The single-nucleotide primer extension assay is in widespread use. It is the basis of a proprietary technique: Pronto (70) (Fig. 4). An amplicon is produced in a conventional PCR and residual dNTPs are eliminated using alkaline phosphatase.
The amplicon is denatured and a specific primer binds to one of the two DNA strands. At its 5' end, the primer has a covalently linked label. Depending on the target sequence, a biotinylated dNTP is added to the 3' end of the primer by DNA polymerase. The now biotinylated primer can bind to streptavidin-coated wells in a 96-well plate, and after washing, a horseradish peroxidase (HRP)-conjugated antibody to the primer label is added. Binding of the HRP-conjugated antibody is manifest by color development in that well and the mutation causing addition of the specific dNTP is detected. Different fluorescent labels can be linked to different dNTPs, such that with an appropriate detector, different mutations at the same nucleotide can be distinguished (such as in K-ras).
6.1. EQUIPMENT FOR PRIMER EXTENSION Aside from a conventional PCR thermocycler, results can be scored either visually or with an enzyme-linked immunosorbent assay (ELISA) plate reader if a numerical output is desired.
6.2. USES OF PRIMER EXTENSION In common with other PCR-based methods of mutation detection, primer extension is suited to the detection of defined mutations. The commercially available tests using Pronto concentrate on common point mutations in both dominant and recessive disorders, in particular those prevalent in the Ashkenazi Jewish population (70-72).
6.3. ADVANTAGES AND LIMITATIONS OF PRIMER EXTENSION Primer extension is a gel-free mutation-detection system of low to high throughput. It only requires commonly available equipment. Although visual scoring is probably adequate for low-throughput applications, medium- to high-throughput benefits from using an ELISA reader.
Limitations include that Pronto kits are currently only manufactured by a single commercial company (http://www. savyondiagnostics.com/), a restricted range of applications is available, mostly based on conditions because of founder mutations in the Ashkenazi Jewish and Mediterranean populations (70-72). However, a large number of in-house primer extension assays have been developed (73,74). Like most PCR-based methods of mutation detection, primer extension is only able to detect defined mutations found by another technique; that is, it is not a method able to screen amplicons for any mutation.
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