DNA Fingerprinting - Simpson Trial
Richard E. Showalter
show at agouron.com
Sun Mar 5 19:04:16 EST 1995
In article <Pine.SUN.3.91.950224084734.6121A-100000 at chuma>, "Janet Hays
(BIO)" <jhays at chuma.cas.usf.edu> wrote:
> On Mon, 20 Feb 1995 U27111 at uicvm.uic.edu wrote:
> > JK> 1. If it is an RFLP test, where did the DNA that's restricted
> > JK> come from? If from a PCR reaction, in my opinion it can be
> > JK> discarded. It could just as easily be from contamination, and
> > JK> in all likelihood is.
> > How's that if it's a positive result with negative internal
> > controls?
> Because the internal controls are irrelevant to whether or not a second
> genome (i.e. a few strands of NBS or RG) have been inadvertantly left on
> a blood sample (i.e. Bronco, OJ blood) in the field during collection.
> Both genomes will be isolated when the DNA is extracted and both genomes
> will be amplified in the PCR reaction. All the internal control tells you
> really is that you haven't contaminated any of your reagents, since the
> blank you run is run without template (no DNA from any of your test
There are also substrate controls that are run to test for the possibility
that there is a DNA source contained on the surface from which you
extracted or removed your sample. The other control generally run is an
unknown control.(What I mean by unknown control is that the criminalist
who conducts the test extracts and amplifies a sample that is chosen at
random from a list of numbered controls. The person doing the test does
not know what DNA type this sample has until the experiment is over and
his results must match the DNA type from a master list held by the lab
supervisor or that test would be invalid.)
> It is even possible through pipette aerosol contamination when
> putting the master mix (no template) into the various tubes with the
> template DNA. You mix a master for several samples, and then apportion it
> between them. If on putting part of the master into one sample, say NBS,
> and then put the next portion into the OJ tube, poor technique may allow
> carryover of the DNA in the first tube to the second tube. At this point
> the second tube will show as a having two genomes though it wasn't
> extracted as such. There are methods and equipment that can avoid this
> kind of error, and perhaps these labs use them, but I for one am
> unwilling to assume that they have.
Generally speaking this type of cross contamination is not really the kind
of problem you are making it out to be. The kind of "contamination" in
PCR reactions that a Criminalist is most concerned about is DNA from a
previous amplification. Amplified DNA is a perfect match for the primers
to react with and it is in tremendous quantity in an amplified sample. UV
lights, bleach, autoclaves, isolated rooms for running the reactions and
testing the products of the PCR reaction are just a few of the precautions
taken while doing these tests. Also remember that just a few strands of
genomic DNA from some kind of "cross contamination" will not necessarily
result in both types being found. Here is a quote from a paper by the
"Validation studies on the Analysis of the HLA-DQalpha Locus using the
Polymerase Chain Reaction" Catherine T. Comey,Ph.D. and Bruce Budowle,
"In conclusion, PCR amplification of DNA from samples exposed to a variety
of insults yields correct DQalpha typing results. The DQalpha alleles
present in a sample at the level of sensitivity of the test were reliably
detected, and no false results are produced as long as the test was
carried out under conditions which prevent allele dropout. The typing
system appears relatively resistant to a variety of environmental insults,
and factors which do influence the test serve to give no results, rather
than false results."
In this paper they coughed and spit and shed scalp dust(eeewww) all over
open tubes that they were extracting DNA samples from and showed that this
handling did not result in incorrect DQalpha types or mixtures from being
called. They mixed two different types of blood to see what ratios of
mixture you need to have in order to see the lesser blood sample. If the
lesser blood sample was 1/100 the major sample. The lesser sample blood
type could be detected but not typed because it's intensity was below an
internal control. Only the major blood type was clearly interpretable.
This is not to say that mixtures or "contamination" of samples is not a
problem to watch out for, its just that you are not going to get it from
brief of chance contact between two genomic sources.
> Just a note, the biggest thing I have learned about being a scientist is
> never, ever, ever trust anyone's results until you have done a thorough
> examination of his materials and methods. Results are meaningless if they
> were not obtained under proper expermental procedures.
I agree fully with this. But, only another scientist would understand.
> Gotta run. Will address the rest of this later.
Richard E. Showalter "Hey Hey Hey Hey it was
Senior Associate Scientist the DNA. Hey Hey Hey Hey
Agouron Pharmaceuticals, Inc. that made me this way."
show at agouron.com Queen-Shear Heart Attack
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