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Degenerative primers = multiple products? - (May/19/2009 )

Hi, im hoping someone can clarify something for me.
We use degenerate primers for cDNA PCR, we get a single band on gel.
If we clone and sequence this band, we get a tree with different groupings of clones
-this means the band was a mix of different product sequences?

we ask because now we have only clones that group together on tree - should we expect more variation?
or does cloning only insert part of PCR product?

Thanks

-Mulletman-

You cloned the products from the degenerative PCR and then sequenced the clones. How many of them have you sequenced? The products may represent a mixture of different sequences with different abundance. sequencing more clones will increase the chance of identify rare sequences in the products.

-pcrman-

Don't forget that polymerases have an error rate, if you used Taq for your PCR then you have an error rate of 1 base in a thousand (0.1%) so the different clones may represent different errors from the polymerase.

-bob1-

Thanks
I guess i was wanting to confirm if degenerate primers is like fishing for unknown sequences - to get a range of potential products of similar size

or should we expect only 1 product..

-Mulletman-

Mulletman on May 20 2009, 06:06 AM said:

Thanks
I guess i was wanting to confirm if degenerate primers is like fishing for unknown sequences - to get a range of potential products of similar size

or should we expect only 1 product..


yes, you should expect only one product.

we prepare degenerate primers when we have amino acid sequence information (not nucleic acid sequence information).

you select a unique region in the aa sequence then determine the theoretical na sequence for the aa sequence. this is where the degeneracy comes into play, there are multiple codons for most amino acids. you may be able to reduce degeneracy based on codon usage for the source species but it will still require a degree of degeneracy.

so, the degenerate primer is still specific for a sequence, hence there should be only one product (there may be allelic variations).

-mdfenko-

If it's a single PCR band giving rise to multiple sequences, you could also be getting splice variants, since your template is cDNA.

-HomeBrew-