Annals of Clinical Trials and Vaccines Research

Minisequencing New

 In the minisequencing technique, a primer extension reaction is performed, starting from a specific primer that is designed to anneal directly adjacent to the mutation site, by the incorporation of a single fluorescent dideoxynucleotide (ddNTP) which is complementary to the variant base in the template. Preimplantation genetic diagnosis (PGD) is presently a valid alternative for couples at high risk of pregnancy with genetic anomalies. PGD enables unaffected embryos generated by IVF to be identified and transferred and it therefore permits couples to avoid termination of affected pregnancies. Protocols for genotyping single cells for monogenic disorders are based on the PCR (Saiki et al., 1985; Li et al., 1988), which represents the only method sensitive enough to detect single gene mutations. Due to its sensitivity, PCR is highly prone to sources of error; thus precautions must be taken in its use for clinical diagnosis. Since the first PCR‐based PGD cases were performed (Handyside et al., 1989; 1990; 1992), several inherent difficulties associated with single cell DNA amplification have become evident. They include potential sample contamination, total PCR failure, allelic drop‐out (ADO, when one of the alleles fails to amplify to detectable levels), and preferential amplification (PA) of one of the alleles.  

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