Alternative dye-labeled primers for automated DNA sequencing

• US 5,861,287 A
• Filed: 10/06/1995
• Issued: 01/19/1999
• 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: Gibbs, Richard A., Metzker, Michael L.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/540,228
• Time in Patent Office: 1,201 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/91.2, 536/24.3-24.33
• US Class Current: 435/91.1
• 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 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. The present invention provides methods for the use of BODIPY® fluorophore-labeled DNA for dye-primer sequencing in which the BODIPY®s are attached to the 5'"'"' end of sequencing primers. 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 DNA sequencing requiring electrophoresis of products of the sequencing reactions, the method comprising the steps of:

forming a first, a second, a third, and a fourth class of polynucleotides by extending from primers a plurality of polynucleotides by means of a DNA polymerase or a reverse transcriptase in the presence of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate, wherein said first class of polynucleotides has a 3'"'"'-terminal dideoxyadenosine and is labeled at a 5'"'"' position with a first fluorophore;

said second class of polynucleotides has a 3'"'"'-terminal dideoxycytidine and is labeled at a 5'"'"' position with a second fluorophore;

each polynucleotide in the third class said third class of polynucleotides has a 3'"'"'-terminal dideoxyguanosine and is labeled at a 5'"'"' position with a third fluorophore; and

said fourth class of polynucleotides has a 3'"'"'-terminal dideoxythymidine and is labeled at a 5'"'"' position with a fourth fluorophore;

wherein at least one of said fluorophores is a 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®

) fluorophore, wherein at least one other fluorophore is used, and, wherein if said first, second, third and fourth fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane, and 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 having a wavelength 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.

4. The method of claims 1, 2, or 3, 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 claims 1, 2, or 3, wherein said BODIPY®

fluorophores have adsorption maxima of about 480 to 650, and an emission maxima of about 480 to 650.

7. The method of claims 1, 2, or 3, wherein said DNA polymerase is selected from the group of Thermosequenase, Klenow fragment, SEQUENASE®

DNA polymerase, Bst DNA polymerase, AMPLITAQ®

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

8. The method of claim 1, 2, or 3, wherein said BODIPY®

fluorophores are coupled to a primer suitable for sequencing by a linker.

9. The method of claim 8, wherein said linker has the formula (CH₂)_n, where n=1-30.

10. The method of claim 9, wherein said linkers are selected from the group of (CH₂)₃, (CH₂)₆, and (CH₂)₁₂.

11. The method of claim 1, 2, or 3, wherein said BODIPY®

fluorophore is attached at the 5'"'"' end of the products of the sequencing reaction and an additional fluorophore is attached at a 3'"'"' position of the product of the sequencing reaction or at one or more internal positions of the products of the sequencing reaction.

12. The method of claim 11, wherein said additional fluorophore is a BODIPY®

fluorophore.

13. The method of claim 11, wherein said additional fluorophore is FAM.

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

forming a first, a second, a third, and a fourth class of polynucleotides by extending from primers a plurality of polynucleotides by means of a DNA polymerase or a reverse transcriptase in the presence of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate, wherein said first class of polynucleotides has a 3'"'"'-terminal dideoxyadenosine and is labeled at a 5'"'"' position with BODIPY®

523/547, BODIPY®

530/550 or 2'"'"',7'"'"'-dimethoxy-4'"'"', 5'"'"'-dichloro-6-carboxy-fluorescein (JOE);

said second class of polynucleotides has a 3'"'"'-terminal dideoxycytidine and is labeled at a 5'"'"' position with BODIPY®

503/512 or 5-carboxy-fluorescein (FAM);

said third class of polynucleotides has a 3'"'"'-terminal dideoxyguanosine and is labeled at a 5'"'"' position with BODIPY®

558/568, BODIPY®

564/570 or N,N,N'"'"',N'"'"'-tetramethyl-6-carboxy-rhodamine (TAMRA); and

said fourth class of polynucleotides has a 3'"'"'-terminal dideoxythymidine and is labeled at a 5'"'"' position with BODIPY®

576/589, BODIPY®

581/591, BODIPY®

589/616, or 6-carboxy-X-rhodamine (ROX);

wherein at least one of said classes is labeled with a BODIPY®

fluorophore;

electrophoretically separating on a gel by size the polynucleotides;

illuminating with an illumination beam bands on the gel, the illumination beam having a wavelength 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.

6. The method of claims 2 or 3, wherein said chain termination method of DNA sequencing is performed by an automated DNA sequencing instrument.

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

forming a first, a second, a third, and a fourth class of polynucleotides by extending from primers a plurality of polynucleotides by means of a DNA polymerase or a reverse transcriptase in the presence of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate, wherein said first class of polynucleotides has a 3'"'"'-terminal dideoxyadenosine and is labeled at a 5'"'"' position with a first BODIPY®

fluorophore;

second class of polynucleotides has a 3'"'"'-terminal dideoxycytidine and is labeled at a 5'"'"' position with a second BODIPY®

fluorophore;

said third class of polynucleotides has a 3'"'"'-terminal dideoxyguanosine and is labeled at a 5'"'"' position with a third BODIPY®

fluorophore; and

fourth class of polynucleotides has a 3'"'"'-terminal dideoxythymidine and is labeled at a 5'"'"' position with a fourth BODIPY®

fluorophore;

wherein said first, second, third and fourth BODIPY®

fluorophores are all different;

electrophoretically separating on a gel by size the polynucleotides;

illuminating with an illumination beam bands of said gel, said illumination beam having a wavelength capable of causing said BODIPY®

fluorophores to fluoresce; and

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

14. The method of claim 14, wherein said additional fluorophore has an adsorption maxima of about 475 to 650.

15. The method of claim 14 wherein said additional fluorophore is BODIPY®

523/547.

16. The method of claim 14, wherein said additional fluorophore is BODIPY®

503/512.

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

forming a first, a second, a third, and a fourth class of polynucleotides by extending from primers a plurality of polynucleotides by means of a DNA polymerase or a reverse transcriptase in the presence of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate, wherein each polynucleotide in the first class has a 3'"'"'-terminal dideoxyadenosine and is labeled at a 5'"'"' position with BODIPY®

523/547 or BODIPY®

530/550;

each polynucleotide in the second class has a 3'"'"'-terminal dideoxycytidine and is labeled at a 5'"'"' position with BODIPY®

503/512;

each polynucleotide in the third class has a 3'"'"'-terminal dideoxyguanosine and is labeled at a 5'"'"' position with BODIPY®

564/570; and

each polynucleotide in the fourth class has a 3'"'"'-terminal dideoxythymidine and is labeled at a 5'"'"' position with BODIPY®

581/591 or BODIPY®

576/589;

electrophoretically separating on a gel by size the polynucleotides;

illuminating with an illumination beam bands on the gel, the illumination beam having a wavelength 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.

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

forming a first, a second, a third, and a fourth class of polynucleotides by extending from primers a plurality of polynucleotides by means of a DNA polymerase or a reverse transcriptase in the presence of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate, wherein each polynucleotide in the first class has a 3'"'"'-terminal dideoxyadenosine and is labeled at a 5'"'"' position with BODIPY®

523/547 or BODIPY®

530/550;

each polynucleotide in the second class has a 3'"'"'-terminal dideoxycytidine and is labeled at a 5'"'"' position with BODIPY®

581/591 or BODIPY®

576/589;

each polynucleotide in the third class has a 3'"'"'-terminal dideoxyguanosine and is labeled at a 5'"'"' position with BODIPY®

503/512; and

each polynucleotide in the fourth class has a 3'"'"'-terminal dideoxythymidine and is labeled at a 5'"'"' position with BODIPY®

564/570;

electrophoretically separating on a gel by size the polynucleotides;

illuminating with an illumination beam bands on the gel, the illumination beam having a wavelength 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.

19. A substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®

) compound as an amidite.