Page 130 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

P1: GMY/GlQ/GLT P2: GRB Final Pages
Encyclopedia of Physical Science and Technology en010k-502 July 16, 2001 16:56

868 Nucleic Acid Synthesis

transmitted to progeny cells, and the genes in these plas-
mids encode distinct proteins which provide growth ad-
vantage or survival to the host bacteria. For example, many
proteinswhichconferdrugresistancebyavarietyofmech-
anisms are encoded by the plasmids, which are invariably
present as double-stranded circular DNA containing sev-
eral to hundreds of kilobase pairs.
The plasmid DNAs are self-replicating genomic units
which are completely dependent on the host bacteria or
yeast for their replication. These are also critical vehi-
cles for recombinant DNA technology based on cutting
and rejoining DNA fragments. Its invention, some three
decades ago, revolutionized molecular biology and is at
the root of nearly all modern breakthroughs in biology.
Restriction endonucleases, which are enzymes char-
acterized by stringent recognition of specific DNA se-
quences, cleave DNA duplexes and often leave identical
terminal sequences in both plasmid DNA and a gene or
segment of a genome. The fragments can then be joined FIGURE 7 Principle of polymerase chain reaction (PCR). A copy
of a relatively short fragment of DNA (0.1–20 kilobase pairs) can
by a DNA ligase. Joining heterologous fragments gen-
be specifically amplified from genomic DNA by PCR. A typical
erates recombinant DNA, for example, a circular plasmid PCR reaction mixture contains genomic DNA; two oligonucleotide
molecule containing foreign genes. These DNA molecules (∼20 bp) primers, which have same sequences as the two ends
can then be introduced into living cells which allow their of the DNA fragment to be amplified; and a thermostable DNA
reproduction, so that a large amount of recombinant plas- polymerase. A cycle of PCR reaction consists of three steps, start-
ing with denaturing the genomic DNA at high temperature (e.g.,
mid can then be generated.
◦
95 C), followed by primer annealing at near Tm (melting temper-
Recombinant plasmids specific for bacteria, yeast, and ature for primer-DNA hybridization), followed by DNA synthesis
even mammalian cells have been generated in the labo- from the primers by the DNA polymerase. Theoretically, the copy
C
ratory and exploited for a variety of basic and applied number of the DNA of interest (N) can be amplified to 2 × N O ,
research applications. Specifically, recombinant expres- where N O is the original copy number and C is the number of PCR
cycles.
sion plasmids can be constructed in order to express the
ectopic protein encoded by the foreign (trans) gene in
the appropriate host cell. Recombinant plasmids of mam- quence can be replicated repeatedly by using an oligonu-
malian cells are based on viruses, rather than on episomal cleotide primer and a DNA polymerase (Fig. 7). After
DNA. Only the DNA replication function of the virus is a duplex DNA molecule is generated, the next cycle is
incorporated into the plasmid, so that the plasmid is repli- carried out by separating the two strands by heating and
cated without producing the active virus. In the case of then starting the next cycle of synthesis after annealing
human cells, simian virus 40 (SV40) is commonly used to oligonucleotide primers to each template strand. Thus,
generate recombinant DNA. the repeated cycles of synthesis, denaturation, and primer
The circularity of the plasmid is essential for E. coli,but annealing to both strands allow synthesis of a specific
not mammalian or yeast cells. This may be consistent with DNA sequence at an exponential rate. Thus, a tiny piece
the circular genome of the bacteria vs linear genomes of of a DNA molecule could be amplified about a million-
eukaryotes. However, plasmid vectors specific for mam- fold after 20 cycles of this chain reaction (assuming 100%
malian cells must be propagated, preferably in E. coli. efficiency of the process; Fig. 7).
Such “shuttle” vectors are therefore required to have a The PCR technology became viable after discovery of
circular configuration. thermostable DNA polymerases derived from bacteria,
such as Thermobacillus aqualyticus (Taq), which grow
at high temperature. The cycles of PCR could then be au-
B. Polymerase Chain Reaction (PCR)
tomatically set in a thermal cycler. PCR does have some
A critical advance in molecular biology came with the in- limitations. The most important of these are: (1) errors in
vention of PCR, based on a remarkably simple principle, DNA replication; (2) less than complete efficiency in each
and revolutionized many important aspects of biomedical step of the reaction; and (3) improper primer annealing
research and medical jurisprudence. The method is based when complex DNA is used. Thus, when amplification of
on the rationale that each strand of a piece of DNA se- a segment of DNA in a complex genome is desired, the