Methods of identifying homologous genes using FISH

• US 10,119,160 B2
• Filed: 01/06/2017
• Issued: 11/06/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Wu, Chao-ting, Beliveau, Brian
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/399,788
• Publication Number:
  US 20170114401A1
• Time in Patent Office: 669 Days
• Field of Search: 435 912
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2543/10 the purpose being "in situ" analysis
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The present invention relates to methods of hybridizing nucleic acid probes to genomic DNA.

Claims

1. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising

identifying a first nucleotide type that is a single nucleotide polymorphism within the first gene,

hybridizing a first primer type directly upstream of the first nucleotide type, extending the first primer type across the first nucleotide type in the presence of a first polymerase, first extension nucleotides and a first labeled extension nucleotide complementary to the first nucleotide type, wherein the first labeled extension nucleotide hybridizes to the first nucleotide type,

identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type,

hybridizing a second primer type directly upstream of the second nucleotide type, extending the second primer type across the second nucleotide type in the presence of a second polymerase, second extension nucleotides and a second labeled extension nucleotide complementary to the second nucleotide type wherein the second labeled extension nucleotide hybridizes to the second nucleotide type, wherein the first gene is differentially labeled from the second gene.

2. The method of claim 1 further including

identifying a third nucleotide type that is a single nucleotide polymorphism within the first gene and which is different from the first nucleotide type and the second nucleotide type,

hybridizing a third primer type directly upstream of the third nucleotide type, extending the third primer type across the third nucleotide type in the presence of a third polymerase, third extension nucleotides and a third labeled extension nucleotide complementary to the third nucleotide type, wherein the third labeled extension nucleotide hybridizes to the third nucleotide type, and wherein the first gene is differentially labeled from the second gene.

3. The method of claim 1 further including

identifying a third nucleotide type that is a single nucleotide polymorphism within the first gene and which is different from the first nucleotide type and the second nucleotide type,

hybridizing a third primer type directly upstream of the third nucleotide type, extending the third primer type across the third nucleotide type in the presence of a third polymerase, third extension nucleotides and a third labeled extension nucleotide complementary to the third nucleotide type, wherein the third labeled extension nucleotide hybridizes to the third nucleotide type, and wherein the first gene is differentially labeled from the second gene,

wherein a specific first nucleotide within the first gene is excluded from being labeled if its corresponding nucleotide on the second gene is of the same nucleotide type as the third nucleotide type on the first gene.

4. The method of claim 1 further including

identifying a third nucleotide type that is a single nucleotide polymorphism within the first gene and which is different from the first nucleotide type and the second nucleotide type,

hybridizing a third primer type directly upstream of the third nucleotide type, extending the third primer type across the third nucleotide type in the presence of a third polymerase, third extension nucleotides and a third labeled extension nucleotide complementary to the third nucleotide type, wherein the third labeled extension nucleotide hybridizes to the third nucleotide type,

identifying a fourth nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, the second nucleotide type, and the third nucleotide type,

hybridizing a fourth primer type directly upstream of the fourth nucleotide type, extending the fourth primer type across the fourth nucleotide type in the presence of a fourth polymerase, fourth extension nucleotides and a fourth labeled extension nucleotide complementary to the fourth nucleotide type wherein the fourth labeled extension nucleotide hybridizes to the fourth nucleotide type, wherein the first gene is differentially labeled from the second gene.

5. The method of claim 1 further including

identifying a third nucleotide type that is a single nucleotide polymorphism within the first gene and which is different from the first nucleotide type and the second nucleotide type,

hybridizing a third primer type directly upstream of the third nucleotide type, extending the third primer type across the third nucleotide type in the presence of a third polymerase, third extension nucleotides and a third labeled extension nucleotide complementary to the third nucleotide type, wherein the third labeled extension nucleotide hybridizes to the third nucleotide type,

identifying a fourth nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, the second nucleotide type, and the third nucleotide type,

hybridizing a fourth primer type directly upstream of the fourth nucleotide type, extending the fourth primer type across the fourth nucleotide type in the presence of a fourth polymerase, fourth extension nucleotides and a fourth labeled extension nucleotide complementary to the fourth nucleotide type wherein the fourth labeled extension nucleotide hybridizes to the fourth nucleotide type, wherein the first gene is differentially labeled from the second gene,

wherein a specific first nucleotide within the first gene is excluded from being labeled if its corresponding nucleotide on the second gene is of the same nucleotide type as the third nucleotide type on the first gene, and

wherein a specific second nucleotide within the second gene is excluded from being labeled if its corresponding nucleotide on the first gene is of the same nucleotide type as the fourth nucleotide type on the second gene.

6. The method of claim 1 wherein the first labeled extension nucleotide or the second labeled extension nucleotide is a modified nucleotide.

7. The method of claim 1 wherein the first labeled extension nucleotide is a first modified nucleotide and the second labeled extension nucleotide is a second modified nucleotide.

8. The method of claim 1 wherein the first labeled extension nucleotide is a first modified nucleotide and the second labeled extension nucleotide is a second modified nucleotide, wherein the first modified nucleotide is attachable only by first modified nucleotides of the same type and wherein the second modified nucleotide is attachable only by second modified nucleotides of the same type.

9. The method of claim 4 wherein the first labeled extension nucleotide is a first modified nucleotide, the second labeled extension nucleotide is a second modified nucleotide, the third labeled extension nucleotide is a third modified nucleotide and the fourth labeled extension nucleotide is a fourth modified nucleotide, wherein the first modified nucleotide and the third modified nucleotide are attachable by the same modified nucleotide.

10. The method of claim 4 wherein the first labeled extension nucleotide is a first modified nucleotide, the second labeled extension nucleotide is a second modified nucleotide, the third labeled extension nucleotide is a third modified nucleotide and the fourth labeled extension nucleotide is a fourth modified nucleotide, wherein the second modified nucleotide and the fourth modified nucleotide are attachable by the same modified nucleotide.

11. The method of claim 4 wherein the first labeled extension nucleotide is a first modified nucleotide, the second labeled extension nucleotide is a second modified nucleotide, the third labeled extension nucleotide is a third modified nucleotide and the fourth labeled extension nucleotide is a fourth modified nucleotide,

wherein the first modified nucleotide and the third modified nucleotide are attachable by the same first attaching modified nucleotide,

wherein the second modified nucleotide and the fourth modified nucleotide are attachable by the same second attaching modified nucleotide, and wherein the first attaching modified nucleotide is different from the second attaching modified nucleotide.

12. The method of claim 1 wherein the first labeled extension nucleotide or the second labeled extension nucleotide is a modified nucleotide to which a detectable label can be added or which can be further modified to distinguish the first gene from the second gene.

13. The method of claim 2 wherein the third labeled extension nucleotide is a modified nucleotide.

14. The method of claim 2 wherein the third labeled extension nucleotide is a modified nucleotide to which a detectable label can be added or which can be further modified to distinguish the first gene from the second gene.

15. The method of claim 4 wherein the first labeled extension nucleotide, the second labeled extension nucleotide, the third labeled extension nucleotide or the fourth labeled extension nucleotide is a modified nucleotide.

16. The method of claim 4 wherein the first labeled extension nucleotide, the second labeled extension nucleotide, the third labeled extension nucleotide or the fourth labeled extension nucleotide is a modified nucleotide to which a detectable label can be added or which can be further modified to distinguish the first gene from the second gene.

17. The method of claim 4 wherein the modified nucleotide of the first labeled extension nucleotide and the third labeled extension nucleotide is different from the modified nucleotide of the second labeled extension nucleotide and the fourth labeled extension nucleotide such that the modified nucleotide hybridized to the first gene is different from the modified nucleotide hybridized to the second gene.

18. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising

identifying a first nucleotide type that is a single nucleotide polymorphism within a first sequence within the first gene,

hybridizing a first labeled complementary sequence to the first sequence and with a first labeled nucleotide of the first labeled complementary sequence hybridizing to the first nucleotide type,

hybridizing a first complementary sequence to the first sequence and adjacent to the first labeled complementary sequence,

ligating the first labeled complementary sequence to the first sequence,

identifying a second nucleotide type that is a single nucleotide polymorphism within a second sequence within the second gene,

hybridizing a second labeled complementary sequence to the second sequence and with a second labeled nucleotide of the second labeled complementary sequence hybridizing to the second nucleotide type,

hybridizing a second complementary sequence to the second sequence and adjacent to the second labeled complementary sequence,

ligating the second labeled complementary sequence to the second sequence,

wherein the first gene is differentially labeled from the second gene.

19. The method of claim 18 wherein the first labeled nucleotide is a first modified nucleotide and the second labeled nucleotide is a second modified nucleotide.

20. The method of claim 18 wherein the first labeled nucleotide is a first modified nucleotide and the second labeled nucleotide is a second modified nucleotide, wherein the first modified nucleotide is only extendable by first modified nucleotides of the same type and wherein the second modified nucleotide is only extendable by second modified nucleotides of the same type.