Could I sequence a gene directly from PCR products, or do I need to use cloning?

Question

I'm trying to learn this stuff from a textbook (without a lab), so it's all pretty abstract...

If I were to want to sequence an entire gene in an individual organism... could I just use PCR to amplify the gene and then sequence that with Sanger Sequencing? Presuming the gene is longer than than 800 nucleotides, is there a way to "primer walk" or "shotgun sequence" using solely the products of PCR?

Or, am I just supposed to clone fragments of the DNA using vectors, and then use those for the sequencing and primer walking?

If I am supposed to clone the fragments, then should I still use PCR to make the original fragment, and then chop THAT up with restriction enzymes to get the smaller fragments... or is PCR an unnecessary step in all this.

I'm kinda confused. Any help would be very much appreciated!

Thank you!!!

Answer 1

Yes, you can sequence directly from PCR products, but it does not always work, may return poor sequence data and you will never get sequence data from the ends of your gene, since you will be using gene specific primers.  You can overcome the last shortcoming of this approach by using sequencing primers that return overlapping sequence data on the ends.  If the gene is in excess of 1000 base pairs you can primer walk on a PCR product as well.

For the best results I would PCR the gene of interest and clone the fragment before sequencing.  You can then produce a lot of template very easily and inexpensively.  Once the fragment is cloned you can sequence using universal primers contained on the vector allowing you to get sequence from the entire inserted fragment.  If your gene is very long you can either primer walk or fragment the gene first using restriction enzymes and then clone each individual fragment.  Once the fragments are clone you can sequence each one using universal vector borne primers.

Answer 2

Yes you can sequence directly from PCR products.

However, cloning vastly improves the purity and quantity of the sample, which is why it's done.  Sequencing from a plasmid is also far better than from a random strand of DNA.  (A plasmid vector is stable, and has a lot of start and end locations for you to prime your sequencing reactions.)

Lol, you're confusing me too.  So I'm not sure if I'm answering your questions but I'll give it a go.  I'm sure I'll be repeating info you know, sorry for that.

Primer walking is used because current sequencing techniques are really only good for 500-800 bp.  For that mater, long-distance PCR isn't always that efficient either (especially with Taq polymerase; more expensive polymerases work quite well on the other hand).  Primer walking just means you do PCR of the gene in several reactions.  So you don't get the whole gene, but small parts of it, each around 500 bp.  So then you get reliable sequencing from each PCR reaction.

When cloning, you still need to do PCR (where are you going to get your insert, otherwise?).  Cloning is done for two reasons: 1. it's far easier to sequence a cloned gene 2. cloning is necessary if you want to do something with the gene later; so you may as well clone it before you sequence it.

The only real reason to sequence a PCR product is because cloning is time consuming (1 day, usually 2).

I'm not sure why you're mentioning restriction enzymes.  I presume you thought you'd cut up a full-length PCR into smaller pieces to sequence?  Well, that's not too smart; if you're sequencing it, you don't know where your cut sites are, and you'd end up with an unknown number of unknown length fragments.  Plus, that doesn't solve the problem of lower PCR efficiencies for long-distance PCR.

Answer 3

It all depends on how much you have.  

If you can get large amounts of the gene, there's no need to do any PCR other than the sequencing itself, although you will have to do several reactions.  Sequencing is only accurate for about 500 nt.

If you can't get much, such as the people trying to sequence the Neanderthal genome, you'd have to clone it.