Fluorescent probes for chromosome painting
View Patent ↗The present invention relates to fluorescent probes which can be used in multicolor fluorescence in situ hybridization, and mainly chromosome painting. The probes intended for labeling a chromosome are such that they are composed of a set of DNA segments which are more represented in certain chromosome bands and which are obtained by IRS-PCR amplification from said chromosomes using PCR primers specific for the repeated and dispersed Alu and LINE DNA sequences. The invention comprises, in addition, methods of producing said probes, multicolor FISH methods which can use said probes as well as diagnostic kits comprising them. Finally, the invention comprises combinations of fluorophores and optical filters.
1. A method of identifying human chromosomes comprising performing a multicolor FISH analysis using a set of nucleic acid probes for labeling one or more human chromosomes and hybridizing said one or more human chromosomes with said set of nucleic acid probes, said set of nucleic acid probes comprising a mixture of two interspersed repeated sequence-polymerase chain reaction (IRS-PCR) amplification products from said chromosome(s), wherein:
the first amplification product is complementary to either a specific primer for Alu DNA sequences or a specific primer for long interspersed element (LINE) sequences;
the second amplification product is complementary to a specific primer for Alu DNA sequences and a specific primer for LINE DNA sequences; and
each of said probes, being specific for one or more human chromosomes, are directly or indirectly labeled with:
a) a fluorophore having an absorption bandwidth wavelength interval spanning 593 nm and an emission bandwidth wavelength interval spanning 613 nm; and
b) a fluorophore having an absorption bandwidth wavelength interval spanning 554 nm and an emission bandwidth wavelength interval spanning 568 nm; and
c) up to five (5) fluorophores selected from the group consisting of:
i) a fluorophore having an absorption bandwidth wavelength interval spanning 494 nm and an emission bandwidth wavelength interval spanning 517 nm;
ii) a fluorophore having an absorption bandwidth wavelength interval spanning 652 nm and an emission bandwidth wavelength interval spanning 670 nm;
iii) a fluorophore having an absorption bandwidth wavelength interval spanning 675 nm and an emission bandwidth wavelength interval spanning 694 nm;
iv) a fluorophore having an absorption bandwidth wavelength interval spanning 743 nm and an emission bandwidth wavelength interval spanning 767 nm;
v) and a fluorophore having an absorption bandwidth wavelength interval spanning 632 nm and an emission bandwidth wavelength interval spanning 658 nm; and
identifying said one or more labeled human chromosomes by fluorescence microscopy, wherein the overlapping of the absorption and emission bandwidth wavelength intervals between the different fluorophores is minimal and there is no overlapping between the absorption and emission maximum of the different fluorophores; and
wherein each of said fluorophores having a specific absorption and emission wavelength is combined with a pair of optical filters, one for absorption and one for emission in a fluorescent microscope.
2. The method according to claim 1 , wherein said set of nucleic acid probes comprises at least one probe for each of the human chromosomes or for a number of them.
3. The method according to claim 1 , wherein said amplification products comprise amplification products obtained from one or more mono-chromosomal rodent-human somatic hybrid.
4. The method according to claim 1 , wherein said first amplification product comprises an amplification product obtained using said primer specific for the Alu DNA sequences.
5. The method according to claim 1 , wherein said first amplification product comprises an amplification product obtained using said specific primer for LINE DNA sequences.
6. The method according to claim 1 , wherein said amplification products comprise amplification products more represented in one type of cytogenetic bands.
7. The method according to claim 6 , wherein said amplification products are more represented in the R bands.
8. The method according to claim 1 , wherein said specific primers comprise a sequence selected from the group consisting of:
SEQ ID No. 1 for the primer specific for the Alu DNA sequences; and
SEQ ID Nos. 2 and 3 for the primer specific for the LINE DNA sequences.
9. The method according to claim 1 , wherein said multicolor FISH analysis is a karyotype analysis.