cDNA sequence differences
Thomas C. Newman
22313tcn at IBM.CL.MSU.EDU
Mon Jun 17 13:15:26 EST 1996
In article <3DDM9R$wn2 at bbs.life.nthu.edu.tw>,
klebs.bbs at bbs.life.nthu.edu.tw (Leo) wrote:
> My question may be a simple one but it is important
> to my research and I will be grateful if someone can
> provide me some information.
> Recently we have isolated a cDNA clone from a Stratagene
> human liver cDNA library. The deduced amino acid sequence of
> the gene product matched very well with that of the enzyme
> we are interested in. Only two substitutions out of 400 or so
> amino acid residues were noted. The nucleotide sequence of
> the cDNA also matched several human EST files in GenBank.
> However, the homology between the cDNA sequence and those of the
> human EST files is only 93%.
Actually the important thing to remember is that EST sequences are one
pass, one strand sequences that are seldom error corrected. Your
sequencing should be more accurate than that of the EST database.
> My questions are:
> 1. Is it possible that the Stratagene cDNA library is not human origin ?
To address the library questions, I would suggest that it is unlikely to
have the wrong origin of library, but don't forget that there is blood,
fat, vessels etc, in liver "tissue".
> 3. How reliable are the commercial cDNA libraries (in terms of
> their source) ? For an example, I have heard a laboratory has
> isolated a bacterial gene from a human cDNA library.
HOWEVER, all library syntheses can have artifacts. I have generated a
library for Arabidopsis (plant) from which we have generated 25000 EST
sequences. I know of 2 clones of bacterial origen, and one that came from
an aphid (plant parasite). These were easy to identify because the library
was directionally cloned and these were in the incorrect orientation as
well as 100% homologous to the other organism over a region > 80 bases.
> 4. How different should I expect to see when comparing cDNA sequences
> of a housekeeping enzyme derived from two different individuals
> (of two different races) ?
In my experience with primates vs human (apolipoprotein E), 98% similarity
is not uncommon. We have found PLANT cell membrane proteins that are 88%
similar to HUMAN cell membrane proteins at the protein level and 72% at the
DNA level. You should be able to find data on allele frequencing of blood
types etc that can give you better indications.
Director of DNA Sequencing Facility
DOE - Plant Research Laboratory
Michigan State University
Dr. Thomas C. Newman
MSU-DOE Plant Research Lab
Manager DNA Sequencing Facility
More information about the Methods