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Hum. Reprod. Advance Access published online on January 21, 2005
Human Reproduction, doi:10.1093/humrep/deh735
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Human Reproduction © European Society of Human Reproduction and Embryology 2005; all rights reserved
Received June 6, 2004
Revised October 10, 2004
Accepted December 9, 2004

Article

Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres

I.E. Agerholm 1*, S. Ziebe 2, B. Williams 3, C. Berg 4, D.G. Crüger 4, G. Bruun Petersen 4, and S. Kølvraa 5

1 The Fertility Clinic, Braedstrup Hospital, DK 8740 Braedstrup, Denmark
2 The Fertility Clinic, Rigshospitalet, DK 2100 Copenhagen, Denmark
3 Applied Biosystem, 35 Wiggins Avenue, Bedford, MA 01730, USA
4 Department of Clinical Genetics, Vejle Hospital, DK 7100 Vejle, Denmark
5 Institute of Human Genetics, University of Aarhus, DK 8000 Aarhus, Denmark and Department of Clinical Genetics, University Hospital of Aarhus, 8000 Aarhus C, Denmark

* To whom correspondence should be addressed.
I.E. Agerholm, E-mail: iag{at}bs.vejleamt.dk


  Abstract

BACKGROUND: The aim was to introduce a new strategy based on peptide nucleic acid (PNA) probes and competitive displacement for using fluorescence in-situ hybridization (FISH) analysis on human blatomeres. METHODS: Sequential FISH analysis with PNA probes and competitive displacement was performed using three different probe sets. The first set consisted of labelled probe only. The second and third sets included labelled as well as unlabelled probe, corresponding to the labelled probes in the previous cycles. The probes for enumeration were for chromosome 1, 13, 16, 17, 18, 21, X and Y. RESULTS: The performance of PNA probes was similar to the established DNA probes. The strategy of competitive displacement resulted in a destabilization of already bound probe before the next FISH cycle at only 50 °C, which allowed for up to five sequential FISH cycles without loss of signal. CONCLUSIONS: PNA probes are a good alternative to DNA probes in the present set-up, since the low temperature required both for binding and destabilization of PNA probes minimizes the loss of signal, and several FISH cycles can therefore be carried out before FISH errors occur.

Keywords: aneuploidy screening; blastomeres; competitive displacement; FISH; PNA probes.
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