Charge tags and the separation of nucleic acid molecules

• US 7,678,542 B2
• Filed: 06/03/2008
• Issued: 03/16/2010
• 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: Prudent, James R., Neri, Bruce P., Hall, Jeff G., Skrzypczynski, Zbigniev, Lyamichev, Victor, Takova, Tsetska Y., Allawi, Hatim T., Wayland, Sarah R.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/132,198
• Publication Number:
  US 20090078574A1
• Time in Patent Office: 651 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/25.3, 536/26.6
• US Class Current: 435/6.1
• 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 method of separating oligonucleotides, comprising the steps of:

a) treating a charge-balanced oligonucleotide containing a charge tag under conditions such that a charge-unbalanced oligonucleotide containing said charge tag is produced, wherein said charge-unbalanced oligonucleotide is contained in a reaction mixture; and

b) separating said charge-unbalanced oligonucleotide from said reaction mixture.

2. The method of claim 1, wherein said conditions comprise treating said charge-balanced oligonucleotide with a reactant, wherein said reactant is selected from the group consisting of an enzyme, a chemical and a device.

3. The method of claim 1, wherein said charge tag is attached to a terminal end of said oligonucleotide, said charge tag attached to a terminal end comprising a phosphate group and a positively charged moiety.

4. The method of claim 1, wherein said charge tag comprises a dye.

5. The method of claim 4, wherein said dye is positioned between said oligonucleotide and said positively charged moiety.

6. The method of claim 4, wherein said positively charged moiety is positioned between said oligonucleotide and said dye.

7. The method of claim 3, wherein said charge tag further comprises a second positively charged moiety.

8. The method of claim 1, wherein said charge tag comprises one or more nucleotides.

9. The method of claim 8, wherein said oligonucleotide comprises a sequence complementary to a target nucleic acid, wherein said one or more nucleotides of said charge tag are not complementary to said target nucleic acid.

10. The method of claim 3, wherein said oligonucleotide 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.

11. The method of claim 1, wherein said charge-balanced oligonucleotide has a net neutral charge and wherein said charge-unbalanced oligonucleotide has a net positive charge.

12. The method of claim 1, wherein said charge-balanced oligonucleotide has a net negative charge and wherein said charge-unbalanced oligonucleotide has a net positive charge.

13. The method of claim 1, wherein said charge tag contains a primary amine.

14. The method of claim 1, wherein said charge tag contains a secondary amine.

15. The method of claim 1, wherein said charge tag contains a tertiary amine.

16. The method of claim 1, wherein said charge tag contains an ammonium group.

17. The method of claim 1, wherein said charge tag comprises a a positively charged moiety added by coupling of a positively charged phosphoramidite.

18. The method of claim 1, wherein said charge tag comprises a a neutral moiety added by coupling of a neutral phosphoramidite.

19. The method of claim 1, wherein said separating comprises capillary electrophoretic separation.

20. The method of claim 1, wherein said separating comprises capillary zone electrophoretic separation.

21. The method of claim 1, wherein said separating occurs in a microchannel.