Degenerate Primers

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c1bl
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Degenerate Primers

Post by c1bl » Tue Jul 31, 2007 3:16 pm

Hello,

I'm very new to molecular biology and I'm helping start up a lab. My supervisor is also new to molecular biology and we are learning as we go so please excuse me if I sound ignorant.

I have a few questions about degenerate primers.
1.) Is there an advantage of using the AA sequence over Nucleotide sequences when aligning sequences for degenerate primer design?

2.) When aligning sequences do we pick the most highly conserved area and design the primers around that area?

3.) Is Vector NTI a good program for this? I have also heard of using ClustralW??

4.) Degenerated Primers are generally the same price as regular primers? Where is a good place to purchase the primers if I'm on the East Coast of Canada.

Any tips or advice would be greatly appreciated as I am very new to this area of research.

Cheers,
Chris

bjfeilmeier
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Post by bjfeilmeier » Tue Jul 31, 2007 5:37 pm

Hi Chris,

Have you figured out your degenerate primers problem yet? If not, here's a bit of help:

1) This depends on how much you know about your organism. Codon usage is the most important factor. If you have a good idea what the amino acid sequence is, then use codon usage tables to decide what codons to use. Nucleotide sequence is usually a pretty poor choice.

2) Yes, I would look at the most highly conserved regions.

3) Vector NTI is great (I use it pretty heavily myself) but there is a steep learning curve with it. It would be worth the time to learn it. The ClustalW algorithm is integrated in Vector NTI. There are versions of ClustalW available on some public websites that can do the alignments for you.

4) I would recommend IDT (Coralville, IA). I've tried many different companies and they are the best.

Hope this helps!

Brad

c1bl
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Degenerate Primers

Post by c1bl » Wed Aug 01, 2007 4:05 pm

Hey Brad,

Thanks for the Reply. I'm half way there to figuring out my Primers. I tried to use Vector NTI yesterday but like you said it has a high learning curve. I'm currently using a 30-day trail of CLC combined workbench for a Mac which is extremely user friendly and efficient.

I've managed to produce degenerate primers from my aligned sequences in a matter of minutes.

For: YGMKGAGGARRACATWASWG
Rev: CAARTTWCCAAYSRYMCCMA

Those are the two primers I got.

Everything except for you ATGC's have an equivalent degeneracy right. I limited my primer search to 256 degeneracy max. What is the maximun degeneracy i should use?

Cheers,
Chris

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