PRIMER EXTENSION PREAMPLIFICATION FOR DETECTION OF MULTIPLE GENETIC-LOCI FROM SINGLE HUMAN BLASTOMERES

A new technology called primer extension preamplification (PEP), which has been applied to single spermatozoa, increases the amount of polymerase chain reaction (PCR) templates by amplifying DNA of the whole genome. The current investigation was aimed at applying PEP to single human blastomeres. Two blastomeres with nuclei from arrested embryos were selected for this study. Using three different PEP protocols (experiments I, II and III), DNA from single blistomeres was amplified using 15-base oligonucleotide random primers. The efficiency of the procedure was determined by further amplifications of aliquots of the PEP products with two specific sequences. Three aliquots from each PEP product were used as PCR templates for the human X chromosome (X) or the exon 10 of the cystic fibrosis gene (CF). PCR amplified products were analysed by gel electrophoresis. In experiment I, when X primers were used, positive signals were detected in all 10 embryos (100%), 90.0% (18/20) of the blastomeres, and in 80.0% (96/120) of the replicates. When CF primers were amplified, all embryos (100%, 10/10), 90.9% (18/20) of the blastomeres and 78.3% (47/60) of the replicates were positive. In experiment II, efficiency was significantly reduced when total time for the procedure was minimized from 8 h to 5 h and 45 min. Although the time was further reduced to 4 h and 40 min in experiment III, the efficiency remained the same as in experiment I when the volume of PEP was reduced from 60 mul (experiments I and II) to 40 mul. One out of 132 control replicates (0.8%) was contaminated. The present study indicates that PEP can be successfully applied to single blastomeres allowing for simultaneous detection of approximately 20 DNA sequences.