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As far as I understand it, PCR can be used to make many copies of one gene. My question is, is it possible to sequence DNA after PCR and is it easier than sequencing it via other methods. If it is possible, how would you do it?

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2 Answers 2

up vote 8 down vote accepted

All sequencing methods, be it classical Sanger sequencing or next-generation sequencing (or even third generation) need a certain amount of DNA to work with.

You either need to extract DNA from a large-ish tissue sample or you need to amplify DNA content from a smaller sample. The first approach is often impractical, or downright impossible (when you want to work with single cells or very small organisms).

So PCR is used routinely to amplify DNA before sequencing. The sequencing itself isn’t influenced by this – you use whatever technique is appropriate. However, the PCR may introduce biases into the data because not all DNA fragments amplify equally efficiently. This needs to be taken care of in the downstream analysis of the sequence data if quantitative information is required (as in RNA-seq or ChIP-seq).

Coming back to your question, PCR isn’t a sequencing method in itself. It just helps getting the necessary amount of raw material in order for sequencing to work. Furthermore, most sequencing methods actually include PCR steps as well, since they rely on the generation of of overlapping DNA fragments to be stitched together.

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We do it all the time. You can use one of your end/flanking primers and use that as a primer for sequencing. Companies will typically have a setup where they can take a "premixed" sample.

Since we tend to use sequetech, here are their details: http://www.sequetech.com/requirements.php?premixed=1

For a second opinion look at Elimbio's: http://www.elimbio.com/Sample_Preparation.htm

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Could you expand your answer with more of the "how"? –  kmm Jul 2 '12 at 15:42
    
Yes, I'm actually more interested in how they sequence the DNA. –  ohblahitsme Jul 2 '12 at 20:10
    
It should work exactly the same way as sequencing a plasmid. Traditional sanger sequencing read by capillary electrophoresis. –  bobthejoe Jul 2 '12 at 20:36

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