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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 7.7Polymerase Chain Reaction: An Alternative to Cloning

An alternative to cloning, called the polymerase chain reaction(PCR), can be used to directly amplify rare specific DNA sequences in a complex mixture when the ends of the sequence are known. This method of amplifying rare sequences from a mixture has numerous applications in basic research, human genetics testing, and forensics.

A typical PCR is outlined in Figure 7-38. Genomic DNA is digested into large fragments using a restriction enzyme and then is heat-denatured into single strands. Two synthetic oligonucleotides complementary to the 3′ ends of the target DNA segment of interest are added in great excess to the denatured DNA, and the temperature is lowered to 50 – 60 °C. The genomic DNA remains denatured, because the complementary strands are at too low a concentration to encounter each other during the period of incubation, but the specific oligonucleotides, which are at a very high concentration, hybridize with their complementary sequences in the genomic DNA. The hybridized oligonucleotides then serve as primers for DNA chain synthesis, which begins upon addition of a supply of deoxynucleotides and a temperature-resistant DNA polymerase such as that from Thermus aquaticus (a bacterium that lives in hot springs). This enzyme, called Taq polymerase, can extend the primers at temperatures up to 72 °C. When synthesis is complete, the whole mixture is heated further (to 95 °C) to melt the newly formed DNA duplexes. When the temperature is lowered again, another round of synthesis takes place because excess primer is still present. Repeated cycles of synthesis (cooling) and melting (heating) quickly amplify the sequence of interest. At each round, the number of copies of the sequence between the primer sites is doubled; therefore, the desired sequence increases exponentially.

Figure 7-38. The polymerase chain reaction.

Figure 7-38

The polymerase chain reaction. The starting material is a double-stranded DNA. Large numbers of primers are added, each with the sequence found in one strand at the end of the region to be amplified. The thermostable Taq polymerase and dNTPs are also (more...)

PCR Amplification of Mutant Alleles Permits Their Detection in Small Samples

Image med.jpgThe sensitivity of procedures used in human genetics testing has been vastly increased by use of PCR amplification. For example, the β-globin gene in a small sample of DNA isolated from an individual can be specifically amplified by the PCR to determine if the person is a carrier of the mutant sickle-cell allele. Quantities of amplified DNA sufficient for sequencing can be prepared rapidly; subsequent sequencing reveals if the mutant allele is present in the sample.

The PCR is so effective at amplifying specific DNA sequences that DNA isolated from a single human cell can be analyzed for mutations associated with various genetic diseases. In one reported case, this approach was used to screen in vitro fertilized human embryos prepared from sperm and ova from a couple who both were carriers of the genetic disorder cystic fibrosis. This disease results from mutation in the CFTR gene, which is located on chromosome 7. The DNA isolated from a single embryonic cell was subjected to PCR amplification and then analyzed for mutations identified in one of the two copies of chromosome 7 in each parent. In this way embryos that had inherited the wild-type chromosome from at least one parent were identified and then transferred to the mother’s uterus. (Removal of a single cell from an in vitro fertilized human embryo has no apparent effect on subsequent development of the embryo after it is implanted in a receptive uterus.) By use of this procedure, carrier couples can be assured of having children that will not be at risk for cystic fibrosis.

Another medical application of the PCR is early detection of infection with HIV, the virus that causes acquired immunodeficiency syndrome (AIDS). The PCR is so sensitive that it can detect HIV at very early stages in the disease (before symptoms appear) when only a few thousand blood cells in a patient are infected with the virus.

DNA Sequences Can Be Amplified for Use in Cloning and as Probes

In basic research, the PCR also has numerous applications. For example, this procedure allows the recovery and rapid amplification of the entire DNA region between any two ends whose sequences are known; the amplified DNA fragment then can be ligated into standard cloning vectors. Fragments of ≈2 kb or less can be amplified readily, and recent refinements of the technique allow amplification of regions of >30 kb.

The PCR also provides an alternative approach for preparing probes to screen genomic or cDNA libraries for clones encoding a protein of interest. The amino acid sequence of two peptides isolated from the purified protein are used to design two degenerate oligonucleotide mixtures containing all possible DNA sequences encoding the two peptides (see Figure 7-19). Rather than using these oligonucleotides as probes for direct screening of a cDNA library, as described previously, they are used as primers in a PCR. First, cDNA is synthesized from total cellular mRNA using reverse transcriptase. The cDNA is then used as the template for a PCR performed with the two degenerate oligonucleotide primers. This reaction amplifies the region of the cDNA between the sequences encoding the peptides used to design the degenerate primers. The PCR procedure effectively selects the correct oligonucleotides for priming DNA synthesis from the degenerate oligonucleotide mixtures, because only DNA synthesized from the correct cDNA template will hybridize to oligonucleotides present in both degenerate primer mixtures. For exponential amplification to take place, priming must occur from both ends of a fragment. Even if an oligonucleotide in one of the degenerate mixtures hybridizes to an incorrect cDNA and primes DNA synthesis, the DNA strand that is synthesized will not be amplified, because it will not contain a sequence complementary to one of the oligonucleotides in the second degenerate primer mixture. The cDNA sequence amplified by this procedure contains the unique sequence of the naturally occurring mRNA encoding the region between the two peptides originally sequenced. This unique DNA sequence can then be radioactively labeled and used as a probe for screening a cDNA or genomic library. A probe prepared in this way gives a much stronger and more specific signal than that obtained by direct use of a degenerate probe.


  •  In the polymerase chain reaction (PCR), the DNA sequence lying between two primers present at high concentration undergoes repeated doublings in an exponential fashion (see Figure 7-38). Large amounts of DNA can be synthesized from just a single initial template DNA molecule by this method. However, the sequence of the ends of the DNA to be amplified must be known.
  • PCR amplification permits highly sensitive detection of mutant alleles in human genetics testing and of very low levels of HIV in infected humans.
  •  In basic research, the PCR is used as both an alternative and an adjunct to standard cloning procedures.
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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21541


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