Charge tags and the separation of nucleic acid molecules

• US 6,780,982 B2
• Filed: 02/06/2001
• Issued: 08/24/2004
• Est. Priority Date: 07/12/1996
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Allawi, Hatim T., Wayland, Sarah R., Takova, Tsetska, Skrzpczynski, Zbigniew, Neri, Bruce P., Lyamichev, Victor
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/777,430
• Publication Number:
  US 20020128465A1
• Time in Patent Office: 1,295 Days
• Field of Search: 536/23.1, 536/25.3, 536/26.6, 536/25.33, 536/25.34
• US Class Current: 536/23.1
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/59 Hydrogenated pyridine rings
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

The present invention relates to novel phosphoramidites, including positive and neutrally charged compounds. The present invention also provides charge tags for attachment to materials including solid supports and nucleic acids, wherein the charge tags increase or decrease the net charge of the material. The present invention further provides methods for separating and characterizing molecules based on the charge differentials between modified and unmodified materials.

Claims

1. A composition comprising a charge tag attached to a terminal end of a nucleic acid molecule, said charge tag comprising a phosphate group and a positively charged moiety, wherein said charge tag further comprises a dye, and wherein said dye is positioned between said nucleic acid and said positively charged moiety.

2. A composition comprising a charge tag attached to a terminal end of a nucleic acid molecule, said charge tag comprising a phosphate group and a positively charged moiety, wherein said charge tag further comprises a dye, and wherein said nucleic acid molecule comprises a sequence complementary to a target nucleic acid, and wherein said charge tag further comprises one or more nucleotides that are not complementary to said target nucleic acid.

19. The composition of claim 2, wherein said dye is positioned between said nucleic acid and said positively charged moiety.

20. The composition of claim 2, wherein said charge tag has a net positive charge of 1 at pH 6-10.

21. The composition of claim 2, wherein said charge tag has a net positive charge of 2 at pH 6-10.

22. The composition of claim 2, wherein said nucleic acid and said charge tag have a combined net neutral charge and wherein said charge tag has a net positive charge.

23. The composition of claim 2, wherein said nucleic acid and said charge tag have a combined net negative charge and wherein said charge tag has a net positive charge.

24. The composition of claim 2, wherein said charge tag contains a tertiary amine.

25. The composition of claim 2, wherein said charge tag contains an ammonium group.

3. A composition comprising a charge tag attached to a terminal end of a nucleic acid molecule, said charge tag comprising a phosphate group and a positively charged moiety, wherein said charge tag further comprises a dye, and wherein said nucleic acid comprises a first portion complementary to a target nucleic acid and a second portion that is not complementary to said target nucleic acid, wherein said second portion comprises said terminal end.

4. A composition comprising a charge tag attached to a terminal end of a nucleic acid molecule, said charge tag comprising a phosphate group and a positively charged moiety, wherein said charge tag further comprises a dye, and wherein said charge tag further comprises a positively charged phosphoramidite.

5. A composition comprising a positively charged phosphoramidite, wherein said phosphoramidite has the structure:

wherein, X is a reactive phosphate group and Y is a protected hydroxyl group.

6. A composition comprising a fluorescent dye directly bonded to a phosphate group, wherein said phosphate group is directly bonded to an amine group, wherein said composition comprises a charge tag, and wherein said fluorescent dye is contained within said charge tag, and wherein said fluorescent dye comprises Cy3.

7. A mixture comprising a plurality of oligonucleotides, each oligonucleotide attached to a different charge tag, each of said charge tags comprising a phosphate group and a positively charged moiety.

8. The mixture of claim 7, wherein said plurality of oligonucleotides comprises four or more oligonucleotides, each attached to a different charge tag.

9. The mixture of claim 7, wherein said plurality of oligonucleotides comprises ten or more oligonucleotides, each attached to a different charge tag.

10. The mixture of claim 7, wherein said plurality of oligonucleotides comprises twenty or more oligonucleotides, each attached to a different charge tag.

11. The mixture of claim 7, wherein said plurality of oligonucleotides comprises fifty or more oligonucleotides, each attached to a different charge tag.

12. The mixture of claim 7, wherein said charge tags comprise a dye.

13. The mixture of claim 7, wherein said charge tags comprise a second positively charged moiety.

14. The mixture of claim 7, wherein said charge tags further comprise one or more nucleotides.

15. The mixture of claim 7, wherein each of said oligonucleotides attached to a charge tag have a combined net neutral charge and wherein said charge tag has a net positive charge.

16. The mixture of claim 7, wherein each of said oligonucleotides attached to a charge tag have a combined net negative charge and wherein said charge tag has a net positive charge.

17. The mixture of claim 7, wherein said charge tags comprise a positively charged phosphoramidite.

18. The mixture of claim 7, wherein said charge tags comprise a neutral phosphoramidite.