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Human Reproduction, Vol. 8, No. 12, pp. 2206-2210, 1993
© 1993 European Society of Human Reproduction and Embryology

review-article

Preimplantation diagnosis: Primer extension preamplification for detection of multiple genetic loci from single human blastomeres

KangPu Xu1, YaXu Tang, Jamie A. Grifo, Zev Rosenwaks and Jacques Cohen

The Gamete and Embryo Research Laboratory, The Center for Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, New York Hospital—Cornell University Medical College New York, NY 10021, USA

Correspondence: 1To whom correspondence should be addressed at: The Gamete and Embryo Research Laboratory, Cornell University Medical College, PO Box 30, 1300 York Avenue, New York, NY 10021, USA

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 blastomeres 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 µl (experiments I and II) to 40 µl. 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.

Key words: blastomeres/DNA amplification/polymerase chain reaction/preimplantation diagnosis


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