DNA Collection

DNA sequencing reactions are just like the PCR reactions for replicating DNA (refer to the previous page DNA Denaturation, Annealing and Replication). The reaction mix includes the template DNA, free nucleotides, an enzyme (usually a variant of Taq polymerase) and a 'primer' - a small piece of single-stranded DNA about 20-30 nt long that can hybridize to one strand of the template DNA. The reaction is initiated by heating until the two strands of DNA separate, then the primer sticks to its intended location and DNA polymerase starts elongating the primer. If allowed to go to completion, a new strand of DNA would be the result. If we start with a billion identical pieces of template DNA, we'll get a billion new copies of one of its strands.



Dideoxynucleotides: We run the reactions, however, in the presence of a dideoxyribonucleotide. This is just like regular DNA, except it has no 3' hydroxyl group - once it's added to the end of a DNA strand, there's no way to continue elongating it. Now the key to this is that MOST of the nucleotides are regular ones, and just a fraction of them are dideoxy nucleotides....



A 'Scan' of one gel lane:

We don't even have to 'read' the sequence from the gel - the computer does that for us! Below is an example of what the sequencer's computer shows us for one sample. This is a plot of the colors detected in one 'lane' of a gel (one sample), scanned from smallest fragments to largest. The computer even interprets the colors by printing the nucleotide sequence across the top of the plot. This is just a fragment of the entire file, which would span around 900 or so nucleotides of accurate sequence.

The sequencer also gives the operator a text file containing just the nucleotide sequence, without the color traces.

Shotgun sequencing: assembly of random sequence fragments

To sequence a BAC, we take millions of copies of it and chop them all up randomly. We then insert those into plasmids and for each one we get, we grow lots of it in bacteria and sequence the insert. If we do this to enough fragments, eventually we'll be able to reconstruct the sequence of the original BAC based on the overlapping fragments we've sequenced!