Mapping cDNAs

Barbara Baker bake at ENZYME.BERKELEY.EDU
Sat Feb 1 04:47:05 EST 1992

> ..there were several suggestions that one should probe the ordered YACs
> with labelled cDNA probes.  This is fine on a small scale. However, if
> we assume that there are about 20,000 cDNAs in the plant, this is an
> unattractive number of hybridizations.

First, my thanks to this group for an informative discussion
on these issues. The discussion has directed me back to a
paper which I found difficult on first reading (Evans, G.
A., and Lewis, K. A. Physical mapping of complex genomes by
cosmid multiplex analysis.PNAS 86, 5030-5034) - if there is
an expert in the house please come forward and correct me on
my interpretation. The question : Does mapping 20,000 cDNA
clones to 700 YACs require 20,000 hybridizations? I think
not. I'm guessing one performs roughly 700, using a 'multiplex'
approach. In this case, the 20,000 clones are 'gridded' into a
100 X 200 matrix. Each row and each column are pooled,
giving 300 pools. Each pool is labelled, and hybridized to
the YAC filter. Therefore, a given cDNA is represented once in a pooled
row and once in a pooled column. If YAC#1 hybridizes
to pool ROW#1 and pool COLUMN#1, then the cDNA at the inter-
section of row#1 and column#1 maps to YAC#1. You can see that
I have oversimplified. Actually, any YAC in *this* experiment will hybridize to 
roughly 20000/700 (30) pools from each dimension,that is we can
tentatively assign 900 possible cDNAs to one YAC (900 being the
number of intersections in a 30 X 30 grid). We may reduce 900 to
the true number by introducing additional dimensions, such as the
two diagonals, and pool members along these 'dimensions' as well 
(adding 200 or 400 additional hybridizations, depending the choice of
diagonals). Now the positive clones lie at the intersection of a row, a
column, and two diagonals drawn on the grid. I think 
the analysis of these sorts of data must be done by computer.
Further, my depiction will be recognized as qualitative -
perhaps I can think more carefully, or more likely, these
ideas have considered and assessed previously by others.
Brian Osborne

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