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except for the terminal 3^0 end base. Thus, if the DNA contains the point mutation only

the primer with the complementary sequence will bind and be incorporated into the

amplified DNA, whereas if the DNA is normal the wild-type primer is incorporated. The

results of the PCR are analysed by agarose gel electrophoresis. A further modification of

ASO–PCR has been developed where the primers are each labelled with a different

fluorochrome. Since the primers are labelled differently a positive or negative result is

produced directly without the need to examine the PCRs by gel electrophoresis.

Various modifications now allow more than one PCR to be carried out at a time

(multiplex PCR), and hence the detection of more than one mutation is possible at the

same time. Where the mutation is unknown it is also possible to use a PCR system with a

gel-based detection method termeddenaturing gradient gel electrophoresis(DGGE). In

this technique a sample DNA heteroduplex containing a mutation is amplified by the PCR

which is also used to attach a GC-rich sequence to one end of the heteroduplex. The

mutated heteroduplex is identified by its altered melting properties through a polyacryl-

amide gel which contains a gradient of denaturant such as urea. At a certain point in the

gradient the heteroduplex will denature relative to a perfectly matched homoduplex and

thus may be identified. TheGC clampmaintains the integrity of the end of the duplex on

passage through the gel (Fig. 6.46). The sensitivity of this and other mutation detection

methods has been substantially increased by the use of PCR, and further mutation

PCR W

Wild-type

primer

Disease gene

Allele-specific

oligonucleotide

primer

Analysis of PCR (M and W) by gel electrophoresis

MW

Homozygous

mutation

Homozygous

wild-type

Heterozygous

Isolate patient sample DNA

PCR M

MWMW

X

Fig. 6.45Point mutation detection using allele-specific oligonucleotide PCR (ASO–PCR).

250 Recombinant DNA and genetic analysis