Characterization of chromosomal inversions using anti-parallel probes
View Patent ↗A method for the characterization of chromosomal inversions using anti-parallel probes is described. Reporter species are attached to oligonucleotide strands designed such that they may hybridize to portions of only one of a pair of single-stranded sister chromatids which may be prepared by the CO-FISH procedure. If an inversion has occurred, these marker probes will be detected on the second sister chromatid at the same location as the inversion on the first chromatid. Further, one or more reporter species are replaced with anti-parallel probes that hybridize at known locations along the second sister chromatid such that the position and size of the inversion may be identified/estimated.
1. A method for detecting inversions in a selected mitotic chromosome, comprising the steps of: generating a pair of single-stranded sister chromatids from said selected chromosome, each sister chromatid having a length of DNA and a series of target DNA sequences that span a portion of the length of the DNA of the chromatid; generating a plurality of non-repetitive probes, wherein each of the probes is single-stranded, unique and identical to at least a portion of a target DNA sequence, each of the probes having at least one label, thereby permitting detection thereof; hybridizing the probes to the sister chromatids; and detecting the hybridized probes; whereby if no inversion exists, all of the probes will hybridize to one of the sister chromatids, and whereby if an inversion exists, at least one of the probes will hybridize to the other sister chromatid at the same location as the inversion, the method further comprising replacing one or more of the non-repetitive probes with a probe(s) having anti-parallel (opposite) orientation to that probe whereby the one or more anti-parallel probes bind the other of the two sister chromatids at a known location along the chromatid, and whereby binding of the anti-parallel probe is used to identify/estimate the position and size of a detected inversion.
2. The method of claim 1 , wherein the portion of the DNA target sequence suitable for locus-specific probes is amplified using polymerase chain reaction.
3. The method of claim 2 , wherein at least one fluorescent label is attached to the DNA target sequence as a fluorescent dNTP by linear polymerase chain reaction.
4. The method of claim 1 , wherein an identical oligonucleotide to the DNA target sequence is synthesized from software-generated tiled arrays.
5. The method of claim 4 , wherein at least one fluorescent label is attached to the oligonucleotide as a fluorescent nucleotide by use of enzymes.
6. The method of claim 1 , wherein said step of generating the pair of single-stranded sister chromatids from the mitotic chromosome is accomplished using CO-FISH.
7. The method of claim 1 , wherein the at least one label of each of the probes comprises a fluorescent label, and said step of detecting the hybridized probes is achieved using fluorescent microscopy.
8. A method for detecting inversions in a selected mitotic chromosome, comprising the steps of: generating a pair of single-stranded sister chromatids from said selected chromosome, each sister chromatid having a length of DNA and a series of target DNA sequences that span a portion of the length of the DNA of the chromatid; generating a plurality of non-repetitive probes, wherein each of the probes is single-stranded, unique and complementary to at least a portion of a target DNA sequence, each of the probes having at least one label, thereby permitting detection thereof; hybridizing the probes to the sister chromatids; and detecting the hybridized probes; whereby if no inversion exists, all of the probes will hybridize to one of the sister chromatids, and whereby if an inversion exists, at least one of the probes will hybridize to the other sister chromatid at the same location as the inversion, the method further comprising replacing one or more of the non-repetitive probes with a probe(s) having anti-parallel (opposite) orientation to that probe whereby the one or more anti-parallel probes bind the other of the two sister chromatids at a known location along the chromatid, and whereby binding of the anti-parallel probe is used to identify/estimate the position and size of a detected inversion.
9. The method of claim 8 , wherein the complementary sequence to the portion of the DNA target sequence suitable for probes is amplified using polymerase chain reaction.
10. The method of claim 9 , wherein at least one fluorescent label is attached to the DNA target sequence as a fluorescent dNTP by linear polymerase chain reaction.
11. The method of claim 8 , wherein a complementary oligonucleotide to the DNA target sequence is synthesized from software-generated tiled arrays.
12. The method of claim 11 , wherein at least one fluorescent label is attached to the oligonucleotide as a fluorescent nucleotide by use of enzymes.
13. The method of claim 8 , wherein said step of generating the pair of single-stranded sister chromatids from the mitotic chromosome is accomplished using CO-FISH.
14. The method of claim 8 , wherein the at least one label of each of the probes comprises a fluorescent label, and said step of detecting the hybridized probes is achieved using fluorescent microscopy.
15. The method of claim 1 wherein the one or more anti-parallel probes is labeled with a different fluorescent label than the other non-repetitive probes.
16. The method of claim 1 wherein the one or more anti-parallel probes is at least two anti-parallel probes and the two probes bind to known locations on the sister chromatid at least 100 Mb apart.
17. The method of claim 1 wherein the one or more anti-parallel probes is at least two anti-parallel probes and the two probes bind to known locations on the sister chromatid at least 10 Mb apart.
18. The method of claim 8 wherein the one or more anti-parallel probes is labeled with a different fluorescent label than the other non-repetitive probes.
19. The method of claim 8 wherein the one or more anti-parallel probes is at least two anti-parallel probes and the two probes bind to known locations on the sister chromatid at least 100 Mb apart.
20. The method of claim 8 wherein the one or more anti-parallel probes is at least two anti-parallel probes and the two probes bind to known locations on the sister chromatid at least 10 Mb apart.