Alternative dye-labeled ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides for automated DNA analysis

• US 5,728,529 A
• Filed: 11/06/1995
• Issued: 03/17/1998
• Est. Priority Date: 06/23/1995
• Status: Expired due to Term

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Metzker, Michael L., Gibbs, Richard A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/553,936
• Time in Patent Office: 862 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/91.2, 536/24.3, 536/25.32, 422/82.07, 422/82.08, 548/405, 252/700
• US Class Current: 435/6.13
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Methods for the use of a class of dyes for improved DNA sequencing by the chain termination method of DNA sequencing, and internal labelling of polynucleotides by enzymatic incorporation of fluorescently-labeled ribonucleotides or deoxyribonucleotides are provided. A new class of dyes, BODIPY® fluorophores, has been described recently. The parent heterocyclic molecule of the BODIPY® fluorophores is a dipyrrometheneboron difluoride compound which is modified to create a broad class of spectrally-discriminating fluorophores. BODIPY® fluorophores have improved spectral characteristics compared to conventional fluorescein and rhodamine dyes. BODIPY® fluorophores have narrower band width, insensitivity to solvent or pH, and improved photostability, thus, BODIPY® fluorophores lead to improved DNA sequencing and/or detection in any method where electrophoresis and detection of DNA is required. Additionally, the spectral properties of the BODIPY® fluorophores are sufficiently similar in wavelength and intensity to be used with conventional equipment known in the art.

Claims

1. A method for distinguishing polynucleotides having different 3'"'"'-terminal dideoxynucleotides in any method of chain termination DNA sequencing, said method comprising the steps of:

forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a 3'"'"'-terminal dideoxyadenosine triphosphate, said 3'"'"'-terminal dideoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

dipyrrometheneboron difluoride linker at a 3 position of a first BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the second class having a 3'"'"'-terminal dideoxycytidine triphosphate, said 3'"'"'-terminal dideoxycytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a second BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the third class having a 3'"'"'-terminal dideoxyguanosine triphosphate, said 3'"'"'-terminal dideoxyguanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a third BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the fourth class having a 3'"'"'-terminal dideoxythymidine triphosphate, said 3'"'"'-terminal dideoxythymidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a fourth BODIPY®

fluorophore that contains at least one reactive functional group;

wherein if said first, second, third and fourth BODIPY®

fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane;

or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;

electrophoretically separating on a gel by size the polynucleotides so as to produce bands on the gel;

illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and

identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

2. The method of claim 1, wherein the BODIPY®

fluorophores are selected from the group of BODIPY®

530/550;

BODIPY®

503/512;

BODIPY®

564/570;

BODIPY®

589/616;

BODIPY®

581/591;

BODIPY®

523/547;

BODIPY®

558/568; and

BODIPY®

576/589.

3. The method of claim 1, wherein said 3'"'"'-terminal dideoxyadenosine triphosphate is labeled with BODIPY®

523/547 or BODIPY®

530/550;

said 3'"'"'-terminal dideoxycytidine triphosphate is labeled with BODIPY®

576/589, BODIPY®

581/591, or BODIPY®

589/616;

said 3'"'"'-terminal dideoxyguanosine triphosphate is labeled with BODIPY®

503/512; and

said 3'"'"'-terminal dideoxythymidine triphosphate is labeled with BODIPY®

558/568 or BODIPY®

564/570.

4. The method of claim 1, wherein said BODIPY®

fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

5. The method of claim 1, wherein said chain termination method of DNA sequencing is performed by an automated DNA sequencing instrument.

6. The method of claim 1, wherein said classes of polynucleotides are formed using a DNA polymerase selected from the group of Klenow fragment, Sequenase®

, Bst DNA polymerase, AmpliTaq®

DNA polymerase, Pfu(exo-)DNA polymerase, Thermosequenase®

, rTth DNA polymerase or Vent(exo-) DNA polymerase, and the reverse transcriptase is selected from the group of AMV-RT or M-MuLV-RT.

7. A method for distinguishing polynucleotides having different ribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, said method comprising the steps of:

forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having an adenosine triphosphate, said adenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a first BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the second class having a cytidine triphosphate, said cytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a second BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the third class having a guanosine triphosphate, said guanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a third BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the fourth class having a uracil triphosphate, said uracil triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a fourth BODIPY®

fluorophore that contains at least one reactive functional group;

wherein if said first, second, third and fourth BODIPY®

fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane;

or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;

electrophoretically separating on a gel by size the polynucleotides;

illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and

identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

8. The method of claim 7, wherein the BODIPY®

fluorophores are selected from the group of BODIPY®

530/550;

BODIPY®

503/512;

BODIPY®

564/570;

BODIPY®

589/616;

BODIPY®

581/591;

BODIPY®

523/547;

BODIPY®

558/568; and

BODIPY®

576/589.

9. The method of claim 7, wherein said adenosine triphosphate is labeled with BODIPY®

523/547 or BODIPY®

530/550;

said cytidine triphosphate is labeled with BODIPY®

576/589, BODIPY®

581/591, or BODIPY®

589/616;

said guanosine triphosphate is labeled with BODIPY®

503/512; and

said uracil triphosphate is labeled with BODIPY®

558/568 or BODIPY®

564/570.

10. The method of claim 7, wherein said BODIPY®

fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

11. The method of claim 7, wherein said internal labelling and distinguishing polynucleotides is performed by an automated GeneScanner.

12. A method for distinguishing polynucleotides having different deoxyribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, said method comprising the steps of:

forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a deoxyadenosine triphosphate, said deoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-3-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a first BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the second class having a deoxycytidine triphosphate, said deoxycytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a second BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the third class having a deoxyguanosine triphosphate, said deoxyguanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-3-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a third BODIPY®

fluorophore that contains at least one reactive functional group;

each polynucleotide in the fourth class having a deoxythymidine triphosphate, said deoxythymidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®

linker at a 3 position of a fourth BODIPY®

fluorophore that contains at least one reactive functional group;

wherein if said first, second, third and fourth BODIPY®

fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane;

or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;

electrophoretically separating on a gel by size the polynucleotides;

illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and

identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

13. The method of claim 12, wherein the BODIPY®

fluorophores are selected from the group of BODIPY®

530/550;

BODIPY®

503/512;

BODIPY®

564/570;

BODIPY®

589/616;

BODIPY®

581/591;

BODIPY®

523/547;

BODIPY®

558/568; and

BODIPY®

576/589.

14. The method of claim 12, wherein said deoxyadenosine triphosphate is labeled with BODIPY®

523/547 or BODIPY®

530/550;

said deoxycytidine triphosphate is labeled with BODIPY®

576/589, BODIPY®

581/591, or BODIPY®

589/616;

said deoxyguanosine triphosphate is labeled with BODIPY®

503/512; and

said deoxythymidine triphosphate is labeled with BODIPY®

558/568 or BODIPY®

564/570.

15. The method of claim 12, wherein said BODIPY®

fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

16. The method of claim 12, wherein said internal labelling and distinguishing polynucleotides is performed by an automated GeneScanner.

17. The method of claim 12, wherein said classes of polynucleotides are formed using a DNA polymerase selected from the group of Klenow fragment, Sequenase®

, Bst DNA polymerase, AmpliTaq®

DNA polymerase, Pfu(exo-)DNA polymerase, Thermosequenase®

, rTth DNA polymerase or Vent(exo-) DNA polymerase, and the reverse transcriptase is selected from the group of AMV-RT or M-MuLV-RT.