Method for analyzing polynucleotides

• US 6,440,705 B1
• Filed: 09/10/1999
• Issued: 08/27/2002
• Est. Priority Date: 10/01/1998
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Nuvelo, Inc. (Arca Biopharma, Inc.)
• Inventors: Verdine, Gregory L., Stanton, Vincent P. Jr., Wolfe, Jia Liu, Kawate, Tomohiko
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/394,457
• Time in Patent Office: 1,082 Days
• Field of Search: 935/6, 935/91.1, 935/91.2, 935/183, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention relates to methods for the analysis of polynucleotides including detection of variance in nucleotide sequence without the need for full sequence determination, full sequence determination of a polynucleotide, genotyping of DNA and labeling a polynucleotide fragment during the process of cleaving it into fragments.

Claims

1. A method for cleaving a polynucleotide, comprising:

a. replacing a natural A, C, G or T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide;

b. contacting the modified polynucleotide with a reagent or reagents that cleave(s) it at greater than 90% of the points of occurrence of the modified nucleotide to give a set of fragments.

2. The method of claim 1, whereby variance in nucleotide sequence among related polynucleotides is detected, further comprising,

c. determining the masses of the fragments obtained from step b; and,

d. comparing the masses of the fragments with the masses of fragments expected from cleavage of a related polynucleotide of known sequence, or

e. repeating steps a-c with one or more related polynucleotides of unknown sequence and comparing the masses of the fragments of the polynucleotide with the masses of fragments obtained from the related polynucleotides.

3. The method of claim 1, whereby a nucleotide sequence of a polynucleotide is determined, further comprising

c. determining the mass of each fragment of the set of fragments;

d. repeating steps a, b and c until each different natural A, C, G, T or U nucleotide in the polynucleotide has been replaced with a modified A, C, G, T or U nucleotide, respectively, each modified polynucleotide has been cleaved to give a set of fragments and the mass of each fragment of each set of fragments had been determined; and

,

e. constructing a nucleotide sequence of the polynucleotide from the masses of all the fragments.

4. The method of claim 1, wherein:

the replaced natural A, C, G, T or U nucleotide is involved in a polymorphism or mutation in the polynucleotide; and

,

the set of fragments obtained after cleavage is analyzed to determine the presence or absence of the polymorphism or mutation.

5. The method of claim 4, wherein analyzing the fragments comprises using electrophoresis, mass spectrometry or FRET detection.

20. The method of any one of claims 1, 6, 8, 10, 14, 15, 16, or 17, wherein natural nucleotides not being replaced with modified nucleotides are replaced with mass-modified nucleotides.

21. The method of any one of claims 1, 6, 8, 10, 14, 15, 16, or 17, wherein the polynucleotide is DNA or RNA.

22. The method of any of claims 1, 6, 8, 10, 14, 15, 16, or 17, wherein detecting the mass of each fragment comprises using a mass spectrometer.

23. The method of claim 22 wherein the mass spectrometer is an electrospray ionization mass spectrometer.

24. The method of claim 22 wherein the mass spectrometer is a matrix assisted laser desorption/ionization mass spectrometer.

31. The method of any one of claims 1, 6, 8, 10, 14, 15, 16, or 17, wherein:

the percentage replacement of a natural A, C, G, T or U nucleotide with a modified A, C, G, T or U nucleotide, the percentage cleavage of a modified A, C, G, T or U nucleotide or both the percentage replacement and the percentage cleavage is greater than 95%.

32. The method of any one of claims 1, 6, 8, 10, 14, 15, 16, or 17, wherein:

the percentage replacement of a natural A, C, G, T or U nucleotide with a modified A, C, G, T or U nucleotide, the percentage cleavage of a modified A, C, G, T or U nucleotide or both the percentage replacement and the percentage cleavage is greater than 99%.

6. A method for cleaving a polynucleotide, comprising:

a. replacing a first natural A, C, G or T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of its points of occurrence in the polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a once-modified polynucleotide;

b. replacing a second, different natural A, C, G, T, or U nucleotide at greater than 90% of its points of occurrence in the once-modified polynucleotide with a second, different modified A, C, G, T or U nucleotide, respectively, to form a twice-modified polynucleotide; and

,

c. contacting the twice-modified polynucleotide with a reagent or reagents that cleaves it at each point where the first modified nucleotide is followed immediately in sequence by the second modified nucleotide.

7. The method of claim 6, further comprising:

d. determining a mass of each fragment of the set of fragments;

e. comparing the mass of each fragment of the set of fragments with a mass of each fragment expected from cleavage of a related polynucleotide of known sequence, or

f. repeating steps a-d with one or more related polynucleotides of unknown sequence and comparing the mass of each fragment of the set of fragments with the mass of each fragment obtained from cleavage of each related polynucleotide.

8. A method for detecting variance in nucleotide sequence among related polynucleotides, comprising:

a. replacing three of four different natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at greater than 90% of their points of occurrence in a polynucleotide with three stabilizing modified A, C, G, T or U nucleotides, respectively, to form a modified polynucleotide having one remaining natural nucleotide;

b. cleaving the modified polynucleotide at greater than 90% of the points of occurrence of the remaining natural nucleotide to give a set of fragments;

c. determining a mass of each fragment of the set of fragments; and

,

d. comparing the mass of each fragment of the set of fragments with the mass of each fragment expected from cleavage of a related polynucleotide of known sequence, or

e. repeating steps a-c with one or more related polynucleotides of unknown sequence and comparing the mass of each fragment of the set of fragments with the mass of each fragment obtained from cleavage of each related polynucleotide.

9. The method of claim 8, further comprising replacing the remaining natural nucleotide with a destabilizing modified nucleotide.

10. A method for detecting variance in nucleotide sequence among related polynucleotides, comprising:

a. replacing two different natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T), nucleotides at greater than 90% of their points of occurrence in a polynucleotide with two modified A, C, G, T or U nucleotides, respectively, each having different cleaving characteristics from the others, to give a modified polynucleotide;

b. cleaving the modified polynucleotide at greater than 90% of the points of occurrence of the first modified nucleotide to give a first set of fragments;

c. determining a mass of each fragment of the first set of fragments;

d. cleaving the first set of fragments into a second set of fragments at greater than 90% of the points of occurrence in the first set of fragments of the second modified nucleotide;

e. determining a mass of each fragment of the second set of fragments; and

,

f. comparing the mass of each fragment of the first set of fragments and the mass of each fragment of the second set of fragments with a mass of each fragment of first and second sets of fragments expected from cleavage of a related polynucleotide of known sequence, or

g. repeating steps a-d with one or more related polynucleotides of unknown sequence and comparing the mass of each fragment of the first set of fragments and the mass of each fragment of the second set of fragments with a mass of each fragment obtained from first and second cleavages of each related polynucleotide.

11. The method of claim 10, wherein the steps are repeated using a modified polynucleotide obtained by replacing different pairs of natural A, C, G, T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides with modified A, C, G, T or U nucleotides, respectively;

that is, replacing A and C with modified A and C, A and G with modified A and G, A and T (or U) with modified A and T (or U), C and G with modified C and G, C and T (or U) with modified C and T (or U) and G and T (or U) with modified G and T (or U).

12. The method of claim 10, wherein detaining the modified polynucleotide and the first set of fragments comprises using a mass spectrometer.

13. The method of claim 12, wherein the mass spectrometer is a tandem mass spectrometer.

14. A method for determining a nucleotide sequence of a polynucleotide, comprising:

a. replacing a natural A, C, G, or T (or, if the polynucleotide is a ribonucleotide, U instead of T) nucleotide at a percentage of points of occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide;

b. cleaving the modified polynucleotide at greater than 90% of the points of occurrence of the modified nucleotide to give a set of fragments;

c. repeating steps a and b until each different natural A, C, G, T or U nucleotide in the polynucleotide has been replaced with a modified A, C, G, T or U nucleotide, respectively;

d. determining a mass of each fragment obtained from each cleavage; and

,

e. constructing the nucleotide sequence of the polynucleotide from the masses of all the fragments, or,

f. generating a sequence ladder from all the fragments obtained in step c; and

,

g. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.

25. The method of any one of claims 14, 15, or 16 wherein generating a sequence ladder comprises gel electrophoresis.

15. A method for determining nucleotide sequence in a polynucleotide, comprising:

a. replacing a natural A, C, G, T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of the points of its occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide;

b. cleaving the modified polynucleotide at a percentage of points of occurrence of the modified A, C, G, T or U nucleotide to give a first set of fragments;

c. repeating steps a and b until each different natural A, C, G,T or U nucleotide in the polynucleotide has been replaced with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide and each modified polynucleotide has been cleaved to give a set of fragments;

d. determining a mass of each fragment of each set of fragments from each cleavage reaction; and

,

e. constructing the nucleotide sequence of the polynucleotide from all the masses;

or,

f. generating a sequence ladder from all the fragments obtained in step c; and

g. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.

16. A method for determining a nucleotide sequence of a polynucleotide, comprising:

a. replacing A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at greater than 90% of each of their points of occurrence in a polynucleotide with four modified A, C, G, T or U nucleotides, respectively, each of which has a different cleaving characteristic from the others, to form a modified polynucleotide;

b. separating the modified polynucleotide into four aliquots;

c. cleaving the modified polynucleotide in each aliquot at greater than 90% of the points of occurrence of a different one of the modified nucleotides such that each aliquot contains a set of fragments resulting from cleavage at a different modified A, C, G, T or U nucleotide;

d. determining a mass of each fragment of each set of fragments in each aliquot; and

,

e. constructing the nucleotide sequence of the polynucleotide from the masses of all the fragments;

or,

f. generating a sequence ladder from all the fragments obtained in step c; and

,

g. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.

17. A method for cleaving a polynucleotide during polymerization, comprising:

providing two or three natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides, and one or two modified A, C, G, T or U nucleotides, four nucleotides in all;

providing one or more polymerases, at least one of which produces or enhances cleavage during polymerization at points where the modified nucleotide is, or the modified nucleotides are, being incorporated into the polynucleotide;

mixing the four nucleotides with the polymerase or polymerases under polymerizing conditions; and

,

running the polymerization with concurrent cleavage.

18. The method of claim 17, wherein two modified nucleotides are used, one being a ribonucleotide and one being a 5′

-amino-2′

,5′

-dideoxynucleotide.

19. The method of claim 18, wherein two polymerases are used, one being Klenow (exo-) polymerase and one being mutant E710A Klenow (exo-) polymerase.

26. A method for cleaving a polynucleotide wherein fragments obtained from the cleavage are labeled, comprising:

a. replacing a natural A, C, G, T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide to form a modified polynucleotide;

b. contacting the modified polynucleotide with a labeled phosphine and a chemical base that cleaves the modified polynucleotide at greater than 90% of the points of occurrence of the modified nucleotide to give a set of fragments in which the labeled phosphine has covalently bonded to the 3′

position of sugar moieties of the modified nucleotides.

27. The method of claim 26, wherein the phosphine is tris(carboxyethyl) phosphine.

28. The method of claim 26, wherein the label is selected from the group consisting of a fluorescent tag and a radioactive tag.

29. A method for determining a nucleotide sequence of a polynucleotide, comprising:

a. replacing natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at greater than 90% of each of their points of occurrence in a polynucleotide with four modified A, C, G. T or U nucleotides, respectively, each of which has a different cleavage characteristic from the others, to form a modified polynucleotide;

b. separating the modified polynucleotide into four aliquots;

c. cleaving the modified polynucleotide in each aliquot at a percentage of points of occurrence of a different modified A, C, G, T or U nucleotide such that each aliquot comprises a set of fragments from cleavage at a different modified nucleotide;

d. creating a sequence ladder from all the fragments; and

,

e. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.

30. A method for determining a nucleotide sequence of a polynucleotide, comprising:

a. replacing natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at a percentage of points of occurrence in a polynucleotide with four modified A, C, G, T or U nucleotides, respectively, each of which has a different cleavage characteristic from the others to form a modified polynucleotide;

b. separating the modified polynucleotide into four aliquots;

c. cleaving the modified polynucleotide in each aliquot at greater than 90% of the points of occurrence of a different one of the modified nucleotides such that each aliquot comprises a set of fragments from cleavage at a different modified A, C, G, T or U nucleotide;

d. creating a sequence ladder from all the fragments; and

,

e. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.