cDNA sequencing/mapping

HAUGE at FRODO.MGH.HARVARD.EDU HAUGE at FRODO.MGH.HARVARD.EDU
Thu Jan 30 08:57:40 EST 1992


In previous messages Rob Last and Chris Somerville have discussed the 
utility of mapping sequenced cDNAs. It is very clear that given the progress
toward genome mapping which is on going in many laboratories, that an
overlapping YAC map of the Arabidopsis genome will be essentially completed
in the near future. One assembled, the YAC clones will provide a minimal
set of clones representing the Arabidopsis genome. The minimal set of clones
can easily be gridded onto a single filter the size of a microtiter dish.
These so called "polytene blots" will then facilitate the mapping of any
clone, both single copy or repetative, by simple hybridization. The ability
to easily map the sequenced cDNAs greatly strengthens argument for 
undertaking a large scale Arabidopsis sequencing effort. With this in mind
Chris points out that a little foresite is required so that the cDNA 
vectors have no significant homology to the YAC vectors. The absence of
homology between the cDNA probes will allow the hybrizations to be carried
out directly without the need to seperate the insert from the vector. The
easist solution as suggested by Brian Osborn is simply to
label the probes using PCR. This is convently done by by amplifying the
insert directly from either a miniprep or crude lysate in the presence of
labeled nucleoside triphospates. By adjusting the concentration of label 
the reactions can be driven to completion so there is no need to separate
the unincorporated label prior to hybridization. Both the reactions
and the subsequent denaturation can easily be done in a microtiter plate
so 96 probings can easily be carried out simultaneously. This will allow
the mapping to keep pace with the sequencing effort. Finally, by using PCR
to label the insert, the signal to noice ratio is increased. This is 
especially important if lambda vectors will be utilized, where the inserts
(probably <4 kb verse 40 some kb of vector) represent less than 10%
of the DNA in the labeling reaction. Brian Hauge
 



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