Page 43 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN004L-956 June 9, 2001 21:7

594 DNA Testing in Forensic Science

no effect on the amount of high-molecular-weight DNA. from the DNA. The latter part is done using a mixture of
This was not a rigorous experiment, but the effects are chloroform (24:1 chloroform:isoamyl alcohol) and phe-
dramatic enough to demonstrate the effect of ultra violet nol (buffered). The phenol–chloroform mixture denatures
light exposure to DNA before stains dry. proteins liberated by the first stage. The major disadvan-
tage of this procedure is the fact that phenol–chloroform
is a hazardous waste and could theoretically pose a risk
II. EXTRACTION OF DNA to pregnant employees. A modern protocol for phenol–
chloroform extraction of various types of evidence can be
As stated previously DNA exists inside of cells. Because found in the literature.
most evidence is in the form of dry stains, the DNA must
be removed from the stain before it can be tested. The
B. Nonorganic Extraction
process of removing DNA from the cells on the evidence
and dissolving it is referred to as extraction. There are In nonorganic extraction the hazardous phenol–chloro-
several procedures available for removing DNA from ev- form protein denaturation step was replaced by a salting
idence so that it can be used. They are referred to as ei- out of proteins. This allowed for the same chemistry to
ther “organic” extraction or “nonorganic” extraction based be used for the initial phase of DNA extraction, and re-
on the nature of the chemicals used. Further, there are placement of the hazardous elements of the procedure
two special types of extraction. The first, called differ- with a nonhazardous alternative. The salting-out proce-
ential extraction, was developed for sexual assault ev- dure has several advantages over the phenol–chloroform
idence to separate the cells that come from the victim extraction. The first is that instead of having two liquid
(epithelial cells from the vagina, rectum, or mouth) from phases (organic and nonorganic) that can occasionally trap
those of the perpetrator (male sperm cells). The second the DNA in the wrong phase (organic) phase, by precip-
method is a specialized “nonorganic” extraction using itating the proteins (e.g., the proteins become insoluble
Chelex beads. Chelex beads can only be used when PCR- and become a solid), there are liquid and solid phases
based DNA testing is going to be used. The basic DNA with the DNA only in the liquid phase (nonorganic).
extraction procedures, whether organic or nonorganic, The second advantage is that the hazardous phenol–
can be adapted for special circumstances such as hair or chloroform is replaced with a harmless salt solution. Com-
tissue. parison of the organic and nonorganic procedures for
blood, and semen indicate that the nonorganic extrac-
tion is on the average as good or better than organic
A. Chloroform–Phenol Extraction
extraction, whether quantitated by yield gel or slot blot
This is the oldest procedure available for extracting DNA (Table II).
from blood and it has been extended to include hair, tissue, Either method of DNA extraction described earlier can
and semen stains. The basic procedure consists of open- be used for both RFLP- or PCR-based DNA testing. Or-
ing up cells with a buffer and an enzyme, usually Pro- ganic DNA extraction is widely used in laboratories doing
tease K, and then denaturing and separating the proteins criminal casework while nonorganic DNA extraction is

TABLE II Comparison of Organic and Nonorganic Extraction of DNA from
Blood and Semen Stains a
Quantitation Blood Blood Semen Semen
method organic nonorganic organic nonorganic

Yield gel
Mean 185 ng 258 ng 175 ng 207 ng
N 21 8 22 8
p .054 .122
Slot blot
Mean 515 ng 908 ng 627 ng 1175 ng
N 22 8 27 8
p .022 .008
a
Data taken from Tables 1 and 2 of Laber et al. (1992). Differences in means
tested by Kruskall–Wallace nonparametric analysis of variance, H statistic with 1 df,
uncorrected p values presented.