Feng, Xiaodong_ Xie, Hong-Guang - Applying pharmacogenomics in therapeutics-CRC Press (2016)

Principles of Pharmacogenetic Biotechnology and Testing in Clinical Practice
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replication, which results in synthesized DNA fragments of variable lengths that
match the positions of the bases substituted by the corresponding dideoxynucleotides
(Sanger et al. 1977). These DNA fragments can be separated by electrophoresis
to reveal the sequence of the DNA template. Sanger sequencing is the
underpinning sequencing technology for the first human genome (Lander et al.
2001) and is still the primary sequencing method in most basic and clinical genetics
laboratories. In this chapter, we briefly review three other major DNA analysis
technologies widely used today.
Polymerase Chain Reaction
An extremely important technique in molecular biology is the polymerase chain
reaction (PCR) proposed in 1983 by Kary Mullis. PCR initiated a new era of
highly efficient gene analysis and manipulation (Saiki et al. 1988). This method utilizes
the basic principle of DNA replication and cycles this process in a test tube,
which allows the generation of millions of copies of a particular DNA sequence.
An ample supply of DNA copies allows easy detection and manipulation of genetic
information encoded in the DNA. The basic setup of a PCR requires the following:
(1) a template that contains the DNA region of interest; (2) two amplification primers
that are complementary to the 3′-ends of the double-stranded DNA template;
(3) deoxynucleoside triphosphates (dNTP), the building blocks used to synthesize new
DNA strands; (4) a thermostable DNA polymerase that synthesizes new DNA strands
comple mentary to the template strands; (5) buffer solution with suitable pH, salt concentrations,
and magnesium or manganese ions (Figure 2.1a). Typically, a PCR consists
of 20–40 cycles of heating and cooling steps. In each cycle, a DNA template is
denatured, annealed to primers, and synthesized into two new copies. Specifically, the
denaturation step occurs at 94–98°C for 10–30 seconds, which disrupts the hydrogen
bonds between complementary bases and unwinds the double-stranded templates into
single strands. At the annealing step, the reaction is cooled down to the annealing
temperature to allow hybridization of the primers to the single-stranded DNA template.
Then the temperature is increased to 68–72°C (the optimal temperature for
thermostable DNA polymerase) to allow new DNA strands to be synthesized. At this
stage, DNA polymerase adds dNTPs to the annealed primers to assemble a nascent
DNA strand complementary to the template strand in the 5′ to 3′ direction. In principle,
DNA polymerase doubles the starting DNA strands at each extension step, leading
to an exponential amplification of a given DNA region.
PCR has been adopted into many variations to fit a variety of applications. For
example, allele-specific PCR is designed to detect allele-specific variations, such as
single nucleotide changes. Prior knowledge of the variations in a DNA sequence is
required to design primers specific for such SNPs. The amplification of a specific allele
is achieved by stringently setting the temperatures for the annealing and elongation
steps. The presence of a mismatch between the template DNA and the complementary
primer would cause failed annealing and subsequent PCR reaction under the stringent
conditions, whereas only a perfect match will lead to an allele- specific amplification.
By identifying allele-specific DNA fragments, SNP information can be easily detected.
Similarly, two pairs of primers can be used in one PCR reaction (a tetra-primer set)