Screening Libraries by PCR

Paul N Hengen pnh at ncifcrf.gov
Thu Aug 8 12:03:48 EST 1996


I am posting bits of a conversation I had with Miguel A. Gomez Lim in hopes
of getting further comments on his suggestion to screen libraries using PCR...

> I have sent several messages to the netters enquiring on the use of PCR alone
> to screen gene libraries (cDNA and genomic)? I have only obtained a couple
> of answers back one positive and one negative.
> I think this is an excellent application of PCR and am surprised people
> have not used it more extensively.
> Miguel.

| I'm not quite sure what exactly you mean by "screening". What are you
| screening for?
| -Paul.

> What I mean by screening is simply the isolation of sequences from gene
> libraries. Using PCR is much more cheap, fast and easy than by
> hybridization. If you could tell me how I can start a discussion of this
> topic in the methods newsgroup I would do it.
> Miguel.

| I'm curious about this topic. Maybe I'm not fully understanding why you
| would want to PCR a gene from an artificial library when you can clone
| the PCR amplified gene directly from genomic DNA. I must be missing something
| important here. I can see that screening this way by PCR would be limited
| in that you cannot be sure your primers will match both sides of the DNA
| fragment cloned within the library. So, why couldn't you screen the colonies
| with a radiolabelled probe DNA instead? Wouldn't this be more efficient for
| recovering all similar sequences regardless of where your primer sequence
| is located?
| -Paul.

> You are right to say that one can screen the colonies with a radiolabelled
> probe DNA. However you can also PCR a phage lysate (for instance) and
> obtain the clone(s) that you want. Your also right to point out that one
> cannot be sure the primers will match both sides of the DNA fragment, but
> this is true for many PCR reactions. Using degenerate primers based on
> heterologous sequences certainly help. As I see it the main advantages of
> this method is the cost (no nylon membranes involved), the fact that no
> radioactivity is needed and the speed. Check this paper  Biotechniques
> 16(1) 98-103. It would be great if you could post it.
> Miguel.

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