Screening for aneuploidies of ten different chromosomes in two rounds of FISH: a short and reliable protocol.
OBJECTIVE
To develop a DNA labelling protocol for the simultaneous detection of five different fluorescent chromosomal DNA probes within one round of hybridisation. In combination with a commercial five-colour probemix for the second round of hybridisation, this results in a fast and reliable Fluorescence in situ Hybridization (FISH) protocol, enabling the detection of 10 chromosomes within a working day. This is especially of use for Preimplantation Genetic Screening (PGS), when only single interphase nuclei are available for analysis and when time is restricted.


METHODS
DNA probes were labelled with four different fluorochromes (Pacific Blue, Alexa Fluor 350, Alexa Fluor 594 and Alexa Fluor 488) using an ARES labelling kit, based on a two-step method. Aminoallyl-dUTPs were incorporated by nick translation, followed by chemical linking of the amino-modified fluorescent dye. The fifth colour was achieved by using two fluorescent dyes in the chemical reaction, resulting in dual labelling of the DNA probe and a fluorescence detectable with a specific filter set. These five probes were simultaneously hybridised in a first FISH round, followed by a second hybridisation with a commercial five-colour probemix, thus allowing the detection of chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. The fixation and pre-treatment procedures of the blastomere nuclei were further optimised.


RESULTS
Using this labelling and FISH protocol, probe hybridisation efficiency, when tested on lymphocyte nuclei, is 95 to 99%. With the fixation protocol, blastomere nuclei maintain good morphology and show condensed and clear signals even after the second round of hybridisation.


CONCLUSIONS
This labelling method in combination with specific epifluorescence filters, enables an independent detection of five different chromosomes in one round of FISH. The whole process of biopsy, fixation and two rounds of hybridisation with the analysis of ten chromosomes can be completed within a day.
Baart
E B
EB
Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus Medical Center, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. e.baart@erasmusmc.nl
Martini
E
E
Van Opstal
D
D
D000782:Aneuploidy; D001757:Blastomeres / Q000648:ultrastructure; D002467:Cell Nucleus / Q000648:ultrastructure; D002877:Chromosomes, Human / Q000648:ultrastructure; D041321:Chromosomes, Human, X / Q000648:ultrastructure; D041322:Chromosomes, Human, Y / Q000648:ultrastructure; D015342:DNA Probes; D005260:Female; D005404:Fixatives; D005456:Fluorescent Dyes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D019836:Preimplantation Diagnosis / Q000379:methods*
D015342:DNA Probes; D005404:Fixatives; D005456:Fluorescent Dyes