A sleazy way to avoid direct PCR Sequencing
sk at lilly.com
Fri Jan 21 10:26:12 EST 1994
In Article <2hml4p$880 at news.u.washington.edu>, findley at u.washington.edu
(Seth Findley) wrote:
>Everybody knows that sequencing a single subclone from a PCR reaction
>isn't too smart because of Taq-mediated mutagenesis.
>And direct sequencing of PCR products can be a technical challenge...
>Can anybody tell me what might be wrong with the following technique
>which I used recently to sequence something I PCR'd?
>In order to increase the complexity of the template I wanted to
>sequence, thereby avoiding individual artefacts I did this:
>After cleaning up my PCR reaction and polishing the ends (I have a very
>efficient protocol for this) I subcloned the fragment into a very well
>phosphatased (blunt) vector and transformed some E. coli.
>Now, rather than pick ONE colony for sequencing, I scraped about one
>*thousand* colonies off the plate and resuspended and mixed (well) the mass
>in a small volume of medium. Then I used a bit to inoculate an overnight
>with. I did an Acid-Phenol miniprep for sequencing and got nice clean
>sequence. (I used an internal primer, so any vector subclones wouldn't
>be primed by my internal primer).
>In this way, I was able to sequence many different PCR products with
>Now I know this looks pretty retrograde, but for somebody who might not
>be able (for whatever reason) to sequence PCR products directly:
>Is a thousand colonies enough to be pretty sure of the sequence?
Yes, there is a serious problem with this approach. All you're getting is
a consensus sequence. You could have a high level of variation in your pool
and not detect it this way. Just as an illustration, let's suppose that
your PCR product is correct at each position in 90% of the molecules. Then
let's say that in the other 10%, all three bases are present at about equal
amounts. You might not detect this extremely high level of heterogeneity,
depending on how you do your sequencing and how you read your gels. If
correctness of the final PCR product is important to your downstream work,
you're better off sequencing a few individual clones rather than spending
weeks or months working with the wrong thing and then finding out that the
sequence is wrong. Of course, if all you want is the sequence, then you can
ignore the comments above. In that case you only need to worry if some
(incorrect) sequence is favored over the correct one during growth. That's
possible, of course, but perhaps less likely.
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