Primed in situ labeling (PRINS) originally was designed by Koch et al. in 1986 for the investigation of minute sequence structures within primate a-satellite DNA (1-3). Since that time, numerous technical variants have been described, and a wide variety of targets have been detected (4,5). PRINS reaction is defined as a nonisotopic in situ labeling method for the rapid and efficient detection of interphase and metaphase DNA. This method is based on the annealing of specific oligonucleotide primers, derived from chromosome-specific subsets of DNA sequences, to the denatured DNA, followed by subsequent primer extension using Taq DNA polymerase. Specific labeling was

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.

Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

obtained in fetal nucleated cells prepared from a sample of maternal peripheral whole blood. Unlike the conventional fluorescence in situ hybridization (FISH) technique, PRINS reaction is a sensitive molecular cytogenetic technique that has empowered the conclusive detection of the presence of the few and rare fetal nucleated cells circulating in maternal blood. We tested the dual-color PRINS reactions to detect sexual chromosomes of fetal cells according to Yan et al. multi-PRINS protocol (6) on blood samples obtained from pregnant women at the second trimester of gestation. The purpose of this study was to determine the exact number of fetal nucleated cells per unit volume of maternal blood. Using specific primers for chromosomes X and Y, we proceeded to the primer-nucleotide-hapten mix annealing stage, followed by specific antibody-fluorophore complex detection. In our previous study, using two simple and efficient methods (FISH and PRINS), we concluded that the exact number of fetal nucleated cells per unit volume of maternal blood (1 mL), fluctuated between 2 and 6 during the second trimester of pregnancy (7). These results suggest that PRINS might be a powerful tool that is more specific, considerably faster, and less expensive than classical FISH techniques. Our experiments show that the PRINS reaction is potentially of great interest as an alternative to FISH for nucleated fetal cell analysis. Furthermore, PRINS could be useful in noninvasive prenatal testing.

Was this article helpful?

0 0

Post a comment