DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible - Insect Molecular Genetics - page 303

of 10 to ≈ 30 nucleotides), deoxynucleotide triphosphates (dNTPs), buffer, and
enzyme (one or more DNA polymerases). These relationships change during the
course of the reaction ( Figure 8.1 ).
The PCR works well with most DNA targets, but adjustments may be needed
in the reaction parameters in order to improve specificity and yield. Parameters
to be modified to optimize the PCR include: the reaction buffer (particularly the
MgCl 2 concentration); relative concentrations of template DNA, primers, dNTPs
and DNA polymerase; annealing time and temperature; and extension time and
temperature ( Carbonari et al. 1993 ). No single protocol is appropriate for all sit-
uations and each new experiment requires optimization. For example, amplify-
ing a 100-bp fragment is not equivalent to amplifying a 10-kb DNA fragment.
Modifying each of the components of the PCR to develop an optimized reaction
can be time consuming and tedious, but kits now are available that allow one to
optimize the PCR with fewer steps.
This chapter describes what we know about the basic PCR, discusses some of
the modifications of the basic method, identifies applications of the PCR, and
provides references to additional information. PCR technology changes and
new applications and methods of significance to entomologists will continue to
become available.
Figure 8.1 The standard, allele-specific PCR protocol. A) Template DNA is isolated and mixed with
primers, dNTPs and Taq DNA polymerase in a buffer with Mg 2 + . The double-stranded template
DNA is heated to denature it so that the primer can anneal to single-stranded target DNA (only
one template stand is shown). Taq synthesizes a new single strand of complementary DNA using the
primer to initiate synthesis. The dNTPs are added in a sequence determined by the template DNA
strand. This initial extension continues on beyond the desired end, as shown by the arrow on the
newly synthesized strand in the lower diagram. The process of denaturation, annealing of the primer
to the template, and DNA synthesis (or extension) is called a cycle.