PCR Is a Powerful Technique in Medical Diagnostics, Forensics, and Molecular

In 1984, Kary Mullis devised an ingenious method called the polymerase chain reaction (PCR) for amplifying specific
DNA sequences. Consider a DNA duplex consisting of a target sequence surrounded by nontarget DNA. Millions of the
target sequences can be readily obtained by PCR if the flanking sequences of the target are known. PCR is carried out by
adding the following components to a solution containing the target sequence: (1) a pair of primers that hybridize with
the flanking sequences of the target, (2) all four deoxyribonucleoside triphosphates (dNTPs), and (3) a heat-stable DNA
polymerase. A PCR cycle consists of three steps (Figure 6.8).
1. Strand separation. The two strands of the parent DNA molecule are separated by heating the solution to 95°C for 15 s.
2. Hybridization of primers. The solution is then abruptly cooled to 54°C to allow each primer to hybridize to a DNA
strand. One primer hybridizes to the 3 -end of the target on one strand, and the other primer hybridizes to the 3 end on
the complementary target strand. Parent DNA duplexes do not form, because the primers are present in large excess.
Primers are typically from 20 to 30 nucleotides long.
3. DNA synthesis. The solution is then heated to 72°C, the optimal temperature for Taq DNA polymerase. This heatstable
polymerase comes from T hermus aq uaticus, a thermophilic bacterium that lives in hot springs. The polymerase
elongates both primers in the direction of the target sequence because DNA synthesis is in the 5 -to-3 direction. DNA
synthesis takes place on both strands but extends beyond the target sequence.
These three steps strand separation, hybridization of primers, and DNA synthesis constitute one cycle of the PCR
amplification and can be carried out repetitively just by changing the temperature of the reaction mixture. The
thermostability of the polymerase makes it feasible to carry out PCR in a closed container; no reagents are added after
the first cycle. The duplexes are heated to begin the second cycle, which produces four duplexes, and then the third cycle
is initiated . At the end of the third cycle, two short strands appear that constitute only the target
sequence the sequence including and bounded by the primers. Subsequent cycles will amplify the target sequence
exponentially. The larger strands increase in number arithmetically and serve as a source for the synthesis of more short
strands. Ideally, after n cycles, this sequence is amplified 2 n -fold. The amplification is a millionfold after 20 cycles and
a billionfold after 30 cycles, which can be carried out in less than an hour.
Several features of this remarkable method for amplifying DNA are noteworthy. First, the sequence of the target need
not be known. All that is required is knowledge of the flanking sequences. Second, the target can be much larger than the
primers. Targets larger than 10 kb have been amplified by PCR. Third, primers do not have to be perfectly matched to
flanking sequences to amplify targets. With the use of primers derived from a gene of known sequence, it is possible to
search for variations on the theme. In this way, families of genes are being discovered by PCR. Fourth, PCR is highly
specific because of the stringency of hybridization at high temperature (54°C). Stringency is the required closeness of the
match between primer and target, which can be controlled by temperature and salt. At high temperatures, the only DNA
that is amplified is that situated between primers that have hybridized. A gene constituting less than a millionth of the
total DNA of a higher organism is accessible by PCR. Fifth, PCR is exquisitely sensitive. A single DNA molecule can be
amplified and detected.
PCR can provide valuable diagnostic information in medicine. Bacteria and viruses can be readily detected with the use
of specific primers. For example, PCR can reveal the presence of human immunodeficiency virus in people who have
not mounted an immune response to this pathogen and would therefore be missed with an antibody assay. Finding
Mycobacterium tuberculosis bacilli in tissue specimens is slow and laborious. With PCR, as few as 10 tubercle bacilli
per million human cells can be readily detected. PCR is a promising method for the early detection of certain cancers.
This technique can identify mutations of certain growth-control genes, such as the ras genes (Section 15.4.2). The
capacity to greatly amplify selected regions of DNA can also be highly informative in monitoring cancer chemotherapy.
Tests using PCR can detect when cancerous cells have been eliminated and treatment can be stopped; they can also
detect a relapse and the need to immediately resume treatment. PCR is ideal for detecting leukemias caused by
chromosomal rearrangements.
PCR is also having an effect in forensics and legal medicine. An individual DNA profile is highly distinctive because
many genetic loci are highly variable within a population. For example, variations at a specific one of these locations
determines a person's HLA type (human leukocyte antigen type); organ transplants are rejected when the HLA types of
the donor and recipient are not sufficiently matched. PCR amplification of multiple genes is being used to establish
biological parentage in disputed paternity and immigration cases. Analyses of blood stains and semen samples by PCR
have implicated guilt or innocence in numerous assault and rape cases. The root of a single shed hair found at a crime
scene contains enough DNA for typing by PCR
DNA is a remarkably stable molecule, particularly when relatively shielded from air, light, and water. Under such
circumstances, large fragments of DNA can remain intact for thousands of years or longer. PCR provides an ideal
method for amplifying such ancient DNA molecules so that they can be detected and characterized (Section 7.5.1). PCR
can also be used to amplify DNA from microorganisms that have not yet been isolated and cultured. As will be discussed
in the next chapter, sequences from these PCR products can be sources of considerable insight into evolutionary
relationships between organisms.