Oligonucleotide tagged nucleoside triphosphates (OTNTPs) for genetic analysis

• US 20040038215A1
• Filed: 08/23/2002
• Published: 02/26/2004
• Est. Priority Date: 08/23/2002
• Status: Active Grant

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Inventors: Nelson, John, Sood, Anup, Kumar, Shiv, Rao, Sudhakar
• Granted Patent: US 7,109,316 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom

Abstract

Oligonucleotide tagged nucleoside triphosphates, OTNTPs, which are substrates for polymerases and or terminal nucleotidyl transferases are provided as well as methods of making these OTNTPs. Further provided are OTNTPs with fluorescent dyes including energy transfer dyes, attached to the oligonucleotide chain, OTNTPs with unnatural bases incorporated in the oligonucleotide sequence and methods for incorporating these OTNTPs in DNA or RNA. Also provided are methods for using the oligonucleotides on OTNTPs for amplifying the oligo sequence on the OTNTP using an amplification method described above. Further provided are reactive bifunctional amidites, methods of making these compounds and methods for detecting single nucleotide polymorphisms using the above OTNTPs. Methods for detecting differential gene expression using the OTNTPs and methods of separating specifically modified DNA or RNA using the OTNTPs are also provided.

Claims

1. An OTNTP comprising the structure

2. The compound according to claim 1 wherein n is selected from 10-100.

3. The compound according to claim 1 wherein n is selected from 2-25.

4. The compound according to claim 1 wherein one or more of the N'"'"'s is labeled with a fluorescent dye.

5. The compound according to claim 1 wherein one of the N'"'"'s is coupled to a donor dye and another N is coupled to an acceptor dye and there is energy transfer between the donor and acceptor when the donor dye is excited.

6. The compound according to claim 1, wherein both R and R′

are H.

10. A method of genetic analysis comprising:

a) mixing a sample of DNA or RNA target with a sequence specific primer in an aqueous buffer;

b) adding a nucleic acid polymerase, a set of four OTNTP terminators, of the structure according to claim 1, bearing unique oligonucleotide sequences to form a mixture;

c) incubating said mixture at a temperature suitable for polymerase activity for a time sufficient to incorporate an OTNTP; and

d) detecting the OTNTP incorporated by hybridization with a labeled probe.

11. The method of claim 10 wherein said sample is an amplified sample.

12. The method according to claim 10 further comprising:

removing the unincorporated OTNTPs prior to the detection step.

13. A method of genetic analysis comprising:

a) mixing a sample of DNA or RNA target with a sequence specific primer in an aqueous buffer;

b) adding a nucleic acid polymerase, a set of four OTNTP terminators, of the structure according to claim 1, bearing a detectable label to form a mixture;

c) incubating said mixture at a temperature suitable for polymerase activity for a time sufficient to incorporate an OTNTP; and

d) detecting the OTNTP incorporated by detecting the label attached to OTNTP.

14. The method of claim 13 wherein said sample is an amplified sample.

15. The method of genetic analysis according to claim 13 wherein said label is a fluorescent moiety.

16. The method according to claim 13 further comprising:

removing the unincorporated OTNTPs prior to the detection step.

17. A method of genetic analysis comprising:

a) mixing a sample of DNA or RNA target with a sequence specific primer in an aqueous buffer;

b) adding a nucleic acid polymerase, a set of four OTNTP terminators, of the structure according to claim 1, bearing a donor and acceptor dye at a distance optimal for energy transfer, each acceptor dye being specific to a particular base and emitting at different wavelengths than other acceptor dyes, to form a mixture;

c) incubating said mixture at a temperature suitable for polymerase activity for a time sufficient to incorporate an OTNTP; and

d) detecting the OTNTP incorporated by exciting the donor dye and measuring the emission from the acceptor dye.

19. The method according to claim 17 further comprising:

removing the unincorporated OTNTPs prior to the detection step.

20. A method of genetic analysis comprising:

a) mixing a DNA or RNA target with a sequence specific primer in an aqueous buffer;

b) adding a polymerase and a set of OTNTP terminators, of the structure according to claim 1, to form a mixture and incubating said mixture at a temperature suitable for polymerase activity for time sufficient to incorporate an OTNTP;

c) amplifying the OTNTP oligonucleotide sequence;

d) identifying the amplified sequence.

21. The method of claim 20 wherein said amplifying is preformed according to a known method selected from the group consisting of PCR, LCR, SDA, 3SR, Qβ

replicase and RCA.

22. The method according to claim 20 further comprising:

removing the unincorporated OTNTPs prior to the amplification step.

23. A method of genetic analysis comprising:

a) mixing a DNA or RNA target with a sequence specific primer in an aqueous buffer;

b) adding a polymerase and a set of OTNTP terminators, of the structure according to claim 1, to form a mixture and incubating said mixture at a temperature suitable for polymerase activity for time sufficient to incorporate an OTNTP;

c) adding a set of 4 single stranded circular DNA each having a portion of the sequence, complementary to one of the four oligonucleotide sequences of the OTNTPs;

d) adding reagents required for RCA;

e) performing RCA by incubating at appropriate temperature for a time sufficient for amplification, and

f) detecting the signal by hybridizing the RCA product With sequence specific ampliflours or molecular beacons.

24. The method according to claim 23 further comprising:

removing the unincorporated OTNTPs prior to the amplification step.

25. A method of genetic analysis comprising:

a) mixing a DNA or RNA target with a sequence specific primer in an aqueous buffer;

b) adding a polymerase and a set of OTNTP terminators, of the structure according to claim 1, to form a mixture and incubating said mixture at a temperature suitable for polymerase activity for a time sufficient to incorporate a terminator;

c) separating unincorporated terminators from the extended primer;

d) adding a set of 4 single stranded circular DNA each having a sequence different from others and in such a manner that the ratio of G, A, T and C for each sequence is substantially different from the others, and at the same time part of the sequence being complementary to one of the four oligonucleotide sequences of the OTNTPs;

e) adding a DNA polymerase suitable for RCA, a set of 4 gamma labeled dNTP'"'"'s and other reagents required for RCA and a phosphatase that is functional under RCA conditions;

f) performing RCA by incubating at an appropriate temperature for a time sufficient for amplification or until all the nucleotides are consumed; and

g) measuring the ratio of dyes released to identify the sequence amplified.

26. A method of genetic analysis comprising:

a) mixing a DNA or RNA target with a sequence specific primer in an aqueous buffer;

b) adding a polymerase and a set of OTNTP terminators, of the structure according to claim 1, where oligonucleotide on each OTNTP is comprised of only three of the four natural bases and the missing base is different for each OTNTP &

incubating the sample at a temperature suitable for polymerase activity for time sufficient to incorporate a terminator;

c) removing unincorporated terminators from the extended primer;

d) adding a set of 4 single stranded circular DNA each having a sequence comprising only 3 of the four natural bases and at the same time part of the sequence being complementary to one of the four oligonucleotide sequences of the OTNTPs;

e) adding a DNA polymerase suitable for RCA, a set of 4 gamma labeled dNTP'"'"'s and other reagents required for RCA and a phosphatase that is functional under the RCA conditions;

f) performing RCA by incubating at an appropriate temperature for a time sufficient for amplification; and

g) measuring the ratio or identity of dyes released, or the ratio and identity of the dyes released, to identify the sequence amplified.

27. The method according to claim 25, wherein each of said oligonucleotide sequences of the OTNTPs and said single stranded circular DNA is composed of one to two of the four natural bases or analogs thereof and the base or the combination of two bases for each set of OTNTP &

single stranded circle is different from other sets of OTNTP &

single stranded circular DNAs.

28. A method of incorporating an OTNTP of the structure according to claim 1, into DNA or RNA using a terminal nucleosidyl transferase, said method comprising:

a) mixing a biological sample containing DNA or RNA or both with an OTNTP and terminal nucleotidyl transferase in a buffer to form a mixture;

b) incubating the mixture at temperature suitable for terminal nucleotidyl transferase activity.

29. A method of incorporating an OTNTP of the structure according to claim 1, into DNA using a DNA polymerase or reverse transcriptase said method comprising:

a) mixing a DNA or RNA template and a primer in an aqueous buffer,

b) adding a DNA polymerase or reverse transcriptase and one or more OTNTP'"'"'s of current invention, and

c) incubating the mixture at a temperature suitable for DNA polymerase or reverse transcriptase activity

30. A method of making an OTNTP of the structure according to claim 1,said method comprising:

a) synthesizing the desired oligonucleotide sequence on a solid support using DNA synthesis using fast deprotecting protecting groups on DNA bases;

b) adding a reactive group to the oligonucleotide using a reactive bifunctional amidite according to any one of claims 7, 8 or 9;

c) incubating the solid support with a functionalized NTP where the functional group is capable of reacting with the second reactive functional group of the bifunctional amidite;

d) cleaving the oligonucleotide from support and deprotecting the bases.

31. A method of making an OTNTP, of the structure according to claim 1, said method comprising:

a) Synthesizing an oligonucleotide of desired sequence on a solid support and attaching a protected amino or thiol modified linker to the 5′

-position of the oligonucleotide or haing a phosphorothioate backbone on the 5′

-end;

b) Cleaving the oligo from the support and deprotecting the bases and the 5′

-amino or thiol group; and

c) Reacting the functionalized oligo with a NTP modified to contain a maleimide, epoxide or NHS ester on the base.

32. A method of incorporating at least one OTNTP, of the structure according to claim 1, into RNA using an RNA polymersase or primase comprising:

a) mixing a DNA template and RNA polymerase or primase in an aqueous buffer;

b) adding at least one of said OTNTP'"'"'s; and

c) incubating the mixture at a temperature suitable for RNA polymerase or primase activity.

33. A method of separating a specific DNA or RNA sequence by

a) incorporating an OTNTP carrying a specific oligonucleotide sequence as an affinity tag into DNA or RNA by the method according to any one of claims 28, 29 or 32;

and

b) incubating with a solid support carrying the complementary oligonucleotide sequence to capture the tagged DNA or RNA; and

c) removing the uncaptured materials by washing.

34. The method of claim 33 further comprising releasing the captured DNA or RNA by washing the captured DNA or RNA with a denaturing buffer.

35. The method according to claim 33, wherein after incorporation, the unincorporated OTNTPs are separated prior to using the affinity tag.

36. A method of quantifying differential gene expression in two different tissues said method comprising:

a) separately making cDNA copies of messages by the method according to claim 29, in two tissue samples using at least one of the OTNTP where the OTNTP has a different oligo tag specific to the each tissue sample;

b) mixing the two samples and hybridizing the cDNA to a DNA or oligonucleotide chip; and

c) detecting the site; and

d) quantifying the amount of each oligonucleotide tag.

7. A compound of the structure

18. The method of claim 7 wherein said sample is an amplified sample.

8. A compound of the structure

9. A compound of the structure