Page 44 - 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

DNA Testing in Forensic Science 595

widely used in laboratories performing paternity testing, be separated based on size. This process of separating
research, and diagnostics. On a worldwide basis nonor- DNA using an electric current is called electrophoresis,
ganic DNA extraction is the more prevalent. With the shift which simply means separation (phoresis) by means of
to PCR-based testing this choice in extraction is increas- electricity (electro).
ingly common. Since DNA is colorless it is not possible to see the DNA
after it has been separated without the use of special dyes
that bind to it. One of the earliest dyes used was ethidium
C. Chelex Extraction bromide, which fluoresces pink when bound to double-
In 1991, a method of DNA extraction was described that stranded DNA and exposed to ultraviolet light. Figure 4 is
was specifically aimed at the extraction of small amounts an ethidium bromide stained yield gel. To quantitate the
of dilute DNA for PCR-based testing using Chelex beads. amount of DNA in the DNA extracts, a set of DNA quan-
The method is simple, relatively fast, and biohazard free. It titation standards are placed on the gel. By visual compar-
is widely used by forensic laboratories doing PCR-based ison of the unknown DNA to the known DNA the amount
typing which has increased the number of laboratories us- of DNA can be approximated. This test provides informa-
ing nonorganic, biohazard-free DNA extraction. The only tion about the relative amount of DNA and whether it is
limitations of Chelex extraction is that it produces a dilute degraded (i.e., the DNA is broken down so that different-
solution of DNA that may need to be concentrated be- size pieces of DNA are present). It does not indicate if the
fore it can be used with some of the newer high-resolution DNA is human, however, since all DNA will fluoresce.
PCR-based typing systems and it cannot be used for RFLP Thus the DNA present may be bacterial as well as hu-
testing. man DNA. For RFLP testing the total amount of DNA in
the sample is the important determinant of how the sam-
ples migrate in the gel. Therefore yield-gel electrophoretic
quantitation of DNA is an appropriate method. Yield-gel
III. QUANTITATION OF DNA
quantitation of DNA for RFLP testing was considered to
be such an integral part of quality assurance that it was
A. Yield-Gel Quantitation
included in the National Institute of Standards, Standard
Whether RFLP- or PCR-based testing is performed it is Reference Material 2390, “DNA Profiling Standard.”
necessary to know how much DNA is present. One of the As with the extraction of DNA using the organic
earliest methods of quantitating small amounts of DNA method, ethidium bromide is potentially hazardous be-
is the use of a yield gel. A small gel is made using a cause the dye is associated with an increased cancer risk.
salt solution to carry electrical current and a supporting Though ethidium bromide is still widely used for the iden-
medium made of agarose (a complex carbohydrate made tification of DNA it is currently being replaced by a new
®
from seaweed). Much like gelatin, the agarose dissolves dye called Sybrr green which is much less carcinogenic
in water that is heated to near boiling and the liquid is and can detect smaller amounts of DNA than ethidium
cooled slightly and poured into a casting tray. A plastic bromide.
comb or well former with rectangular teeth is placed in
the liquid agarose. Once the agarose gels, the comb is
B. Slot-Blot Quantitation
removed leaving behind rectangular wells in the agarose
gel. The DNA to be tested is mixed with loading buffer, In contrast to RFLP, for PCR-based testing, the amount
and placed in the wells. Loading buffer is a mixture of a of human DNA and not the total amount of DNA is an
large amount of sugar and dye. The high concentration of important determinant in how likely it will be to obtain
sugars makes the mixture heavier than the salt solution results. A slot blot does not rely on electrophoresis to sep-
so that the DNA sinks to the bottom of the well. The arate the DNA but rather on the ability of denatured (sep-
dye allows the migration of the DNA to be monitored. arated DNA strands) DNA to bind to homologous com-
The agarose was melted in water containing salt. When plementary sequences. The ability to quantitate human
electrical current is applied to the gel, electricity flows DNA requires sequences of DNA that are common in the
through the gel because of the salt and moves (migrates) human genome so that a single DNA sequence can recog-
from the negative electrode (cathode) toward the positive nize them and bind to them. The repeated DNA sequence
electrode (anode). Since all DNA has a negative charge, called D17Z1 is the basis for all human DNA slot-blot
and was placed in the wells at the cathodal end of the gel, quantitation systems. There are several of these proce-
the negatively charged DNA will migrate out of the wells dures commercially available. In one of the most widely
toward the positive end of the gel. If the DNA is broken used tests, the quantitation requires that denatured DNA
into pieces that are different sizes, the smaller pieces will is applied to a membrane using a slotted plastic appa-
move through the gel faster than the larger pieces and will ratus. The denatured DNA binds to the membrane. The