Taq Polymerase 45
tissues of cancer metastases. We would like to restrict this chapter to
some examples with characteristic features of PCR application in
clinical diagnosis.
The reasons for the success of the PCR as a standard method in clinical
diagnosis are its outstanding sensitivity, reliability, and versatility. Diag-
nostic screening for mutations by the PCR should always be comple-
mented with a statistical analysis; the probability of false diagnosis is on
the order of 1% for a single-locus analysis in single cells (50).
The extreme sensitivity of the technique can trace one target sequence
in a background of 105-106 cells (3). The PCR makes affirmative state-
ments on the genetic information of single somatic or sperm cells possible
(36). In a serial examination, 700 different sperms were typified, demon-
strating a frequency of recombination of 0.16 in one individual (51). An
extension of this test series could allow the detection of recombination
frequencies <0.001; hence, data would be available that are more exact
than information from linkage analysis of pedigree studies.
Multiple gene loci can be amplified in parallel. Six domains of the
Duchenne Muscle Dystrophy (DMD) gene were amplified in a single
experiment (45). Thus, it is possible to monitor by PCR 70% of the
deletions in the 700-kb DMD gene. A simple test for 13-thalassemia
was developed by combining the PCR amplification with subsequent
dot blot hybridization to allele specific oligodeoxynucleotides (52).
The PCR was established to amplify fragments up to 3000 bp to amounts
detectable by staining (24,53). The amplification of larger fragments
is feasible, although with little efficiency: Hypervariable minisatellite
alleles up to 5-10 kb were faithfully amplified (15). Up to several micro-
grams of product can be achieved by the PCR, amounts sufficient as
substrate in binding experiments, e.g., footprint analysis (54).
4.2. PCR Applications in Combination
with Nonradioactive Detection Techniques
The use of radioactive probes should be avoided in routine screen-
ing tests. Even DNA of low abundance can be analyzed by the PCR
followed by restriction analysis of the products (55). The definite
sequence is enhanced to high copy numbers; therefore, radioactive
detection systems are dispensable. Product identification can be easily
done, for example, by hybridization to biotinylated oligodeoxy-
nucleotides and subsequent detection via avidin-linked enzymes (56).