Method for rapid base sequencing in DNA and RNA

• US 4,962,037 A
• Filed: 10/07/1987
• Issued: 10/09/1990
• Est. Priority Date: 10/07/1987
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Government of the United States of America
• Inventors: Stewart, Carleton C., Ratliff, Robert L., Martin, John C., Keller, Richard A., Moyzis, Robert K., Jett, James H., Shera, E. Brooks
• Primary Examiner(s): YARBROUGH, AMELIA
• Application Number:
  US07/105,375
• Time in Patent Office: 1,098 Days
• Field of Search: 435/6, 435/41, 435/75, 436/501, 436/94, 436/175, 436/800, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/626 being a flow cytometer
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials

Abstract

A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

Claims

1. A method for DNA and RNA base sequencing, comprising the steps of:

isolating a single fragment of DNA or RNA;

introducing said single fragment into a moving sample stream;

sequentially cleaving the end base from the DNA or RNA fragment with exonuclease to form a train of said bases; and

detecting said bases in said train in sequential passage through a detector which detects single molecules.

2. A method according to claim 1, wherein each said base of said single fragment is modified to contain a tag having an identifiable characteristic for said base.

3. A method according to claim 2, where said bases are modified prior to said cleavage.

4. A method according to claim 2 further including the step of enzymatically synthesizing a strand of DNA complementary to a DNA or RNA strand to be characterized, where each nucleotide forming said synthesized strand contains a tag characteristic of that nucleotide.

5. A method according to claim 2, wherein said tag is separated from the nucleotide by a linker arm effective for said cleavage.

6. A method according to claim 1, wherein said cleaved bases are detected optically.

7. A method according to claim 6, wherein each said tag is a fluorescent dye characteristic of one type of said nucleotides.

8. A method according to claim 7, further including the step of exciting each said fluorescent dye and detecting the fluorescence spectrum of said dye.

9. A method according to claim 1 wherein said step of isolating said single fragment of DNA or RNA includes the step of hybridizing said fragment to a substrate having a site effective for said hybridization.

10. A method according to claim 9, further including the step of selecting said DNA or RNA fragments from a heterogeneous collection of DNA or RNA fragments wherein said site is a biotinylated probe effective to hybridize with DNA or RNA fragments to be selected.

11. A method according to claim 9, wherein said isolating said single fragment includes the step of providing said substrate with a single site effective to hybridize with a single DNA fragment.

12. A method for base sequencing of DNA or RNA fragments, comprising the steps of:

forming said fragments with bases having identifiable characteristics;

sequentially cleaving single identifiable bases from a single one of said fragments to form a train of said identifiable bases; and

identifying said single, cleaved bases in said train.

13. A method according to claim 12, further including the step of attaching a characteristic identifiable fluorescent dye to each said base.

14. A method according to claim 12, wherein the steps of forming said fragments includes the steps of forming by enzymatic synthesis a complementary strand of said DNA or RNA to be sequenced from said bases having identifiable characteristics and thereafter base sequencing said complementary strand.

15. A method according to claim 14, further including the step of attaching a characteristic identifiable fluorescent dye to each said base.

16. A method according to claim 13, wherein said step of identifying said single, cleaved bases includes the step of exciting each said fluorescent dye and detecting the fluorescence spectrum of said dye.

17. A method according to claim 15, wherein said step of identifying said single, cleaved bases includes the step of exciting each said fluorescent dye and detecting the fluorescence spectrum of said dye.

18. A method for DNA or RNA base sequencing, comprising the steps of;

modifying each nucleotide effective for DNA or RNA synthesis to attach a fluorescent dye characteristic of that nucleotide with a linker arm effective to enable DNA or RNA synthesis and exonuclease cleavage;

synthesizing from said modified nucleotides a strand of DNA complementary to a DNA or RNA strand having a base sequence to be determined;

cleaving each said modified nucleotide sequentially from a single fragment containing said complementary DNA strand; and

fluorescing each said characteristic dye to identify said sequence of nucleotides.

19. A method according to claim 18, wherein the step of fluorescing said dyes further comprises the steps of:

exciting each said modified nucleotide with a laser effective to fluoresce said characteristic dye; and

detecting said fluorescence to sequentially identify said nucleotides and generate said sequence of said DNA or RNA.

20. A method according to claim 18, further including the step of suspending a single fragment of synthesized DNA or RNA strand in a laminar flow stream.

21. A method according to claim 18, wherein each synthesized DNA or RNA fragment is manipulated by hybridizing said fragment to a microsphere having a site effective for hybridization.