Compositions and methods for polynucleotide sequence determination Number:6,803,201 from the United States Patent and Trademark Office (PTO) owispatent

Title: Compositions and methods for polynucleotide sequence determination

Abstract: The present invention relates to a method for identifying a nucleotide at a predetermined location on a target polynucleotide. The method involves single nucleotide extension reaction comprising an oligonucleotide primer comprising a first sequence and a second sequence or a tag. The method may further comprises a probe which hybridizes to the second sequence or an anti-tag molecule which interacts with the tag, where the hybridization or interaction causes a detectable signal transfer which is indicative of the identity of the nucleotide base at the predetermined location. The invention further provides compositions and kits for performing the subject method of the invention.

Patent Number: 6,803,201 Issued on 10/12/2004 to Sorge,   et al.

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Inventors:	Sorge; Joseph A. (Wilson, NY); Arezi; Bahram (Encinitas, CA); Hogrefe; Holly (San Diego, CA)
Assignee:	Stratagene (La Jolla, CA)
Appl. No.:	10/056,598
Filed:	January 24, 2002

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Current U.S. Class:	435/6 ; 435/91.1; 435/91.2; 536/23.1; 536/24.3; 536/24.32; 536/25.32; 536/26.6
Current International Class:	C12Q 1/68 (20060101)
Field of Search:	435/6,91.1,91.2,810,24.33 536/23.1,24.3,24.33,24.32,25.32,26.6,243

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References Cited [Referenced By]

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U.S. Patent Documents

	5888819		March 1999		Goelet et al.
	5945283		August 1999		Kwok et al.
	6013431		January 2000		Soderlund et al.
	6177249		January 2001		Kwok et al.
	6287778		September 2001		Huang et al.
	6316200		November 2001		Nadeau et al.

Foreign Patent Documents

	0 648 280		May., 1999		EP
	WO 01/32887		May., 2001		WO
	WO 01/32887		May., 2001		WO

Other References

International Search Report of International Application No. PCT/US03/02117..

Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Wilder; Cynthia B.
Attorney, Agent or Firm: Palmer & Dodge LLP Williams; Kathleen M.

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Claims

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What is claimed is:

1. A composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said composition comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to said target polynucleotide immediately 3' of said nucleotide, and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence; and (b) an oligonucleotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer, said oligonucleotide probe labeled with a first member of a pair of interactive labels.

2. The composition of claim 1, further comprising a first polynucleotide chain terminator, which is incorporated in a template-dependent manner into said oligonucleotide primer by a polynucleotide synthesis enzyme.

3. The composition of claim 2, further comprising one or more of a second, a third and/or a fourth polynucleotide chain terminator, wherein said first, second, third and fourth polynucleotide terminators are not identical.

4. The composition of claim 2, wherein said first polynucleotide chain terminator is labeled with a second member of said pair of interactive labels.

5. The composition of claim 4, wherein said first and second members of said pair of interactive labels interact with each other to generate a signal by fluorescent resonance energy transfer.

6. The composition of claim 1, further comprising a template-dependent polynucleotide synthesis enzyme for incorporating in a template-dependent manner a complementary polynucleotide chain terminator into said oligonucleotide primer.

7. The composition of claim 6, wherein said polynucleotide synthesis enzyme is a JDF-3DNA polymerase.

8. The composition of claim 2, wherein said oligonucleotide primer comprises a separation moiety that permits separation of said oligonucleotide primer and/or said oligonucleotide probe hybridized to said primer from unincorporated polynucleotide chain terminator, and oligonucleotide probe which is not hybridized to said oligonucleotide primer.

9. The composition of claim 8, further comprising a target moiety specific for said separation moiety, wherein said separation moiety binds to said target moiety to permit said separation.

10. The composition of claim 9, wherein said target moiety is attached to a solid support.

11. The composition of claim 4, wherein said first and second members of said pair of interactive labels are fluorescent molecules which interact with each other to generate a signal by fluorescent resonance energy transfer.

12. A composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said composition comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3' of said nucleotide, and is covalently attached to a tag molecule; and (b) an anti-tag molecule which binds to said tag molecule, said anti-tag molecule labeled with a first member of a pair of interactive labels.

13. The composition of claim 12, wherein said tag molecule is located on the 5' terminal of said oligonucleotide primer.

14. The composition of claim 13, wherein said tag molecule is a first member of a specific binding pair which comprises said first member and a second member.

15. The composition of claim 14, wherein said anti-tag molecule is said second member of said specific binding pair.

16. The composition of claim 15, wherein said specific binding pair is a biotin-streptavidin pair.

17. The composition of claim 1, wherein said second sequence is at the 5' terminal of said first sequence.

18. The composition of claim 1, further comprising a labeled conventional deoxynucleotide, and the other three unlabeled chain terminators, wherein said labeled conventional deoxynucleotide is incorporated into the oligonucleotide primer at a position corresponding to the predetermined nucleotide of the target polynucleotide.

19. The composition of claim 1, wherein one member of the pair of interactive labels is a quencher molecule.

20. A kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said kit comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to said target polynucleotide immediately 3' of said nucleotide, and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence; (b) an oligonucleotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer, said oligonucleotide probe labeled with a first member of a pair of interactive labels; and (c) packaging materials therefore.

21. The kit of claim 20, further comprising a polynucleotide chain terminator, which can be incorporated in a template-dependent manner into said oligonucleotide primer by a polynucleotide synthesis enzyme.

22. The kit of claim 21, further comprising one or more of a second, a third and/or a fourth polynucleotide chain terminator, wherein said first, second, third and fourth polynucleotide terminators are not identical.

23. The kit of claim 21, wherein said polynucleotide chain terminator is labeled with a second member of said pair of interactive labels.

24. The kit of claim 20, further comprising a template-dependent polynucleotide synthesis enzyme for incorporating in a template-dependent manner a complementary polynucleotide chain terminator into said oligonucleotide primer.

25. The kit of claim 24, wherein said polynucleotide synthesis enzyme is a JDF-3 DNA polymerase.

26. A kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, said kit comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3' of said nucleotide, and is covalently attached to a tag molecule; (b) an anti-tag molecule which binds to said tag molecule, said anti-tag molecule being labeled with a first member of a pair of interactive labels; and (c) packaging materials therefore.

27. The kit of claim 26, wherein said tag molecule is a first member of a specific binding pair which comprises said first member and a second member.

28. The kit of claim 27, wherein said anti-tag molecule is said second member of said specific binding pair.

29. The kit of claim 28, wherein said specific binding pair comprises a biotin-streptavidin pair.

30. A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising: (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer comprising a first sequence which hybridizes to said target polynueleotide immediately 3' of said nucleotide and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence, an oligonuolcotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer, said oligonucleotide probe labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of said pair of interactive labels, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer, and permits said oligonucleotide probe to hybridize to said oligonucleotide primet to permit said pair of interactive labels to generate a signal; and (b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.

31. A method of identifying the presence of a nucleotide at a predetermined position of a target polynuclceotide, said method comprising the steps: (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer comprising a first sequence which hybridizes to said target polynucleotide immediately 3' of said nucleotide and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence and a polynucleotide chain terminator labeled with a second member of a pair of interactive labels, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer; (b) inctibating the oligonucleotide primer comprising said second member of said pair of interactive labels with an oligonucleotide probe comprising said third sequence which hybridizes to said second sequence of said oligonucleotide primer and said probe labeled with a first member of said pair of interactive labels, such chat formation of a hybrid between said oligonucleotide probe and said primer permits said pair of interactive labels to a generate a signal; and (c) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.

32. The method of claim 30 or 31, wherein said signal is generated by fluorescent resonance energy transfer.

33. The method of claim 30 or 31, wherein said oligonucleotide primer comprises a first sequence which hybridizes to said target polynucleotide and a second sequence which does not hybridize to said target polynucteotide in the presence of a third sequence.

34. The method of claim 33, wherein said oligonucleotide probe comprises said third sequence which hybridizes to said second sequence of said oligonucleotide primer.

35. The method of claim 30 or 31, wherein said polynucleotide chain terminator is incorporated by a polynucleotide synthesis enzyme.

36. The method of claim 30 or 31, wherein said reaction mixture further comprises one or mote of a second, a third and/or a fourth polynucleotide chain terminator, wherein said first, second, third and fourth polynucleotide terminators are not identical.

37. The method of claim 35, wherein said polynucleotide synthesis enzyme is a JDF-3 DNA polyrnerase.

38. The method of claim 33, wherein said second sequence is at the 5' terminal of said first sequence.

39. The method of claim 30 or 31, wherein said oligonucleotide primer comprises a separation moiety that permits separation of said oligonucleotide primer from said reaction mixture.

40. The method of claim 39, wherein a target moiety is provided for said separation moiety to form a specific binding pair for separation.

41. The method of claim 40, wherein said target moiety is attached to a solid support.

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Description

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FIELD OF THE INVENTION

This invention relates to the field of polynucleotide sequence determination, in particular, relates to determine the identity of a single nucleotide in a target polynucleotide sequence, e.g., single nucleotide polymorphism ("SNP") analysis.

BACKGROUND

Techniques for the analysis of polynucleotide sequences have found widespread use in basic research, diagnostics, and forensics. Single nucleotide detection is applied in processes including the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.

Single nucleotide detection can be accomplished by a number of methods. Most methods rely on the use of the polymerase chain reaction (PCR) to amplify the amount of target DNA. One of the first developed PCR-dependent methods is restriction site polymorphism detection, where the PCR product is cleaved by a restriction enzyme and then analyzed by electrophoresis. Another early method is allele-specific PCR in which one of the PCR primers is designed such that it will discriminate at its 3' end between DNA targets having a sequence that perfectly matches the primer from those targets not perfectly matching the primer.

TaqMan was the first homogenous assay capable of detecting single nucleotide polymorphisms (U.S. Pat. No. 5,723,591). In this assay, two PCR primers flank a central probe oligonucleotide. The probe oligonucleotide comprises two fluorescent moieties. During the polymerization step of the PCR process, the polymerase cleaves the probe oligonucleotide. The cleavage causes the two fluorescent moieties to become physically separated, which causes a change in the wavelength of the fluorescent emission. As more PCR product is created, the intensity of the novel wavelength increases. While TaqMan accomplishes the goal of single nucleotide detection in a homogenous assay, it has two disadvantages. The first is that each nucleotide to be detected requires a different oligonucleotide probe comprising two different fluorescent moieties. Such probes must be custom-synthesized and are thus expensive. The second disadvantage is that TaqMan probes are not very discriminating for single nucleotide differences. Thus there can be significant false-positive signals.

Molecular Beacons are an alternative to TAQMAN (U.S. Pat. Nos. 6,277,607; 6,150,097; 6,037,130). Molecular Beacons undergo a conformational change upon binding to a perfectly matched template. The conformational change of the Beacon increases the physical distance between a fluorophore moiety and a quencher moiety on the Beacon. This increase in physical distance causes the effect of the quencher to be diminished, thus increasing the signal derived from the fluorophore. Molecular Beacons are more discriminating of single nucleotide differences, as compared with TaqMan probes. However they still require the synthesis of a custom oligonucleotide (the Beacon) having two different fluorescent moieties for each target sequence being examined. Thus the technology is expensive.

There are several other fluorescent and enzymatic PCR technologies, such as SCORPIONS.TM., SUNRISE.TM. primers, and DNAzymes. Not all of these are suitable for single nucleotide detection, and most of them require the synthesis of a custom, fluorescently labeled oligonucleotide for each target nucleotide.

Hybridization to a "DNA chip" is another way of detecting single nucleotide differences (U.S. Pat. No. 5,856,104). Typically oligonucleotides that are complementary to the suspected target DNAs are synthesized on a solid surface ("chip" or "oligonucleotide array"). The target DNA is PCR amplified, labeled, and then hybridized to the oligonucleotide array. Ideally, perfectly matched PCR fragments will hybridize to the array, but mismatched fragments will not. While the technology, in theory, offers the opportunity to look at many different loci simultaneously, in practice the need to amplify the target DNA using PCR limits the degree to which the assay can be multiplexed. In addition the start-up costs for designing an oligonucleotide microarray can be very expensive. Lastly, the frequency of false-positive and false-negative spots is very high, and necessitates the use of many surface-bound oligonucleotides for each target DNA sequence.

There currently are two non-PCR based technologies capable of detecting single nucleotide changes in complex genomes. The Invader-Squared method (U.S. Pat. No. 6,001,567) utilizes a cascade of DNA cleavage reactions. While sensitive, it requires the synthesis of several long, target-specific oligonucleotides in addition to several detection oligonucleotides. The rolling circle detection method (Lizardi et al., Nature Genetics 19: 225-232) utilizes a target nucleotide-specific ligation reaction to create a circular template that is then replicated with a polymerase in rolling-circle fashion. One of the advantages is that the reaction does not require thermal cycling. One drawback is that ligation reactions are not highly specific for single nucleotide detection.

Single base extension ("SBE"; also called minisequencing) is a technology that uses dideoxy chain terminators in combination with a DNA polymerase to determine the identity of a single nucleotide in a target DNA sample that has been PCR amplified (Syvanen et al., 1990, Genetics 8:684-642; U.S. Pat. No. 5,888,819; Euoropean patent application EP 0648280 A1, each of which is incorporated herein by reference). The technology uses a DNA primer that is hybridized to a target polynucleotide in the presence of dideoxy chain terminators, but typically in the absence of deoxynucleotide triphosphates. A DNA polymerase will add a single dideoxy chain terminator to the 3' end of a primer that is reasonably hybridized to the DNA target. The polymerase incorporates the appropriate dideoxy terminator determined by the complementary sequence in the target polynucleotide. Thus, the identity of the dideoxy terminator that is incorporated reflects the identity of the nucleotide within the target polynucleotide that is immediately adjacent to the target nucleotide that is hybridized with the 3' nucleotide of the primer.

There are a number of patents and patent applications for SBE. In U.S. Pat. No. 6,013,431, the dideoxy chain terminators would be labeled with reporter moieties, such as fluorescent molecules, and the incorporation of a label into a primer is measured by gel electrophoresis. The method described in U.S. Pat. Nos. 6,015,675; 5,582,989; 5,578,458 relates to placing the primer on a solid surface, such as a chip. The chip is exposed to a solution containing the target polynucleotide plus fluorescently labeled dideoxy chain terminators and polymerase. When a single labeled base is added to the bound primer, the probe begins to fluoresce.

Fluorescence polarization has been used to perform SBE. With this approach the chain terminators are fluorescently labeled as with other methods. However rather than separating the labeled primers by gel electrophoresis or physical separation, the incorporated chain terminators are generated by shining polarized light on the sample, and then detecting the polarization of the emitted fluorescent light. Fluorescent light emitted by unincorporated terminators will not be polarized because these small molecules are rapidly moving in solution. However labeled terminators that have been incorporated onto the end of a primer will be moving more slowly and tend to emit polarized light. Thus the degree to which the emitted light is polarized reflects the degree to which there has been incorporation of a dideoxy chain terminator onto the end of a primer. The color of the polarized emitted light reflects the particular dideoxy terminator (A, C, G, or T) that was incorporated onto the 3' end of the primer. The advantage to the fluorescent polarization method is that it is homogeneous (all done in a single test tube). However the input target DNA is typically a PCR fragment, and the PCR reaction needs to be performed prior to SBE. Moreover the PCR product needs to be separated from the PCR primers and deoxynucleotides of the PCR reaction prior to performing the SBE reaction.

Another homogenous method has been described in U.S. Pat. No. 6,177,249. This patent uses fluorescence resonance energy transfer ("FRET") (Wittwer, et al., 1997, Biotechniques 22:130-138; Bernard, et al., 1998, Am. J. Pathol. 153:1055-1061). FRET occurs when two fluorescent molecules are in close physical proximity (e.g., 10-100 .ANG.), and one of the fluorescent molecules can absorb light of a wavelength that is emitted by the other fluorescent molecule. For example, suppose the first fluorescent molecular is stimulated by blue light and emits green light, and the second fluorescent molecule is stimulated by green light and emits red light. If, for example, an oligonucleotide contains both fluorescent molecules and the primer is illuminated with blue light, it will emit red light without emitting much green light. In U.S. Pat. No. 6,177,249 (supra), the SBE primer contains one fluorescent molecule. The dideoxy chain terminators contain another (up to 4 different) fluorescent molecules. Upon addition of a terminator to the 3' end of a primer, FRET can occur. As per the FRET example above, stimulating blue light would be converted to green light by the fluorophore on the primer, and then would be further converted to red light after a terminator has been added to the primer. The emission of red light would be used to monitor the degree to which terminators have been added to the primer. One would use 4 terminators with 4 different emission spectra, but all capable of being stimulated by the wavelength released by the primer-bound fluorophore. The advantage to this method is that it is a homogenous assay, although still requiring a PCR amplification pre-SBE step for complex genomes. The disadvantage is that the user must synthesize an expensive, custom oligonucleotide primer for each target DNA locus being examined.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for the detection of nucleotides at predetermined locations on a polynucleotide of interest. The embodiments of the invention include compositions and methods in which a primer extension reaction is designed to extend a single nucleotide (single base extension, SBE) and the incorporation of a labeled chain terminator is determined by signal transfer.

The invention provides a composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the composition comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3' of the nucleotide, and a second sequence which does not hybridize to the target polynucleotide in the presence of a third sequence; and (b) an oligonucleotide probe comprising the third sequence which hybridizes to the second sequence of the oligonucleotide primer, the oligonucleotide probe labeled with a first member of a pair of interactive labels.

The second sequence of the oligonucleotide primer is preferably located at the 5' terminal of the first sequence.

The composition of the invention may also comprise a first polynucleotide chain terminator, which is incorporated in a template-dependent manner into the oligonucleotide primer by a polynucleotide synthesis enzyme.

The composition of the invention may further comprise one or more of a second, a third and/or a fourth polynucleotide chain terminator, where the first, second, third and fourth polynucleotide terminators are not identical.

The composition of the invention may still further comprises a template-dependent polynucleotide synthesis enzyme for incorporating in a template-dependent manner a complementary polynucleotide chain terminator into the oligonucleotide primer.

Preferably, the first polynucleotide chain terminator of the subject composition is labeled with a second member of the pair of interactive labels.

In a preferred embodiment, one member of the pair of interactive labels is a quencher molecule.

In one embodiment of the invention, the first and second members of the pair of interactive labels interact with each other to generate a signal by fluorescent resonance energy transfer.

Preferably, the first and second members of the pair of interactive labels are fluorescent molecules which interact with each other to generate a signal by fluorescent resonance energy transfer.

Also preferably, the polynucleotide synthesis enzyme of the subject composition is a JDF-3 DNA polymerase.

In one embodiment of the invention, the oligonucleotide primer comprises a separation moiety that permits separation of the oligonucleotide primer and/or the oligonucleotide probe hybridized to the primer from unincorporated polynucleotide chain terminator, and oligonucleotide probe which is not hybridized to the oligonucleotide primer.

Preferably, the composition of the subject invention also provides a target moiety specific for the separation moiety, where the separation moiety binds to the target moiety to permit the separation.

The target moiety of the composition is preferably attached to a solid support.

The invention provides another composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the composition comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3' of the nucleotide, and is covalently attached to a tag molecule; and (b) an anti-tag molecule which binds to the tag molecule, the anti-tag molecule labeled with a first member of a pair of interactive labels.

The tag molecule of the subject composition is preferably located on the 5' terminal of the oligonucleotide primer.

Preferably, the tag molecule is a first member of a specific binding pair which comprises the first member and a second member.

Also preferably, the anti-tag molecule is the second member of the specific binding pair.

In one embodiment, the specific binding pair is a biotin-streptavidin pair.

The invention provides a kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the kit comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3' of the nucleotide, and a second sequence which does not hybridize to the target polynucleotide in the presence of a third sequence; (b) an oligonucleotide probe comprising the third sequence which hybridizes to the second sequence of the oligonucleotide primer, the oligonucleotide probe labeled with a first member of a pair of interactive labels; and (c) packaging materials therefore.

The kit of the subject invention may also comprise a polynucleotide chain terminator, which can be incorporated in a template-dependent manner into the oligonucleotide primer by a polynucleotide synthesis enzyme.

The kit of the subject invention may further comprise one or more of a second, a third and/or a fourth polynucleotide chain terminator, where the first, second, third and fourth polynucleotide terminators are not identical.

The polynucleotide chain terminator of the kit is preferably labeled with a second member of the pair of interactive labels.

The kit of the subject kit may still further comprise a template-dependent polynucleotide synthesis enzyme for incorporating in a template-dependent manner a complementary polynucleotide chain terminator into the oligonucleotide primer.

Preferably, the polynucleotide synthesis enzyme is a JDF-3 DNA polymerase.

The invention provides a kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the kit comprising: (a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3' of the nucleotide, and is covalently attached to a tag molecule; (b) an anti-tag molecule which binds to the tag molecule, the anti-tag molecule being labeled with a first member of a pair of interactive labels; and (c) packaging materials therefore.

The tag molecule of the subject kit is preferably a first member of a specific binding pair which comprises the first member and a second member.

Preferably, the anti-tag molecule is the second member of the specific binding pair.

In one embodiment of the invention, the specific binding pair comprises a biotin-streptavidin pair.

The invention provides a method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, the method comprising: (a) incubating the target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to the target polynucleotide immediately 3' of the nucleotide, an oligonucleotide probe which hybridizes to the oligonucleotide primer and labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of the pair of interactive labels, where the incubating permits the polynucleotide chain terminator to be incorporated into the oligonucleotide primer, and permits the oligonucleotide probe to hybridize to the oligonucleotide primer to permit the pair of interactive labels to generate a signal; and (b) detecting the signal, where the detection is indicative of the presence of the nucleotide in the target polynucleotide.

The invention also provides a method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, the method comprising the steps: (a) incubating the target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to the target polynucleotide immediately 3' of the nucleotide and a polynucleotide chain terminator labeled with a second member of a pair of interactive labels, where the incubating permits the polynucleotide chain terminator to be incorporated into the oligonucleotide primer; (b) incubating the oligonucleotide primer comprising the second member of the pair of interactive labels with an oligonucleotide probe labeled with a first member of the pair of interactive labels, such that formation of a hybrid between the oligonucleotide probe and the primer permits the pair of interactive labels to a generate a signal; and (c) detecting the signal, where the detection is indicative of the presence of the nucleotide in the target polynucleotide.

In one embodiment of the invention, the signal is generated by fluorescent resonance energy transfer.

In a preferred embodiment, the oligonucleotide primer comprises a first sequence which hybridizes to the target polynucleotide and a second sequence which does not hybridize to the target polynucleotide in the presence of a third sequence.

Preferably, the second sequence on the oligonucleotide primer is located at the 5' terminal of the first sequence.

Also preferably, the oligonucleotide probe comprises the third sequence which hybridizes to the second sequence of the oligonucleotide primer.

In one embodiment, the polynucleotide chain terminator is incorporated by a polynucleotide synthesis enzyme.

The reaction mixture of the subject method may also comprise one or more of a second, a third and/or a fourth polynucleotide chain terminator, where the first, second, third and fourth polynucleotide terminators are not identical.

Preferably, the polynucleotide synthesis enzyme is a JDF-3 DNA polymerase.

The oligonucleotide primer of the subject method may comprise a separation moiety that permits separation of the oligonucleotide primer from the reaction mixture.

Preferably, a target moiety is provided in the subject method for the separation moiety to form a specific binding pair for separation.

In one embodiment, the target moiety is attached to a solid support.

The invention provides a method for identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, the method comprising: (a) incubating the target polynucleotide in a reaction mixture comprising an anti-tag molecule labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of the pair of interactive labels, and an oligonucleotide primer which hybridizes to the target polynucleotide immediately 3' of the nucleotide, the oligonucleotide primer covalently coupled to a tag molecule, where the incubating permits the polynucleotide chain terminator to be incorporated into the oligonucleotide primer, and the incubating also permits the anti-tag molecule to interact with the tag molecule on the oligonucleotide primer, so that the pair of interactive labels generate a signal; and (b) detecting the signal, where the detection is indicative of the presence of the nucleotide in the target polynucleotide.

In a preferred embodiment, the signal is generated by fluorescent resonance energy transfer.

In another preferred embodiment, one member of the pair of interactive labels is a quencher molecule.

Preferably, the tag molecule is located at 5' terminal of the oligonucleotide primer.

The tag molecule of the subject method may comprise a first member of a specific binding pair which comprises the first member and a second member.

The anti-tag molecule may comprise the second member of the specific binding pair.

In one embodiment, the specific binding pair is a biotin-streptavidin binding pair.

The chain terminator of the invention may be one selected from the group consisting of: a dideoxynucleotide triphosphate, a ribofuranose analog, a reversible nucleotide terminator, and an acyclic terminator.

The target polynucleotide of the invention may present in a sample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the hybridization of an oligonucleotide probe comprising a third sequence which hybridizes to an oligonucleotide primer comprising a first and a second sequences and an incorporated chain terminator in one embodiment of the invention. The probe is labeled with a first member of a pair of interactive labels. Chain terminators (L1 to L4) are used, each labeled with a different second member of the pair of interactive labels. Each terminator will emit a different signal (e.g., color) when stimulated by the stimulus (F) coming from the oligonucleotide probe. The signal form each terminator is generated by FRET.

FIG. 2 illustrates the use of a tag and anti-tag pair to replace the primer-probe interaction of FIG. 1 in one embodiment of the invention.

FIG. 3 illustrates the use of an oligonucleotide probe which is fully complementary to the oligonucleotide primer in one embodiment. FRET signal is generated between two members (dye 1 and dye 2) of a pair of interactive labels present on ddNTP and the probe.

FIG. 4 illustrates that the positive control (A4 well) shows a ROX signal increase due to FRET from Fluorescein compared to the negative control (A3 well) according to one embodiment of the invention.

FIG. 5 illustrates that the positive control (B2 well) shows a ROX signal increase due to FRET from Fluorescein compared to the negative control (B1 well) according to one embodiment of the invention.

FIG. 6 illustrates the use of a quencher molecule according to one embodiment of the invention.

FIG. 7 demonstrates a ROX signal decrease for the positive control due to quenching of ROX fluorescence by BHQ2 upon incorporation of ROX-ddC according to one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Target polynucleotide" refers to a polynucleotide having a sequence, to which the presence or absence or identity of at least one nucleotide is to be determined, i.e., by primer extension, conventional sequencing or mini-sequencing. In the context of a preferred application of the method according to the present invention, a target polynucleotide comprises a nucleotide at a predetermined position of the target polynucleotide whose presence or absence or identity in the target polynucleotide is to be determined. The terms "nucleotide" and "nucleotide base" are used interchangeably. A target polynucleotide may be a length between 10 kb and 10 base pairs, e.g., 1 kb-50 base pairs, or 500 base pairs-100 base pairs. A target polynucleotide of the invention may be a naturally occurring polynucleotide (i.e., one existing in nature without human intervention), or a recombinant polynucleotide (i.e., one existing only with human intervention).

According to the invention, a nucleotide can be modified, biotinylated, radiolabeled, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and the like. The term "nucleotide" includes the derivatives and analogs thereof and includes dNTPs and ddNTPs.

A nucleotide "position" as used herein refers to the location of a given single base within a polynucleotide, including an oligonucleotide.

A "polynucleotide" is a covalently linked sequence of nucleotides (i.e., ribonucleotides for RNA and deoxyribonucleotides for DNA) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next. "Polynucleotide" includes, without limitation, single- and double-stranded polynucleotide. The term "polynucleotide" as it is employed herein embraces chemically, enzymatically or metabolically modified forms of polynucleotide. "Polynucleotide" also embraces a short polynucleotide, often referred to as an oligonucleotide.

A polynucleotide or an oligonucleotide (e.g., the oligonucleotide primer or the oligonucleotide probe) has a "5'-terminus" (5' end) and a "3'-terminus" (3' end) because polynucleotide phosphodiester linkages occur to the 5' carbon and 3' carbon of the pentose ring of the substituent mononucleotides. The end of a polynucleotide at which a new linkage would be to a 5' carbon is its 5' terminal nucleotide. The end of a polynucleotide at which a new linkage would be to a 3' carbon is its 3' terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3'- or 5'-terminus. As used herein, a polynucleotide sequence, even if internal to a larger polynucleotide (e.g., a sequence region within a polynucleotide), also can be said to have 5'- and 3'-ends.

Oligonucleotides are typically less than 150 nucleotides long (e.g., between 5 and 150, preferably between 10 to 100, more preferably between 15 to 50 nucleotides in length), however, as used herein, the term is also intended to encompass longer or shorter polynucleotide chains. Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides, therefore serving as primers for polynucleotide chain extension. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.

As used herein, an "oligonucleotide primer" is an oligonucleotide comprising a sequence complementary to a target polynucleotide. An oligonucleotide, according to the invention, hybridizes to a target polynucleotide through base pairing so to initiate an elongation (extension) reaction to incorporate a nucleotide into the oligonucleotide primer. An "oligonucleotide primer" according to the present invention, may comprise a first sequence that hybridizes to a target polynucleotide immediately 3' of a nucleotide at a predetermined location. An "oligonucleotide primer" may comprise a first sequence which hybridizes to a target polynucleotide and a second sequence which does not hybridize to the target polynucleotide in the presence of a third sequence. The first sequence or the second sequence of an oligonucleotide may be between 10 to 100 nucleotides in length, preferably between 15-50 nucleotides in length. A common second sequence may be used for a number of oligonucleotide primers comprising the same first sequence. An oligonucleotide primer useful in the present invention may be covalently coupled to a tag molecule.

An "oligonucleotide probe" is an oligonucleotide comprising a third sequence which is complementary to the oligonucleotide primer. One or more oligonucleotide probes can be made, each comprising a different sequence complementary to the oligonucleotide primer. An "oligonucleotide probe" according to the invention, may be between 10 to 100 nucleotides in length, preferably between 15-50 nucleotides in length. When an oligonucleotide probe is designed to complement to a common second sequence on a number of oligonucleotide primers, the oligonucleotide probe is also referred to as a universal probe for the number of oligonucleotide primers.

As used herein, an "oligonucleotide hybridizing to a target polynucleotide immediately 3' of a nucleotide" is an oligonucleotide comprising a first sequence that is complementary to the target polynucleotide. The oligonucleotide has a 3' terminal nucleotide complementary to the nucleotide next to the 3' end of the nucleotide, with no nucleotides in between the position of the 3' terminal nucleotide of the oligonucleotide and the position of the 3' end of the nucleotide. The hybridization of the oligonucleotide to the immediately 3' of the nucleotide of the target polynucleotide allows the incorporation of a nucleotide or a nucleotide analog (e.g., a ddNTP), in a template dependent manner, into the oligonucleotide at the position corresponding to the predetermined nucleotide of the target polynucleotide.

A "tag molecule" refers to a molecule covalently coupled to an oligonucleotide primer. An "anti-tag molecule" refers to a molecule which interacts with the tag molecule through specific binding. An anti-tag molecule useful in the invention may be further labeled with a member of a pair of interactive labels. The tag and anti-tag molecule pair allows the interaction of a labeled anti-tag molecule with an oligonucleotide primer which may comprise an incorporated labeled polynucleotide chain terminator. A tag molecule and its corresponding anti-tag molecule, according to the invention, can be members of a specific binding pair. It is not critical for either a tag molecule or an anti-tag molecule to be a specific member of a specific binding pair, so long as it permits the binding between the members of the specific binding pair.

As used herein, a "specific binding pair" refers to two different molecules, where one molecule has an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The two molecules of a specific binding pair may also comprise complementary sequences and form the specific binding through base-pairing. A "specific binding pair", according to the invention, include, but are not limited to members of an immunological pair such as antigen-antibody, or an operator-repressor, nuclease-nucleotide, biotin-streptavidin, ligand-receptor pair, polynucleotide duplexes, IgG-protein A, DNA-DNA, DNA-RNA.

A specific binding pair can be used to separate an oligonucleotide primer or an oligonucleotide probe from a target polynucleotide when desired. The two different molecules in such a specific binding pair can also be referred to as a separation moiety and a target moiety. As used herein, a "separation moiety" is the molecule of a specific binding pair which is coupled to the oligonucleotide primer or the oligonucleotide probe. A "target moiety" refers to the other molecule of the specific binding pair which is optionally coupled to a solid support. "Separation", as used herein refers to physically separating one molecule from another molecule, for example, separating an oligonucleotide primer or an oligonucleotide primer/probe duplex from an unincorporated chain terminator or from an unhybridized oligonucleotide probe.

As used herein, a "solid support" refers to a porous or non-porous water insoluble material. The support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross-linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like. A "solid support" also include magnetic particle such as magnetic beads and such as disclosed in U.S. Pat. Nos. 5,898,071 and 5,705,628. Natural or synthetic assemblies such as liposomes, phospholipid vesicles and cells can also be employed.

Binding of a specific binding pair molecule to a support or surface may be accomplished by well-known techniques, commonly available in the literature. See, for example, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface can have any one of a number of shapes, such as strip, rod, particle or bead.

As used herein, "non-conventional nucleotide" refers to a) a nucleotide structure that is not one of the four conventional deoxynucleotides dATP, dCTP, dGTP, and dTTP recognized by and incorporated by a DNA polymerase, b) a synthetic nucleotide, c) a modified conventional nucleotide, or d) a ribonucleotide (since they are not normally recognized or incorporated by DNA polymerases) and modified forms of a ribonucleotide. Non-conventional nucleotides include but are not limited to those listed in Table 1, which are commercially available, for example, from New England Nuclear.

"Complementary" refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand. It is known that an adenine base of a first polynucleotide region is capable of forming specific hydrogen bonds ("base pairing") with a base of a second polynucleotide region which is antiparallel to the first region if the base is thymine or uracil. Similarly, it is known that a cytosine base of a first polynucleotide strand is capable of base pairing with a base of a second polynucleotide strand which is antiparallel to the first strand if the base is guanine. A first region of a polynucleotide is complementary to a second region of the same or a different polynucleotide if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide of the first region is capable of base pairing with a base of the second region. A first polynucleotide that is 100% complementary to a second polynucleotide forms base pair at every nucleotide position. A first polynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70% complementary) contains mismatched nucleotides at one or more nucleotide positions.

As used herein, a "detectable marker" or a "detectable label" refers to a molecule capable of generating a detectable signal. A "detectable marker" may be detected directly or detectable through a specific binding reaction that generates a detectable signal. The label can be isotopic or non-isotopic, usually non-isotopic, and can be a catalyst, such as an enzyme (also referred to as an enzyme label), a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule (also referred to as a fluorescent label), chemiluminescer (also referred to as a chemiluminescent label), coenzyme, enzyme substrate, radioactive group (also referred to as a radiolabel), a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye (also referred to as a colorimetric label), catalyst or other detectable group, and the like. The label may be a directly detectable label or may be a member of a signal generating system, and thus can generate a detectable signal in context with other members of the signal generating system, e.g., a biotin-avidin signal generation system. The label can be bound directly to a nucleotide or a polynucleotide sequence or indirectly via a linker.

The preferred labels, according to the invention, are members of a pair of interactive labels. The members of a pair of "interactive labels" generates a detectable signal when brought in close proximity. The signals generated is preferably detectable by visual examination methods well known in the art, preferably by a fluorescence resonance energy transfer assay (FRET) (Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300).

First and second members of a pair of interactive labels may be a donor and an acceptor, a receptor and a quencher, or vice versa. As used herein, the term "donor" refers to a fluorophore which absorbs at a first wavelength and emits at a second, longer wavelength. The term "acceptor" refers to a fluorophore, chromophore or quencher with an absorption spectrum which overlaps the donor's emission spectrum and is able to absorb some or most of the emitted energy from the donor when it is near the donor group (typically between 1-100 nm). If the acceptor is a fluorophore capable of exhibiting FRET, it then re-emits at a third, still longer wavelength; if it is a chromophore or quencher, then it releases the energy absorbed from the donor without emitting a photon. Although the acceptor's absorption spectrum overlaps the donor's emission spectrum when the two groups are in proximity, this need not be the case for the spectra of the molecules when free in solution. Acceptors thus include fluorophores, chromophores or quenchers that, following attachment to either a chain terminator or to an anti-tag molecule, show alterations in absorption spectrum which permit the group to exhibit either FRET or quenching when placed in proximity to the donor through the binding interactions of the anti-tag molecule and a tag molecule comprising the chain terminator.

As used herein, a "reporter molecule" is a molecule capable of generating a fluorescence signal. A "quencher molecule" is a molecule capable of absorbing the fluorescence energy of an excited reporter molecule, thereby quenching the fluorescence signal that would otherwise be released from the excited reporter molecule. In order for a quencher molecule to quench an excited fluorophore, the quencher molecule must be within a minimum quenching distance of the excited reporter molecule at some time prior to the reporter molecule releasing the stored fluorescence energy.

According the invention, a pair of interactive labels may comprise more than one second member, each second member can interact with the same first member of the pair of interactive labels and generate a distinguishable signal transfer which is indicative of the identity of each of the second member.

As used herein, references to "fluorescence" or "fluorescent groups" or "fluorophores" include luminescence and luminescent groups, respectively.

As used herein, the term "hybridization" is used in reference to the pairing of complementary polynucleotide strands. Hybridization and the strength of hybridization (i.e., the strength of the association between polynucleotide strands) is impacted by many factors well known in the art including the degree of complementarity between the polynucleotides, stringency of the conditions involved affected by such conditions as the concentration of salts, the Tm (melting temperature) of the formed hybrid, the presence of other components (e.g., the presence or absence of polyethylene glycol), the molarity of the hybridizing strands and the G:C content of the polynucleotide strands.

As used herein, the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which polynucleotide hybridizations are conducted. With "high stringency" conditions, polynucleotide pairing will occur only between polynucleotide fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak" or "low" stringency are often required when it is desired that polynucleotides which are not completely complementary to one another be hybridized or annealed together. The art knows well that numerous equivalent conditions can be employed to comprise high or low stringency conditions.

As used herein, "high stringency conditions" refer to temperature and ionic condition used during polynucleotide hybridization and/or washing. The extent of "high stringency" is nucleotide sequence dependent and also depends upon the various components present during hybridization. Generally, highly stringent conditions are selected to be about 5 to 20 degrees C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature defined by the following equation: T.sub.m =69.3+0.41.times.(G+C)%-650/L, wherein L is the length of the probe in nucleotides. "High stringency conditions", as used herein, refer to a washing procedure including the incubation of two or more hybridized polynucleotides in an aqueous solution containing 0.1.times.SSC and 0.2% SDS, at room temperature for 2-60 minutes, followed by incubation in a solution containing 0.1.times.SSC at room temperature for 2-60 minutes. "High stringency conditions" are known to those of skill in the art, and may be found in, for example, Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory and Schena, ibid.

As used herein, "low stringency conditions" refer to a washing procedure including the incubation of two or more hybridized polynucleotides in an aqueous solution comprising 1.times.SSC and 0.2% SDS at room temperature for 2-60 minutes.

As used herein, the term "Tm" is used in reference to the "melting temperature". The melting temperature is the temperature at which 50% of a population of double-stranded polynucleotide molecules becomes dissociated into single strands. The equation for calculating the Tm of polynucleotides is well-known in the art. The Tm of a hybrid polynucleotide is often estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating Tm for PCR primers: .brket open-st.(number of A+T).times.2.degree. C.+(number of G+C).times.4.degree. C..brket close-st., see, for example, C. R. Newton et al. PCR, 2nd Ed., Springer-Verlag (New York: 1997), p. 24. This formula was found to be inaccurate for primers longer that 20 nucleotides. Other more sophisticated computations exist in the art which take structural as well as sequence characteristics into account for the calculation of Tm. A calculated Tm is merely an estimate; the optimum temperature is commonly determined empirically.

"Polynucleotide chain terminator", or "chain terminator", or "terminator" means any nucleotide that when incorporated into a primer extension product prevents the further extension of such primer extension product. One requirement of a nucleotide terminator is that when the nucleotide terminator includes a ribofuranose sugar portion, the 3'-position must not have a hydroxy group capable of being subsequently used by a polymerase to incorporate additional nucleotides, e.g., dideoxyadenosine triphosphate (ddATP), dideoxycytosine triphosphate (ddCTP), dideoxyguanosine triphosphate (ddGTP), dideoxythymidine triphosphate (ddTTP), or dideoxyuridine triphosphate (ddUTP). Alternatively, a ribofuranose analog could be used, such as arabinose. Exemplary nucleotide terminators include 2',3'-dideoxy-.beta.-D-ribofuranosyl, .beta.-D-arabinofuranosyl, 3'-deoxy-.beta.-D-arabinofuranosyl, 3'-amino-2',3'-dideoxy-.beta.-D-ribofuranosyl, and 2',3'-dideoxy-3'-fluoro-.beta.-D-ribofuranosyl (Chidgeavadze). Nucleotide terminators also include reversible nucleotide terminators (Metzker) and acyclic terminators.

"Primer extension reaction" or "chain elongation reaction" means a reaction between a target-primer hybrid and a nucleotide which results in the addition of the nucleotide to a 3'-end of the primer such that the incorporated nucleotide is complementary to the corresponding nucleotide of the target polynucleotide. Primer extension reagents typically include (i) a polymerase enzyme; (ii) a buffer; and (iii) one or more extendible nucleotides. Both conventional sequencing and mini-sequencing act as primer extension reactions until a nucleotide terminator is incorporated. Mini-sequencing reagents, according to the present invention may comprise an extendible nucleotide.

As used herein, "polymerase chain reaction" or "PCR" refers to an in vitro method for amplifying a specific polynucleotide template sequence. The PCR reaction involves a repetitive series of temperature cycles and is typically performed in a volume of 50-100 .mu.l. The reaction mix comprises dNTPs (each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP), primers, buffers, DNA polymerase, and polynucleotide template. One PCR reaction may consist of 5 to 100 "cycles" of denaturation and synthesis of a polynucleotide molecule.

As used herein, "polynucleotide polymerase" refers to an enzyme that catalyzes the polymerization of nucleotide. Generally, the enzyme will initiate synthesis at the 3'-end of the primer annealed to a polynucleotide template sequence, and will proceed toward the 5' terminal of the template strand. "DNA polymerase" catalyzes the polymerization of deoxynucleotides. Useful DNA polymerases include, but are not limited to, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108:1; U.S. Pat. No. 5,556,772, incorporated herein by reference), Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32), Thermococcus litoralis (Tli) DNA polymerase (also referred to as Vent DNA polymerase, Cariello et al., 1991, Polynucleotides Res, 19: 4193), Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239), Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (Patent application WO 0132887), and Pyrococcus GB-D (PGB-D) DNA polymerase (Juncosa-Ginesta et al., 1994, Biotechniques, 16:820). The polymerase activity of any of the above enzyme can be defined by means well known in the art. One unit of DNA polymerase activity, according to the subject invention, is defined as the amount of enzyme which catalyzes the incorporation of 10 nmoles of total dNTPs into polymeric form in 30 minutes at optimal temperature.

DNA polymerases used in the present invention are preferred to have reduced discrimination against non-conventional nucleotides.

As used herein, "discrimination" refers to the tendency of DNA polymerase to not carry out the incorporation of non-conventional nucleotides into the nascent DNA polymer. DNA polymerase has the ability to sense nucl