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


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 degrees 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.
Agerholm
I E
IE
The Fertility Clinic, Braedstrup Hospital, DK 8740 Braedstrup, The Fertility Clinic, Rigshospitalet, DK 2100 Copenhagen, Denmark. iag@bs.vejleamt.dk
Ziebe
S
S
Williams
B
B
Berg
C
C
Crüger
D G
DG
Petersen
G Bruun
GB
Kølvraa
S
S
D000782:Aneuploidy; D001757:Blastomeres / Q000502:physiology*; D002467:Cell Nucleus; D002877:Chromosomes, Human; D015342:DNA Probes; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009691:Nucleic Acid Denaturation; D020135:Peptide Nucleic Acids; D019836:Preimplantation Diagnosis / Q000379:methods*; D013696:Temperature
D015342:DNA Probes; D020135:Peptide Nucleic Acids