MultiStar 24 FISH consists of four DNA probe mixes each hybridizing to six different chromosomes using six different fluorochromes. It can be applied in lymphocytes, sperm and blastomeres. The fully optimized protocol consists of three 15-30 minutes hybridizations followed by an overnight one. In between the individual hybridizations, the preceding probes are washed off after imaging the results. The morphology of the cell types is retained despite repeated denaturation, hybridization and post hybridization washes. Moreover the entire protocol can be completed within 24 hours, which fits the window for clinical PGS application.
This novel method eliminates the bottleneck perceived when using FISH by omitting key chromosomes relevant to implantation failure not covered in a limited panel.
Metaphase preparation from male lymphocyte cells visualizing all 24 chromosomes.
The probes required to provide information for 24 chromosomes comprises four panels each containing 6 chromosome-specific identifier sequences each labelled with a different fluorochrome. The first 3 panels use centromeric sequences (Panel 1: chromosomes 1,3,4,6,7,8; Panel 2: chromosomes 9,10,11,12,17,20 and Panel 3: chromosomes 2,15,16,18,X,Y) . In contrast, Panel 4 used for the final round of hybridization uses unique sequence probes for chromosomes 5,13,14,19,21,22 since centromeric sequences were not available for these chromosomes. A combination of separate probe denaturation for 10 minutes followed by a short co-denaturation between probe and sample is used. Panels 1-3 require short hybridization times (15 minutes) whereas Panel 4 requires overnight hybridization.
Image kindly provided by Prof. D. Griffin, University of Kent, United Kingdom
Setup of the different layers
All DNA probes are labeled with PlatinumBright™ based on the Universal Linkage System (ULS™), KREATECH´s proprietary non-enzymatic labeling technology capable of linking fluorescent labels or haptens to any nucleic acid of interest.
MultiStar 24 FISH consists of four DNA probe mixes each hybridizing to six different chromosomes using six different fluorochromes. It can be applied in lymphocytes, sperm and blastomeres. The fully optimized protocol consists of three 15-30 minutes hybridizations followed by an overnight one. In between the individual hybridizations, the preceding probes are washed off after imaging the results. The morphology of the cell types is retained despite repeated denaturation, hybridization and post hybridization washes. Moreover the entire protocol can be completed within 24 hours, which fits the window for clinical PGS application.
This novel method eliminates the bottleneck perceived when using FISH by omitting key chromosomes relevant to implantation failure not covered in a limited panel.
Metaphase preparation from male lymphocyte cells visualizing all 24 chromosomes.
The probes required to provide information for 24 chromosomes comprises four panels each containing 6 chromosome-specific identifier sequences each labelled with a different fluorochrome. The first 3 panels use centromeric sequences (Panel 1: chromosomes 1,3,4,6,7,8; Panel 2: chromosomes 9,10,11,12,17,20 and Panel 3: chromosomes 2,15,16,18,X,Y) . In contrast, Panel 4 used for the final round of hybridization uses unique sequence probes for chromosomes 5,13,14,19,21,22 since centromeric sequences were not available for these chromosomes. A combination of separate probe denaturation for 10 minutes followed by a short co-denaturation between probe and sample is used. Panels 1-3 require short hybridization times (15 minutes) whereas Panel 4 requires overnight hybridization.
Image kindly provided by Prof. D. Griffin, University of Kent, United Kingdom
Setup of the different layers
All DNA probes are labeled with PlatinumBright™ based on the Universal Linkage System (ULS™), KREATECH´s proprietary non-enzymatic labeling technology capable of linking fluorescent labels or haptens to any nucleic acid of interest.