Advantages of realtime PCR protocols for PND

The ability to carry out rapid DNA analysis for genotype characterization has become an increasingly important requirement for the clinical diagnostic laboratory. A wide variety of methods exist for detecting point mutations in a DNA molecule, including ASO, PCR-RFLP, ARMS, DGGE,

Table 17.2 Sequences of primers included in the first-round multiplex PCR for PGD protocol

Primers Sequence of primers Product size, bp

(70373-70393)** R 5'-CAT CAA GGG TCC CAT AGA CTC-3' 689

(70560-70580)** R 5'-TAA AAG AAA CTA GAA CGA TCC-3' 637

GABRB3

D13S314

* For amplifying the P-globin gene, include either Set1 or Set2 in first-round multiplex

PCR, with subsequent use of either LC1 primers or LC2 primers (as appropriate) for mutation analysis on the LightCycler® (see Figure 17.1).

** Co-ordinates of primers based on GenBank NG_000007.

*** 5' fluorescently labeled with Cy5.5.

F: forward primer.

R: reverse primer.

and direct sequencing. However, these methods require several hours and sometimes days to complete a diagnosis, and, consequently, there is a need for more rapid, high-throughput assays.

When applied for PND, the LightCycler® real-time PCR method is very rapid and accurate. Once the parental mutations are known, PND requires less than 3 hours for completion, including the stage of DNA extraction from the fetal sample. The distribution of mutations means that many cases require only one PCR reaction (including two LightCycler® mutation detection probe sets if necessary), minimizing both the time and cost of the PND procedure. Furthermore, in cases where the two parental mutations differ but are under the same detection probe, the melting curve of a normal allele is always distinguished when present, thus preventing a misdiagnosis.

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