Charge tags and the separation of nucleic acid molecules

• US 7,384,746 B2
• Filed: 06/23/2004
• Issued: 06/10/2008
• 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:
  US10/875,094
• Publication Number:
  US 20050130179A1
• Time in Patent Office: 1,448 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/24.33, 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 plurality of charge-balanced oligonucleotides, each containing a different charge tag, under conditions such that two or more charge-unbalanced oligonucleotides containing different charge tags are produced, wherein said charge-unbalanced oligonucleotides are contained in a reaction mixture; and

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

2. The method of claim 1, wherein said separating comprises separating said charge-unbalanced oligonucleotides such that charge-unbalanced oligonucleotides containing different charge tags are separated from one another.

3. The method of claim 1, wherein said plurality of charge-balanced oligonucleotides comprise four or more charge-balanced oligonucleotides comprising different charge tags.

4. The method of claim 1, wherein said plurality of charge-balanced oligonucleotides comprise ten or more charge-balanced oligonucleotides comprising different charge tags.

5. The method of claim 1, wherein said plurality of charge-balanced oligonucleotides comprise twenty or more charge-balanced oligonucleotides comprising different charge tags.

6. The method of claim 1, wherein said plurality of charge-balanced oligonucleotides comprise fifty or more charge-balanced oligonucleotides comprising different charge tags.

7. 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.

8. The method of claim 1, wherein one or more of said different charge tags is attached to a terminal end of one or more of said plurality of oligonucleotides, said one or more of said charge tags attached to a terminal end comprising a phosphate group and a positively charged moiety.

9. The method of claim 1, wherein one or more of said different charge tags comprises a dye.

10. The method of claim 1, wherein one or more of said different charge tags comprises a positively charged moiety added by coupling of a positively charged phosphoramidite.

11. The method of claim 1, wherein one or more of said different charge tags comprise a neutral moiety added by coupling of a neutral phosphoramidite.

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

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

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