ABSTRACT
The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (nâ=â36); trisomy 18 (nâ=â6); trisomy 13 (nâ=â4); 45, X (nâ=â5); 47, XXX (nâ=â3); 48, XXYY (nâ=â2); and unaffected controls (nâ=â40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.
Subject(s)
Aneuploidy , Chromosome Disorders/diagnosis , Fluorescent Dyes/chemistry , Polymerase Chain Reaction/methods , Prenatal Diagnosis/methods , Trisomy/genetics , Automation , Chromosome Mapping , DNA Primers/chemistry , DNA Primers/genetics , Electrophoresis, Capillary , Female , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Male , Reproducibility of Results , Retrospective Studies , Sensitivity and SpecificityABSTRACT
The objective of the present study was to construct a human chromosome 21 specific DNA library for further use in research of genetic disease. Human chromosome 21 microdissected from the peripheral blood cells were subjected to repeatedly incubation in gradient temperature bath to release DNA. The library of chromosome 21 was constructed using the DNA fragment of 100-500 bp and 500-2 000 bp recovered from the products of DOP-PCR. Florescence in situ hy-bridization (FISH) and dot blotting analyses were carried out to assess the chromosome 21 specificity of the DNA library. The results indicated that DNA of chromosome 21 was released easily after repeatedly incubation in gradient temperature bath. Recovery of DNA fragments from DOP-PCR in different size ranges improved the efficiency of cloning of large fragments. Both FISH and dot blotting analyses revealed that the DNA library constructed in this study was chromosome 21-specific. This DNA library facilitates identification and investigation of the chromosome 21 related abnormality.