Sep 28 04:15:19 2010 said:
Hi,
I'm trying to design degenerate PCR primers and have hit an unexpected snag using iCODEHOP. I don't understand how the reverse primers generated by the program are supposed to be read. I would assume that in the output from the program they are written 5'-3' (from left to right) - complementary to the reverse strand - as is the convention. However, several confusing bits of information seem to contradict this simple assumption.
First is "iCODEHOP help" 's cryptic description: "the primer's direction; 'forward' primers run 5' to 3' along the selected sequences while 'reverse' primers are designed to hybridize to the anti-sense strand of your target and are shown as if hybridized to their reverse complement beneath the blocks region" - no further explanation is given.
Second, the iCODEHOP primers are, as far as I understand, designed so they have a non-degenerate 5' "clamp" region (CAPITALS) and a degenerate 3' "core" region (lowercase). For example a forward primer: CGGCGGCGGCcayathaayca. In contrast, the output for a reverse primer looks like this: accctygtrcgGATGATGGACGTCATGTTC. Is that 3' - 5' or the conventional 5' - 3'? Is it the true sequence or the complementary sequence?
As I'm ordering degenerate primers the sequence direction and complementarity is naturally important.
Help that sheds light on this mystery is much appreciated!
Cheers,
/Aucklander
Well do you know the non-degenerate clamp sequence they are supposed to be binding to? If so you can just compare the output sequence with what it is supposed to be binding to and work out manually which way round it must be binding?
Also if the sequence in Caps is supposed to be the clamp, then surely that must always be at the 5' end of the oligo, while the lower-case degenerate part is always at the 3' end? meaning that the reverse primer is indeed displayed there in 3'-5' direction.
I must admit that is a very unclear description in the help section.