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Biotin-labelled dNTP

Fig. 4. Pronto assay. A conventional PCR is carried out, followed by enzymatic degradation of unincorporated dNTPs. A 5'-labeled primer is then annealed to the PCR and DNA polymerase and a biotinylated dNTP added. The biotinylated dNTP is only added to the primer if it matches the target amplicon. The reaction mix is then added to a streptavidin-coated well and biotinylated primers are thus bound; unbound primers are washed away. A HRP-linked antibody to the 5' primer label is then added and detected by color development measured visually or by an ELISA plate reader.

Fig. 4. Pronto assay. A conventional PCR is carried out, followed by enzymatic degradation of unincorporated dNTPs. A 5'-labeled primer is then annealed to the PCR and DNA polymerase and a biotinylated dNTP added. The biotinylated dNTP is only added to the primer if it matches the target amplicon. The reaction mix is then added to a streptavidin-coated well and biotinylated primers are thus bound; unbound primers are washed away. A HRP-linked antibody to the 5' primer label is then added and detected by color development measured visually or by an ELISA plate reader.

In MLPA, genomic DNA is denatured and a mixture of oligonucleotide probes is hybridized to the DNA (Fig. 3). Each MLPA probe consists of two oligonucleotides that take part in a one-cycle OLA reaction. The two oligonucleotides have generic sequences attached to the 5' end of the upstream primer and to the 3' end of the downstream primer. Thus, after the initial OLA step, a PCR is then carried out using primers complementary to the generic sequences on the ends of the OLA primers. In this way, multiple different target sequences can be interrogated, but which are all amplified using the same PCR primers, with equal efficiency. As one of the generic primers is fluorescently labeled, then the fluorescence resulting from a particular amplicon (MLPA probe) is proportional to the amount of starting target DNA, and the copy number can be determined. The lengths of the various probes are engineered so that individual probes differ by a few basepairs and can be easily separated on a fluorescent DNA analyzer (66).

5.2. ADVANTAGES AND USES OF MLPA Multiplex ligation-dependent probe amplification is quick, inexpensive and relatively simple. Probes can be easily designed for novel applications, and a wide range of clinically useful kits are commercially available (see http://www.mrc-holland.com/). The use of MLPA in detecting exon copy number changes in DNA mismatch repair genes in hereditary non-polyposis colorectal cancer (HNPCC) has been described (67-69).

5.3. LIMITATIONS OF MLPA Kits are currently manufactured by only a single commercial company, although they are open to suggestions for new applications. Like any dosage technique, MLPA is sensitive to DNA quality and quantity. It is best to perform tests in duplicate and be suspicious of discordant results. Control probes are included in MLPA kits from MRC-Holland and notice should be taken of them and the manufacturer's instructions.

If a point mutation or polymorphism occurs within an MLPA probe-binding site, it might result in failure of ligation and, hence, appear as a deletion of a whole exon. The deletion of two or more contiguous exons is unlikely to be the result of this effect, but apparent deletion of a single exon might be. For this reason, another complementary technique should be used to confirm the nature of the mutation if it appears to be deletion of a single exon. This could be by using an alternative probe (pair of MLPA primers), real-time or long-range PCR, Southern blot, or mRNA analysis. It might also be useful to sequence the exon in question.

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