person is homozygous or heterozygous, respectively. The range of variation shown in the patterns from different persons depends on how many different alleles exist at the particular target locus, e.g., how many different tandem repeats are in the population as a whole (see Figure 1-3B).

An alternative to the use of a single-locus probe is the use of a multilocus probe that hybridizes to many different VNTR sites in the genome. The resulting patterns in a single person contain many bands of varied intensity; the patterns have been compared with barcodes. The approach was developed by Jeffreys and colleagues.2,3,16,17 Because of the complexity of the patterns, interpretation can be difficult. Consequently, the use of multiple single-locus probes is favored.

Polymerase Chain Reaction for Amplifying DNA

Use of the polymerase chain reaction (PCR) allows a million or more copies of a short region of DNA to be made. It is a method of DNA amplification. For DNA typing, one amplifies a genetically informative sequence, usually 100-2,000 bp long, and detects the genotype in the amplified product. Because many copies are made, genetic typing can rely on nonisotopic methods. With PCR amplification, very small samples of tissue or body fluids—theoretically even a single nucleated cell—can be used to study DNA.18,19

The PCR process (Figure 1-6) is simple; indeed, it is analogous to the process by which cells replicate their DNA.20,21,22,23 Two short oligonucleotides are hybridized to the opposite strands of a target DNA segment in positions flanking the sequence region to be amplified; the two oligonucleotides are oriented so that their 3' ends point toward each other. (The ends of a DNA segment are referred to as 5' and 3'; synthesis of new chains proceeds from the 3' end.) The two oligonucleotides serve as primers for an enzyme-mediated replication of the target sequence. The PCR amplification process itself consists of a three-step cycle:

  1. The double-stranded template DNA is dissociated into single strands by incubation at high temperature, typically 94°C.

  2. The temperature is lowered to allow the oligonucleotide primers to bind to their complementary sequences in the DNA that is to be amplified.

  3. A DNA polymerase extends the primers from each of the two primer-binding sites across the region between them, with the target sequence as template.

Because the extension products of one primer bind the other primer in successive cycles, there is in principle a doubling of the target sequence in each cycle. However, the efficiency of amplification is not 100%, and the yield from a 30-cycle amplification is generally about 106-107 copies of the



The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement