The sensitivity of real-time PCR is expected to maximize the detection of amplified DNA copies, even when present in low numbers. This potentially reduces the frequency of amplification failure and allele drop-out. Furthermore, even if allele drop-out does occur, it is monitored by the use of the nested real-time PCR, which will detect both alleles contributing to the genotype. Embryos with 'impossible' genotypes are considered to have allele drop-out and are not recommended for transfer. Only those embryos in which a normal allele is definitely detected are recommended for transfer.
The protocol as designed for the P-hemoglobinopathies is capable of detecting a wide range of mutations and genotype interactions simply through selecting the appropriate P-globin gene primers and LightCycler® hybridization probe(s) (Table 17.1 and Figure 17.1). This is an important consideration when offering PGD for a common disease with a wide spectrum of mutation interactions, as it precludes the need to standardize case-specific protocols each time a new couple requires PGD.
The incorporation of real-time PCR into the PGD protocol means that the entire procedure for genotype analysis is very rapid, requiring less than 6 hours for completion, and well within the 24-hour time limit between blastomere biopsy and embryo transfer.
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