Page 46 - 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 597

FIGURE 5 DNA polymerase copying one strand of a portion of double-stranded DNA. (From Schanfield, M. S. (2000).
Deoxyribonucleic Acid/Polymerase Chain Reaction. In “Encyclopedia of Forensic Sciences” (Siegel, J. A., Saukko,
P. J., and Knupfer, G. C., eds.), Academic Press, London, p. 516.)

unique sequence of DNA. For a primer to recognize a the target DNA defined by the primers. This is called the
unique sequence in the genome it must be long enough extension step. This completes one cycle of the PCR pro-
for no other sequence to match it by chance. This can usu- cess. To make enough copies of the target DNA to detect
ally be achieved with a sequence of 20 to 25 nucleotides. the process is repeated from 25 to 40 times. This is done
These manufactured pieces of DNA are called “primers” using a device called a thermalcycler. The process is illus-
and they are complementary to the start and stop areas trated in Fig. 6. If the process were perfect 30 cycles would
defined earlier. The “forward primer” is complementary create over a billion copies of the original target DNA.
to the beginning sequence on one strand of DNA, usually The heating and cooling of the tubes are done in an
called the positive strand. The “reverse primer” is comple- electromechanical device call a “thermalcycler,” which in
mentary to the stop sequence on the opposite or negative general, consists of an aluminum blocks with wells de-
strand of DNA. signed to fit the plastic PCR reaction tubes. The aluminum
block has heating and cooling elements controlled by a mi-
croprocessor that can raise and lower the temperature of
B. Multiplexing PCR Reactions the block and the plastic PCR reaction tubes in the block.
One of the advantages of PCR is that more than one region In the thermal cyclers that were first made, the plastic
can be amplified at a time. Although it is necessary to reaction tubes extended above the thermal block. This al-
select carefully primers that cannot bind to each other, the lowed cooling to take place above the reaction. The water
only limitation is how many pairs of primers can be placed in the reaction mixture would evaporate and condense at
together is the ability to detect the amplified product. the top of the tube, changing the concentration of reac-
tants and affecting the success of the amplification. To
limit the evaporation, mineral oil was placed on top of the
C. PCR Process reaction mixture. New thermal cyclers have heated lids on
top of the block to prevent or minimize evaporation. The
To perform a PCR reaction several ingredients are needed.
microprocessor can store many sets of instructions such
They include PCR reaction buffer, which is basically a salt
that different programs can be kept in the microprocessor
solution at the right pH for the enzyme being used, the
to amplify different sequences of DNA.
four nucleotides (DNA building blocks), primers, a ther-
mostable DNA polymerase (Taq, Pfu, Vent, Replinase, D. Detection of PCR Products
etc), and template DNA. These reactants are placed in
small plastic reaction tubes. The process consists of There are many methods for detecting PCR products.
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heating a solution of DNA to greater than 90 C. Double- Since large amounts of product are produced there is no
stranded DNA comes apart or melts to form single- need to use techniques such as radioactive detection, al-
stranded DNA at this temperature. This is called the denat- though it has been used in some clinical setting. In forensic
uration step. The solution is then cooled down to between testing, one of the advantages of PCR-based testing is that
50 and 65 C the primers will bind to their complemen- it does not require the use of hazardous materials to detect
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tary locations. This is called the annealing or probe hy- it. There is normally enough product so that if the PCR
bridization step. Finally, the solution temperature is raised products are run on a yield gel and stained with ethidium
to 72 C at which point the polymerase makes a copy of bromide or Cyber green, there is normally enough DNA
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