🚀 go-pugleaf

Oky.

First, the Brookhaven group did NOT invent primer walking -- just
a new twist on it (see one of the most recent Science's for details).

The idea of primer walking is that you will sequence a gene in steps --
much like using stepping stones to cross a stream you can't jump.

Suppose your DNA of interest looks like:

0)  kkkkkkk????????????????????????????????????????????????????

where a "k" represents a block of known sequence, and ???? the unknown
sequence you wish to acquire.  You synthesize a short (~20nt) piece
of DNA which will hybridize within k and provide a free 5' OH group pointing
towards the unknown region, and acquire sequence using dideoxy sequencing:

        p--------------->
1)  kkkkkkk????????????????????????????????????????????????????

You have now extended the known region, and so you can repeat the
process until you have "walked" as far as you wish.

So what are the problems?  Since you can't make the 2nd primer until
the results are in from the first round (2 days minimum to get data
plus 1 day synth time), you must have many such walking points to get
anywhere quickly. Worse, if one reaction fails you are delayed an extra
round, since that deprives you of the new knowledge (If your stepping
stone falls into an unseen deep spot, you have to find another one and
try again).

The Brookhaven group claims to eliminate the delay from the synthesis 
by using 3 6nt sequences lined up end-to-end instead of a custom-synthesized
primer.  The advantage is that someone can synthesize all the possible 6nt
sub-primers and you can draw them from a stock, eliminating the extra day's
wait for the synthesis.  IT DOES NOTHING ABOUT ANYTHING ELSE!  So, failures
still cost you dearly.

Another variant on primer walking can be found in:

Ohara, O., RL Dorit, W Gilbert.  1989.  PNAS 86:6883-6887.
	Direct genomic sequencing of bacterial DNA: The pyruvate kinase I gene 
	of Escherichia coli.

This method is actually being used to completely sequence the genome
of a small bacterium.


Keith Robison
Harvard University
Department of Cellular & Developmental Biology
Department of Genetics / HHMI

robison@biosun.harvard.edu