Biology Reference
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8.3 The Basic Polymerase Chain Reaction (PCR)
The PCR involves combining a DNA sample (the template) with oligonucleotide
primers, deoxynucleotide triphosphates (dNTPs), and a DNA polymerase in a
buffer. The specificity of the basic PCR depends on complementary base pairing
by the two primers to the template (target) DNA ( Figure 8.1A ).
The primers , single-stranded (ss) sequences that flank the DNA to be ampli-
fied, anneal to the template DNA that has been denatured by heating it so that
it is single-stranded. Repeated PCR cycles involve heat denaturation to separate
the template DNA strands, cooling to allow annealing of primers to the comple-
mentary DNA sequences of the ss template DNA, and “extension” (or replica-
tion) of new (product) DNA strands by DNA polymerase. The base sequence of
the new strand is determined by the sequence of the ss template DNA. DNA syn-
thesis proceeds across the region between the annealed primers ( Figure 8.1B ).
This mixture is repetitively heated and cooled until the desired amount of tem-
plate DNA has been amplified, usually after 25-30 cycles.
8.3.1 The First Few Cycles are Critical
All cycles begin by denaturing the template DNA (and any previously synthe-
sized product) by heating it so that the template DNA and newly synthesized
DNA become single-stranded ( Figure 8.1A ). As the temperature is lowered, the
primers anneal to the complementary sequences of the single strands of DNA.
The annealing step in the early cycles requires the primers to “scan” the tem-
plate DNA for the correct target sequences to which they can anneal ( Ruano
et al. 1991, Dieffenbach 1995, Harris and Jones 1997 ). Because much of the tem-
plate DNA will not have the correct complementary sequence, annealing during
the early cycles may not be as efficient as it is during the middle cycles. Improper
interactions of primers with template in the first few cycles can lead to nonspe-
cific products and reduced yields. The PCR product will be specific only if the
two primers bind to sites on the complementary strand of the DNA and, for the
standard PCR, these sites are not more than 1-2kb apart. Thus, the first few
cycles are very important if accurate, and high, yields are to be produced.
During the middle cycles, the DNA product previously synthesized is the pre-
ferred template for the primers, so this target template is perfectly demarcated
( Figure 8.1B ). Finally, in the late cycles, denatured amplified products that are
present in high concentration can hybridize to themselves, thus blocking the
primers from their complementary sites. In theory, DNA sequences up to 10 kb
in length can be synthesized by the standard protocol, but sequences of 2 kb, or
less, are more readily obtained.
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