DNA in invertebrates HELP!
svetlov at oncology.wisc.edu
Mon Nov 17 11:41:20 EST 1997
In article <Pine.SOL.3.96.971111170752.21365A-100000 at suma3.reading.ac.uk>,
Clive Boorman <sau95cb at reading.ac.uk> wrote:
> Hi anyone who can help,
> We have been looking at invertebrate crustacea genes and have
> isolated a cDNA sequence for the gene in question. Our problem is, getting
> any further. The genomic DNA is causing us many problems probably caused by
> polysaccharides. Having extracted DNA from whole tissue and purified it we
> have to re-extract it using CTAB, however this has not cured our problems.
> Primers which work on mRNA don't work on genomic DNA.
> Has anyone been working on DNA extraction and analysis in
> invertebrates who can help solve the possible polysaccharide or other
> peculiarities of crustacean DNA ?
> Is there any other reason why we may be getting no PCR product from
> genomic DNA, when we can get a product from mRNA (RT-PCR) everytime ?
You might be dealing with several problems at the same time, namely
efficient isolation of genomic DNA and running PCR from genomic DNA. I
found for meself that any method of genomic DNA isolation that is good for
DNA fingerprinting is good for PCR (reverse is not always true). However,
the efficiency of PCR amplification from the genomic DNA usually drops as
the length of the product increases. I'd try to order an additional primer
located about 200 bp away from one of the ones you already have and run a
PCR reaction with it and your genomic DNA - see if the size is the problem.
Other things to beware is the kind of polymerase you use - some of them are
less processive than others or can exhibit differential sensitivity to
various inhibitors/contaminants (on some genomic templates I get good
amplifications with the Pwo, but little or now product with Pfu etc.). If
you suspect that the length can be a factor, try using different amounts of
templates and several enzymes/combinations thereof.
Carbohydrates are often a problem in invertebrates and in the DNA
fingerprinting times (where every living thing was getting its genomic DNA
isolated) there were quite a few objects, refractory to conventional DNA
isolation techniques. Two things I recall that worked quite well on
carbohydrate-enriched material were chitinase treatment and incubation with
hypochloride - before the lysis. Somebody reported using complex enzymatic
mixtures such as Novozyme 254 or Zymolyase to strip the cells of, well,
pretty much everything, but I've found that the nuclease levels in some of
the bathches are so high that DNA also gets degraded if the timing is not
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
More information about the Methods