Primer Design

[Sr_08]

I am trying to amplify a region for a strain tht has not been sequenced so far.
I designed primers using the the sequence information of the conserved domain.
The size of my amplicon expected is around 600bp. But, I was only able to amplify a 200bp product.

The region tht was conserved was around the region between 120-480 bp.

Can anyone suggest any other ways to design the primers ?

thnx!

[Irideus]

Maybe you could design your primers inside the region that is well conserved, instead of using flanking regions?
What´s your departure material? gDNA? cDNA?
Is it a bacterial, yeast or viral gene you are trying to catch?

[Sr_08]

I used gDNA as my template and used the inside region which was well conserved.
It is a bacterial gene I am trying to catch.I have highlighted the conserved regions I used for my Forward and Reverse primer design.
I am lost; any thoughts how how to get the complete gene amplified?


gi 50593059 13 ARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFN 92
Cdd:TIGR01323 1 ARAKALEQVLKSKGLIPEGAVDQLTSLYENEWGPENGAKVVAKAWVDPEFRALLLKDATAACAQFGYTGPQGEYIVALEN 80

90 100 110 120 130 140 150 160
....*....|....*....|....*....|....*....|....*....| ....*....|....*....|....*....|
gi 50593059 93 DSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADPRGVLkRDFG FDIPDEVEVRVWDSSSEIRYIVIPERPAGT 172
Cdd:TIGR01323 81 TPGVHNVVVCTLCSCYPWPVLGLPPEWYKGFEYRARLVRDPRGVL-REFGTELPSDVEIRVWDSSAESRYLVLPQRPAGT 159

170 180
....*....|....*....|....*.
gi 50593059 173 DGWSEDELAKLVSRDSMIGVSNALTP 198
Cdd:TIGR01323 160 EHMSEEQLQQLVTRDSLIGVSLPRTP 185

[Irideus]

OK. According to the sequence you pasted, your amplicon should be around 372 bp (124 aminoacids*3), so I´m confused about you saying you should expect a 600 bp amplicon?
Besides, now I see you are using degenerate primers (and since you lack nucleotide sequence info, these are REALLY degenerate). That could explain why you are getting an unexpected amplicon: it could be due to a simple mispriming.

Several strategies could help in minimizing off-target amplification. These are:

1. "Hot-start" (keeping your tubes in ice, and placing them in the cycler block only after it has reached >80 degrees celsius).
   
2. Raising your annealing temperature (Tann) an empirical number of degrees (2-5), or
   
3. A "touch-down" protocol (starting with an annealing temperature which is ten degrees above your normal Tann, and lowering one degree per cycle through ten cycles (consult your cycler manual or brochure to do this) before letting the rest of the program cycle using the usual Tann.

Alternatively, you can redesign your primers.

The 3´end of your forward primer looks quite nice. A lysine in the 3´end is a good deal: only two possible codons coding for it (i.e. fewer degeneration). But I still would have continued to the tryptophan (only one possible codon) to enhance specificity. Thus, the sequence to add to the 3´end of your current forward primer would be: DSIUGG (Ser/Ala + Trp). I think this could enhance your primer specificity a lot.
With respect to the reverse primer, it looks like you´ve chosen many aminoacids with wide codon redundance. This greatly hampers your primer specificity.

I would use the following region to design a reverse primer:

gi 50593059 175 WSEDEL
Cdd:TIGR01323 172 MSEEQL

The REVERSE primer thus being:

5´-IARRTSITCYTCISWCKW-3´

I hope this helps at least a little.
Good luck with your experiments.

[Sr_08]

HI Irideus,
Thank u very much. Sorry to get back late to ur posts though...
will surely keep u updated.

[Sr_08]

Does this region seems ok for Forward and reverse?

FP

ARTKAIE

RP

RDSMIGVS

[Irideus]

Yep. They look pretty cool.
Now is my turn to apologize for the delay in getting back to you.

Have you had any advances in your cloning?

Regards,

Leo.