target sequence. The primers become physically incorporated into the amplification products.

Both the amplification and the genetic typing can be completed in a day. The efficiency can be improved by amplifying several different products in the same reaction mix; this is termed multiplex amplification.

Several methods have been coupled with PCR for the detection of genetic variation in the amplified DNA; some are listed in Table 1.2. Detection of variation in DNA with PCR-amplified material is fundamentally no different from detection with unamplified samples. The difference is technical: the availability of the larger quantity of pure material produced with PCR affords more options for means of detection.

The use of allele-specific oligonucleotide (ASO) probes is the most generalized approach to the detection of alleles that differ in sequence.33 The sequence-specific probe is usually a short oligonucleotide, 15-30 nucleotides long, with a sequence exactly matching the sequence of the target allele. The ASO probe is mixed with dissociated strands of PCR reaction product under such conditions that the ASO and the PCR product strands hybridize if there is perfect sequence complementarity, but do not if there are mismatches in sequence.

The usual format for the use of ASO probes is to spot dissociated PCR product strands onto a nitrocellulose or nylon membrane and probe the membrane with labeled ASO. That is analogous to Southern blotting; because the samples are spotted as a "dot" on the membrane, the method is referred to as "dot blotting" (Figure 1-7). An alternative method uses an array of ASO probes immobilized on a test strip.34 The test strip is immersed in a solution of labeled PCR product; the PCR product hybridizes only to its complementary probe. This procedure has been called ''reverse dot blotting" or "blot dotting." A commercial kit based on the reverse dot blot principle has been released (Cetus Corporation).

TABLE 1.2 Some PCR-Based Systems for the Detection of Genetic Variation

Sequence-based detection systems

Allele-specific oligonucleotide (ASO)24

Allele-specific priming of PCR25,26,27

Oligonucleotide-ligation assay (OLA)28

Restriction-site-specific cleavage (Amp-FLPs)29

Denaturing-gradient gel electrophoresis30

Chemical cleavage of mismatched heteroduplexes31

Length-variation systems

Simple insertions and deletions

VNTR polymorphisms32

Analysis of nucleotide sequences



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