Finding 5' sequence of cDNA

Amy Boedeker kierlab1 at vetmed.tamu.edu
Thu Oct 17 09:39:34 EST 1996


Hello,

I am working on the Hageman Factor gene in mice and cats.  I have 
recently been trying to retrieve a correct 5' gene sequence (exons 
1-3) from a cat cDNA library (gt11, made by Clonetech using cat liver 
in 1991).  I have retrieved the rest of the cDNA successfully (exons 
4-14).

The size of hageman factor cDNA is around 2kb.  I have the complete 
sequences of human, bovine and guinea pig to compare with- they have a 
lot of similarity with one another.  I also have some mouse hageman 
factor sequence.

I have retrieved 5 lambda clones from the cDNA library using a probe 
containing exons 10-14 (600bp).  I tried sequencing one of these 
clones after subcloning a fragment that I determined should contain 
the 5' sequence I'm looking for.  The sequence "got weird" around exon 
9 as I went upstream from exon 14.  The sequence going downstream from 
the EcoRI site (the insert site of gt11) was not homologous to 
anything I have.  This should have been exons 1-3 based on the size of 
the fragment I subcloned...

I have tried PCR on the remaining lambda clones with not much more 
success.  I used primers that I designed using the human, bovine, and 
guinea pig homology regions.  Is lambda tricky to PCR?  I tried 
lowering the annealing temp to 42 degrees Celcius but got bands of 
incorrect sizes.

I also tried 1st strand PCR on RNA I isolated from my limited supply 
of cat liver.  I did not have any luck with this.

I did not think that sequencing the 5' end of this gene would be this 
difficult.  If anyone has any suggestions, please let me know.  Also, 
regarding lambda clones, what percent will contain an entire cDNA 
sequence of a 2kb size???  

Thank you in advance,
Amy



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