CA2283789C - Dna polymerases having improved labeled nucleotide incorporation properties - Google Patents

Abstract

The present invention relates to mutant DNA polymerases that exhibit reduced discrimination against labeled nucleotides into polynucleotides. The DNA polymerases of the invention have at least one mutation in the nucleotide label interaction region of the enzyme such the mutation results in reduced discrimination against labeled nucleotides. The nucleotide label interaction regions is located at portions of the O-helix, (ii) the K-helix, and (iii) the inter O-P helica l loop of Taq DNA polymerase or analogous positions in other DNA polymerases. In addition to providing novel mutant DNA polymerases, the invention also provides polynucleotides encoding the subject mutant DNA polymerases. The polynucleotides provided may comprise expression vectors for the recombinant production of the mutant polymerases. The invention also provides host cells containing the subject polynucleotides. The invention also includes numerous methods of using the subject DNA polymerases, including uses for chain termination sequencing and PCR. Another aspect of the invention is to provide kits for synthesizing fluorescently labeled polynucleotides in accordance with the methods of the invention. Kits of the invention comprise a mutant DNA polymerase of the invention and a fluorescently labele d nucleotide that exhibits reduced discrimination with respect to the mutant DNA polymerase in the kit.

Description

DNA POLYMERASES HAVING IMPROVED LABELED

Inventors: John Brandis, Curtis Bloom, and Jack Richards Field of the Invention The invention is related to DNA polymerises having mutations that alter the ability of the enzyme to incorporate labeled nucleotides into a poiynucleotide molecule.
Back round DNA polymerises are enzymes that synthesize the formation of DNA molecules from deoxynucleotide triphosphates using a template DNA strand and a complementary synthesis primer annealed to a portion of the template. A detailed description of DNA
polymerises and their enzymological characterization can be found in Kornberg, DNA Replication Second Edition, W. H. Freeman ( 1989).
DNA polymerises have a variety of uses in molecular biology techniques suitable for both research and clinical applications. Foremost among these techniques are DNA
sequencing and nucleic acid amplification techniques such as PCR (polymerise chain reaction).
The amino acid sequence of many DNA polymerises have been determined.
Sequence comparisons between different DNA polymerise have identified many regions of homology between the different enzymes. X-ray diffraction studies have determined the tertiary structures of Klenow fragment, T7 DNA polymerise, and Taq DNA
polymerise.
Studies of the tertiary structures of DNA polymerises and amino acid sequence comparisons have revealed numerous structural similarities between diverse DNA
polymerises. In general, DNA polymerises have a large cleft that is thought to accommodate the binding of duplex . DNA. This cleft is formed by two sets of helices, the first set is referred to as the "fingers"
region and the second set of helices is referred to as the "thumb" region. The bottom of the cleft is formed by anti-parallel ~i sheets and is referred to as the "palm"
region. Reviews of DNA polymerise structure can be found in Joyce and Steitz , Ann. Rev. Biochem 63 :777-822 (1994). Computer readable data files describing the three-dimensional structure of some SUBSTITUTE SHEET ( rule 26 ) DNA polymerises have been publicly disseminated.
Fluorescently labeled nucleotides have greatly simplified and improved the utility of many procedures in molecular biology. The use of fluorescently labeled nucleotides for labeling polynucleotides in synthesis procedures, has to a large extent replaced the use of radioactive labeling. Fluorescentiy labeled nucleotides have been widely used in DNA
sequencing, see Smith et al Nature 321:674-679 ( 1986), in PCR, and other forms of polynucleotide fragment analysis.
A major problem with using fluorescently labeled nucleotides is the ability of DNA
polymerises to discriminate against the incorporation of fluorescently labeled nucleotides.
For example, the inventors have discovered that in competition assays between a TET (6 carboxy-4,7,2',7'-tetrachlorofluorescein) labeled 2' 3' dideoxynucieotide and the corresponding unlabeled dideoxynucleotide, Taq DNA polymerise incorporates the unlabeled dideoxynucleotide into DNA at least 85 times more frequently than the corresponding unlabeled nucleotide. This discrimination between labeled and unlabeled nucleotides has profound effects on procedures using DNA polymerises to label DNA. For example, much larger amounts of fluorescently labeled nucleotide must be used in sequencing reactions. This large amount of fluorescently labeled nucleotide is expensive and can generate excessive background fluorescence, thereby reducing the yield of sequence information.
In view of the problems arising from the ability of DNA polymerises to discriminate against the incorporation of fluorescently labeled nucleotides, the inventors have developed several novel DNA polymerises that have reduced discrimination against the incorporation of one or more fluorescently labeled nucleotides into DNA.
Summary Naturally occurring DNA polymerises preferentially incorporate unlabeled nucleotides over corresponding fluorescently labeled nucleotides into polynucleotides.
This ability of DNA polymerises to discriminate against fluorescently labeled nucleotide has undesirable effects on many molecular biology procedures that require the enzymatic addition of fluorescently labeled nucleotides, e.g., labeled dideoxy terminator sequencing. The present invention relates to mutant DNA polymerises that exhibit reduced discrimination against fluorescently labeled nucleotides into polynucleotides.
The DNA polymerises of the invention have at least one mutation in the nucleotide SUBSTITUTE SHEET ( rule ?f ) label interaction region of the enzyme such that the mutation results in reduced discrimination against fluorescently labeled nucleotides. The nucleotide label interaction region of a DNA
polymerise is formed by portions of the O-helix, (ii) the K helix, and (iii) the inter O-P helical loop of Taq DNA polymerise or analogous positions in other DNA polymerises.
Amino acid residues within the nucleotide label interaction region as defined by TET (II) ~ddC are E520.
A531, L522, 8523, E524, A525, H526, P527, I 528, V529, E530, K531, I532, 8536, E537, 8573, Q582, N583, V586, 8587, P589, Q592, 8593, 8595, D610, T612, Q613. E615, 8636, D637, T640, F647, V654, D655, P656, L657, 8659, 8660, T664, E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820, L828, K831, and E832. The sites at 8660, T664, and E681 are of prefered sites for introducing mutations. In a preferred embodiment of the invention for use with fluorescein-type dyes, a mutation is present at position 681 converting an E (glutamic acid) to M {methionine), i.e., E681M.
In a preferred embodiment of the invention for use with fluorescein- fluorescein energy transfer dyes a mutation is present at position 657 converting an L (leucine) to a G
(glycine). In addition to providing mutant Taq DNA polymerises having reduced discrimination against labeled nucleotides, the invention includes mutants derived from a wide variety of DNA
polymerises, both thermostable and otherwise.
In addition to providing novel mutant DNA polymerises, the invention also provides polynucleotides encoding the subject mutant DNA ~polymerases. The polynucleotides provided may comprise expression vectors for the recombinant production of the mutant polymerises. The invention also includes host cells containing the subject polymerise polynucleotides.
The invention also includes numerous methods of using the subject DNA
polymerises.
The subject methods involve synthesizing a fluorescently labeled polynucleotide by means of a polynucleotide synthesis reaction catalyzed by a mutant DNA polymerise that has reduced discrimination against incorporating labeled nucleotides into polynucleotides.
The subject methods of polynucleotide synthesis include the step of extending a primed polynucleotide template with at least one fluorescent labeled nucleotide, wherein the extension is catalyzed by a DNA polymerise that has deduced discrimination against labeled nucleotides into polynucleotides. The subject methods of synthesizing a fluorescently labeled polynucleotide may be used in a variety of methods such as Singer sequencing and the polymerise chain reaction (PCR).

SUBSTITUTE SHEET ( rule 26 ) Another aspect of the invention is to provide kits for synthesizing fluorescently labeled polynucleotides in accordance with the methods of the invention. Kits of the invention comprise a mutant DNA polymerise of the invention and a fluorescently labeled nucleotide that exhibits reduced discrimination with respect to the mutant DNA polymerise in the kit.
According to one aspect of the invention, there is provided a DNA polymerise having at least one mutation in the nucleotide label interaction region, said nucleotide label interaction region being selected from the group consisting of (i) the O-helix, (ii) the K helix, and (iii) the inter O-P helical loop, wherein the mutation is at an amino acid residue position selected from the group consisting of E520, A531, L522, 852:3, E524, A525, H526, P527, I528, V529, E530, K531, I532, 8536, E537, 8573, Q582, N583, V586, 8587, P589, Q592, 8593, 8595, D610, T612, Q613, 8636, D637, T641), F647, V654, D655, P656, L657, 8659, 8660, T664, E681, L682, A683, I684, P68:p, E688, F692, Q754, H784, L817, E820, L828, K831, and E832, wherein the position of said amino acid residue within the DNA polymerise being defined with respect t:o Taq DNA polymerise and wherein the DNA polymerise has reduced discrimination for a fluorescein-type dye labeled nucleotide as compared with the naturally occurring DNA polymerise.
According to a further aspect of the invention, there is a polynucleotif.e encoding a DNA polymerise according to the above described DNA polymerise, an expression vector having a promoter, wherein the vector comprises said polynucleotide in functional combination with the promoter, sand a host cell comprising said expression vector.
Brief Description of the Drawings Figure 1 is a computer model of DNA bound to Taq DNA polymerasc:.
Amino acid residues that form the nucleotide label interaction site are highlighted in orange. The rest of the polymerise is indicated in green. The template is indicated in blue. The dye moiety of the labeled nucleotide is red. The remainder of the tabled nucleotide is white.
Figure 2 is plot of a next nucleotide effect assay.

Figure 3 is plot of a next nucleotide effect assay.
Figure 4 is a representation of the structure of the fluorescently labele;d nucleotide "TET(lI). ddCTP."
Detailed Description of Specific Embodiments of the Invention.
Terminology Positions of amino acid residues within a DNA polymerase are indicated by either numbers or number/letter combinations. The numbering starts at the amino terminus residue. The letter is the single letter amino acid code for the amino acid residue at the indicated position in the naturally occurring enzyme from which the mutant is derived. Unless specifically indicated otherwise, an amino acid residue position designation should be construed as referring to the analogous position in a.ll DNA polymerases, even though the single letter amino acid code specifically relates to the amino acid residue at the indicated position in Taq DNA polymerase.
Individual substitution mutations are indicated by the form of a letter/number/letter combination. The letters are the single letter code for amino acid residues. The numbers indicate the amino acid residue position of the mutation site.
The numbering system starts at the amino terminus residue. The numbering of the residues in Taq DNA polymerase is as described in U.S. Patent No. 5,079,352 (Gelfand). Amino acid sequence homology between different DNA polymerases permits corresponding positions to be assigned to amino acid residues for DN.A
polymerises other than Taq. Unless indicated otherwise, a given number 4a refers to position in Taq DNA polymerise. The first letter, i.e., the letter to the left of the number, represents the amino acid residue at the indicated position in the non-mutant enzyme.
The second letter represents the amino acid residue at the same position in the mutant enzyme. For example, the term "R660D" indicates that the arginine at position 660 has been replaced by an aspartic acid residue.
The term "discrimination" as used herein refers to the property of a DNA
polymerise to preferentially incorporate unlabeled nucleotides over corresponding fluorescently labeled nucleotides into DNA, i.e., the DNA polymerise discriminates against the fluorescently labeled nucleotide. Preferential incorporation may be measured in an assay in which a fluorescentiy labeled 2'3' dideoxynucleotide and a corresponding unlabeled 2'3' dideoxynucleotide compete for incorporation into the same site of a polynucleotide. An example of such an assay can be found below in example 2.
The term "reduced discrimination" as used herein refers to reduction in discrimination against incorporation of a fluorescently labeled nucleotides in a mutant DNA
polymerise as compared to the parent enzyme. A reduction in discrimination may be described quantitatively by reference to the selectivity assays in Example 2 or reference to other assays providing for measurement of the same properties of the polymerise. A
reduction in selectivity number as measured by the selectivity assays is a reduction in discrimination and may be expressed by a ratio of selectivity numbers. For example, a mutant DNA
polymerise with a selectivity number of 8 would have a 10-fold reduction in discrimination when compared with a parent DNA polymerise having a selectivity number of 80.
The term "parent" or "parent enzyme" is used to distinguish a mutant DNA
polymerise from the DNA polymerise that the mutant enzyme was derived from.
Thus any naturally occurring DNA polymerise may be referred to as parent enzyme. A
first DNA
polymerise having mutations with respect to a naturally occurring enzyme is also be referred to as a parent enzyme with respect to a second DNA polymerise having additional mutations.
The term "discrimination reducing mutations" refers to mutations in the nucleotide label interaction region of a DNA polymerise that result in reduced discrimination against the incorporation of fluorescently labeled nucleotides. The term is used to distinguish mutations in a DNA polymerise, including mutations in the nucleotide label interaction region, that do not reduce discrimination against fluorescently labeled nucleotides from mutations that do reduce discrimination.
-S-SUBSTITUTE SHEET { ruie 26 ) The term "nucleotide" as used herein, unless specifically noted otherwise, is used broadly to refer to both naturally occurring nucleotide and a variety of analogs including 2',3'dideoxynucleotides.
The term "fluorescein-type dyes" refers to a class of xanthene dye molecules which include the following fused three-ring system:
HO / O / O
/ /
Y
where a wide variety of substitutions are possible at each deoxy ring position. A
particularly preferred subset of fluorescein-type dyes include the 4,7,-dichorofluoresceins (Menchen}. Examples of fluorescein-type dyes used as fluorescent labels in DNA sequencing methods include 6-carboxyfluorescein (6-FAM), S-carboxyfluorescein (5-FAM), 6-carboxy-4,7,2',7'-tetrachlorofluroscein (TET), 6-carboxy-4,7,2',4',5',7'-hexachlorofluorescein (HEX), 5-(and 6)carboxy-4',5'-dichloro-2'7'-dimethoxyfluorescein (JOE), and 5-carboxy-2',4',5',7'-tetrachlorofluorescein (ZOE). Many times the designation -1 or -2 is placed after an abbreviation of a particular dye, e.g., HEX-1. The "-1" and "-Z" (or "1" and "ll") designations indicate the particular dye isomer being used. The 1 and 2 isomers are defined by the elution order (the 1 isomer being the first to elute) of free dye in a reverse-phase chromatographic separation system utilizing a C'-8 column and an elution gradient of 1 S% acetonitrile/85% 0.1 M triethylammonium acetate to 35%
acetonitrile / 65% 0.1 M triethylammonium acetate.
The term "alkynylamina type linker" refers to an alkynylamino linker of the type as described in U.S. Patent No. 5,047,519 (Hobbs), U.S. Patent No.

5,151,507 (Hobbs), and U.S. Patent No. 5,821.356 (Khan). Additional alkynylamino type liners are described in U. S. Patent No. 5,770,716 (Khan'I.
The term "TET(II)~ddCTP" refers to the fluorescently labeled nucteotide of the structure indicated in figure 4, The term "fluorescence energy transfer dye" refers to dye moieties joined by a linker that permits fluorescence energy transfer between the two dye moieties.
For use in chain termination sequencing, the linker is sufficiently small and of the proper shape and orientation to permit a :DNA polymerise to incorporate a nucleotide triphosphate labeled with the dye interest. Examples of energy transfer dyes can be found in U.S. Patent No.
5,863,727 (Lee) and U.S. Patent No. 5,80(),996 (Lee).
The term "mutation" as used herein refers to a change in amino acid residue at a specific location of a protein. The change in amino acid residue is a change defined with respect to a naturally occurring protein. A protein having a mutation may be referred to as a "mutant" protein.
Embodiments of the invention The present invention relates to DNA polymerises containing mutations that reduce the ability of the polymerise to discriminate against the incorporation of fluorescently labeled nucleotides into polynucleotides, These mutations are in a region of the DNA polymerise molecule referred to herein as "the nucleotide label interaction region." The nucleotide label interaction region is formed by portions of three regions of the DNA polymerise. These three regions are located in (i) the O-helix, (ii) the K helix, and (iii) the inter O-P helical loop of Taq DNA
polymerise or analogous positions in other DNA polyrnerases. DNA polymerises having reduced discrimination against fluorescently labeled nucleotides are particularly useful for chain termination DNA sequencing using 2'3'dideoxynucleotides, i.e. Singer type sequencing.
Enzyme kinetic experiments (described in examples 2 and 3) performed with Taq DNA. polymerise and fluorescently labeled dideoxynucleotides support a theory that Taq DNA polymerise and other DNA polymerises, undergo a conformational shift upon the binding of nucleotides during I)NA synthesis. This predicted conformational shift suggests a set of amino acid residues that interact with fluorescent labels joined by a linker to the nucleic acid base of a nucleotide, thereby resulting in discrimination against nucleotides that are fluorescently labeled. This set of amino acid residues forms the nucleotide label interaction region. The specific molecular model for the binding of fluorescently labeled nucleotide to a DNA
polymerise proposed by the applicants is used to predict the amino acid residues that form the nucleotide label interaction region of a given DNA polymerise.
Applicants model for a conformational shift in DNA polymerise during DNA synthesis is offered as a explanation of how the nucleotide label interaction region was determined. The _7_ ~ I

model provides guidance in making mutations in DNA polymerise that reduce the ability of a DNA polymerise to discriminate against the incorporation of fluorescently labeled nucleotides into polynucleotides. Figure 1 is a computer model showing how DNA
and Taq DNA polymerise interact in the model. Whether or not the true mechanism of DNA
polymerise-nucleotide interaction is the same or different as the model used to determine the parameters of the nucleotide label interaction region is not determinative to the operability of the invention described herein.
The mutant DNA polymerises of the invention exhibit reduced discrimination against nucleotides labeled with a fluorescein-type dye. In other words, the mutant DNA
polymerises of the invention contain at least one mutation that increases the ability of the polymerise to incorporate a fluorescein-type dye labeled nucleotide relative to the corresponding unlabeled nucleotide. In addition to reduced discrimination against nucleotides labeled with fluorescein-type dyes, the mutant DNA polymerises of the invention may also exhibit reduced discrimination against nucleotides labeled with other fluorescent dyes that are not fluorescein-type dyes, as well as reduced discrimination against other detectable moieties.
The fluorescently labeled nucleotides for which a given embodiment of the mutant DNA
polymerises of the invention exhibit reduced discrimination may vary with respect to the particular fluorescent label, the linker used to attach the fluorescent label to the nucleotide, the site of attachment for the tinker on the fluorescent label, the specific nucleotide base that is selected, and the site of attachment for the linker on the nucleotide. The precise degree of reduction in discrimination against a fluorescently labeled nucleotide will vary in accordance with the specific mutation or mutations introduced into the DNA polymerise.
The precise degree of reduction in discrimination will also vary in accordance with the specific fluorescently labeled nucleotide assayed, e.g., variations in base, dye, or linker. Mutant DNA
polymerise of the invention may exhibit anywhere from i slight reduction in discrimination against fluorescently labeled nucleotides to a complete elimination in discrimination, i.e., the mutant enzyme does not significantly differ with respect of rate of incorporation of labeled or unlabeled nucleotides. It is preferable to use embodiments of the subject mutant DNA
polymerises that have at least a two-fold reduction in discrimination against one or more fluorescein type dye labeled nucleotides.
It will be appreciated by persons skilled in the art of molecular biology that the nucleotide label interaction region of a given DNA polymerise is defined with respect to a _g-SUBSTITUTE SHEET ( rule 26 ) specific fluorescently labeled nucleotide. Changes in one or more of the following parameters of the structure of a fluorescently labeled nucleotide may alter the identity of the amino acid residues that form the nucleotide label interaction site of a given DNA
polymerise: ( 1 ) identity of the base, (2) the site of attachment on the nucleotide base, (3) the identity of the S linker joining the base to the florescent dye, and (4) the identity of the fluorescent dye. The nucleotide labeled interaction region of Taq def ned with respect to TET(II)~ddCTP
comprises the amino acid residues E520. A531, L522, 8523, E524, A525, H526, P527, I
528, V529, E530, K531, I532, 8536, E537, 8573, Q582, N583, V586, 8587, P589, Q592, 8593, 8595, D610, T612, Q613, E615, 8636, D637, T640, F647, V654, D655, P656, L657, 8659, 8660, T664, E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820, L828, K831, and E832. The sites at 8660, T664, and E681 are of prefered sites for introducing mutations. Given that the 3-dimensional structure of Taq DNA
polymerise (and other DNA polymerises) is well known and the three dimensional structure of TET(II)~ddCTP is understood with a high degree of certainty, the location of the amino acid 1 S residues that constitute the labeled nucleotide interaction region with respect to TET(II)~ddCTP may be translated to a different set of amino acid residues to accommodate structural differences between TET(II~ddCTP and other fluorescently labeled nucleotides so as to define the labeled nucleotide interaction site with respect to those other nucleotides. For example, increasing the length of the linker between the base and the fluorescent label and the base may predictably alter the identity of amino acid residues that form the labeled nucleotide interaction site, even though the base, base attachment site, and fluorescent dye are the same.
In many embodiments of the subject polymerises, the set of amino acid residues that form the labeled nucleotide interaction site with respect to a given fluorescently labeled nucleotide will overlap with the set of amino acid residues that form the labeled nucleotide interaction site as defined with respect to a second fluorescently labeled nucleotide.
Embodiments of the invention include mutant DNA polymerises that exhibit reduced discrimination against nucleotides labeled with fluorescein-type dyes, wherein the fluorescein type dye is joined to the nucleotide base by an alkynylamino-type linker. The fluorescein-type ' dye may be a fluorescent energy transfer dye, comprising a fluorescein-type dye moiety as a component of the energy transfer dye. In addition to reduced discrimination against fluorescently labeled nucleotides comprising an alkynylamino-type linker, the mutant DNA
polymerises of the invention may also exhibit reduced discrimination against nucleotides SUBSTITUTE SHEET ( rule 26 ) ~ I

comprising other types of linker. In order to minimize stearic interference between the poiynucleotide and the fluorescent label, purines are usually labeled at position 7 and pyrimidines are usually labeled at position 5.
Mutant DNA polymerises of the invention have one or more discrimination reducing mutations at amino acid residue positions within the nucleotide label interaction region of a given DNA polymerise. Discrimination reducing mutations are usually, although not necessarily, substitution mutations. Several different amino residues may be substituted at a given position of a parent enzymes so as to give rise to a discrimination reducing mutations.
The amino acid residues at a given residue position within the nucleotide label interaction reeion may be systematically varied so as to determine which amino acid substitutions result in the reduction of discrimination against the fluorescein-type dye labeled nucleotide dye of interest and the degree of such a reduction in discrimination. The extent to which a particular mutation (or set of mutations) reduces discrimination may be measured by a selectivity assay as described in example 2. The substitution mutation is preferably, although not necessarily, 1 S a mutation that reduces the size of the amino acid residue side chain of the amino acid residue present in the parent DNA polymerise. Mutations are preferably, although not necessarily, conservative so as to maintain the specific polar or non-polar character of the amino acid residue at the analogous position parent molecule. The mutations in the nucleotide label interaction region of a DNA polymerise preferably result in the substitution of the amino acid residue of the parent enzyme with the amino acid residue at the corresponding position of phage T7 DNA polymerise (provided that a difference exists between the amino acid residues at that position in T7 polymerise and the parent enzyme).
Discrimination reducing mutations are in the nucleotide label interaction region of DNA polymerises. The nucleotide label interaction region is formed by portions of three regions of the DNA polymerise. These three regions are located in (i)the O-helix, (ii) the K
helix, and (iii) the inter O-P helical loop of Taq DNA polymerise or analogous positions in other DNA polymerises. Positions in Taq DNA polymerise that form the nucleotide label interaction region are positions E520. A531, L522, 8523, E524, A525, H526, P527, I 528, V529, E530, K531, I532, 8536, E537, 8573, Q582, N583, V586, 8587, P589, Q592, 8593, 8595, D610, T612, Q613, E615, 8636, D637, T640, F647, V654, D655, P656, L657, 8659, 8660, T664, E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820, L828, K831, and E832. Analogous positions in DNA polymerises other than Taq are also form SUBSTITUTE SHEET ( rule 26 ) a nucleotide Label interaction region. Preferred positions for substitution mutations are 8595, D655, 8660, and E681. A particularly preferred position for mutations is E681, with the preferred substitution at position 681 being M. Other suitable substitution mutations at E681 are as follows (listed in order of decreasing preference, excpt where note by a equal sign to denote approximate equivalence"): M>I>W>L>V>P>H=K=G=T=S>D=A=N>Y=C. A
preferred substitution mutation at position 8660 is R660D.
The specific amino acid residues that form the nucleotide interaction region will vary in accordance with the particular DNA polymerise selected as a parent enzyme for the introduction of discrimination reducing mutations. The determination of analogous amino acid residues positions between different DNA polymerises may easily be achieved by the person skilled in the art because of the large number of DNA polymerise amino acid sequences that have been determined and the many regions of homology have been found between these different DNA polymerises. For example, a large compilation of the amino acid sequences of DNA polymerises from a wide range of organism and homology alignments 1 S between the sequences can be found in Braithwaite and Ito, Nucl. Acids Res. 21 (4):787-802 (1993). Examples of amino acid residues within the nucleotide label interaction regions of phage T7 polymerise and E. toll DNA polymerise are provided in Table 1. In addition to providing mutant DNA polymerises having reduced discrimination for fluorescein type dyes in Taq, T7 and E. toll DNA polymerise I, the invention provides mutant DNA
polymerises from many other organisms. In general, the teachings of the invention may used to produce mutant DNA polymerises having reduced discrimination for fluorescein type dyes from any DNA polymerise that shares sufficient amino acid sequence homology to Taq DNA
polymerise to permit a person of ordinary skill in the art to identify one or more amino acid residue positions in the DNA polymerise that are analogous to positions E520.
A531, L522, 8523, E524, A525, H526, P527, I 528, V529, E530, K531, 1532, E537, 8573, V586, 8587, P589, Q592, 8593, 8595, D610, T612, Q613, E615, 8636, T640, F647, V654, D655, P656, L657, 8659, 8660, T664, E681, L682, A683, I684, P685, E688, F692, Q754, L817, E820, L828, K831, and E832 in Taq DNA polymerise. Parent DNA polymerises that may be modified to contain discrimination reducing mutations in the nucleotide label interaction region include, but are not limited to, DNA polymerises from organisms such as Thermus flavus, Pyrococcus furiosus, Thermotoga neapolitana, Thermococcus litoralis, Sulfolobus solf'ataricus, Thermatoga maritima, E. toll phage T5, and E toll phage T. The DNA
-I I-SUBSTITUTE SHEET ( rule 26 ) ~ I

polymerises may be thermostable or not thermostable. It will be appreciated that the present invention enables persons skilled in the art to introduce fluorescein-type dye discrimination reducing mutations in to DNA polymerises from a wide variety of organisms, including DNA
polymerises that have not been isolated at the time of the filing of this application provided.
Additionally, embodiments of the invention includes some purified naturally-occurring DNA
polymerises that have the desired low degree of discrimination against fluorescently labeled nucleotides. Such naturally-occurring DNA polymerises are structurally and functionally analogous to the mutant DNA polymerises explicitly described herein.
The amino acid residues that constitute the nucleotide label interaction region of a given DNA polymerise vary in accordance with the specific fluorescently labeled nucleotide that is used to define the nucleotide label interaction region. Similarly, the mutations that are discrimination reducing mutations may vary in accordance with the specific fluorescently labeled nucleotide that is used to define the labeled nucleotide interaction region.
Additionally, the degree of discrimination reduction achieved by the mutation (or mutations) I S in the labeled nucleotide interaction site may vary with the specific labeled nucleotide of interest. For example, E681M is the preferred discrimination reducing mutation in Taq with respect to TET(II)~ddCTP resulting in a 47x reduction in discrimination and a significantly lower reduction in discrimination against a second fluorescently labeled nucleotide.
Conversely, an E681T mutation may result in a high level reduction in discrimination against the second fluorescently labeled nucleotide and only a low level of reduction in discrimination against TET(II)~ddCTP.
Given that a mutant DNA polymerise of the invention may have discrimination reducing mutation in the nucleotide label interaction region resulting in a significant degree of reduction in discrimination for a specific fluorescently labeled nucleotide and little or no reduction in the degree of reduction of discrimination against another fluorescently labeled nucleotide (assuming there is significant discrimination against that fluorescently labeled nucleotide by the parent DNA polymerise), a given mutant DNA polymerise may be said to be "receptive" with respect to one or more given fluorescently labeled nucleotide. A specific mutant DNA polymerise is referred to as "receptive" with respect to a specific fluorescently labeled nucleotide if a discrimination reducing mutation in the nucleotide label interaction site in the specific enzyme of interest results in at least a five fold reduction in discrimination against that given fluorescently labeled nucleotide. A mutant DNA polymerise of the SUBSTITUTE SHEET ( rule 26 ) invention may be receptive with respect to more than one fluorescently labeled nucleotide.
Conversely, a specific fluorescently labeled nucleotide may be "receptive"
with respect to a given mutant DNA polymerise of the invention.
In embodiments of the subject mutant DNA polymerises comprising more than one S discrimination reducing mutation in the nucleotide label interaction region, the mutation site may be in the same or different region of the three regions of a polymerise that form the nucleotide label interaction region, In general, mutant DNA polymerises of the invention will have 1, 2, or 3 discrimination reducing mutations. However, the invention also provides mutant DNA polymerises having more than 3 discrimination reducing mutations.
By combining multiple discrimination reducing mutations, greater levels of reduction in labeled nucleotide discrimination may be achieved. However, in many embodiments of the invention, mutant DNA polymerises have levels of reduced labeled nucleotide discrimination that are the same or less than the levels of DNA polymerise with single discrimination reduction mutations in the nucleotide label interaction region. Preferred combinations of mutations in a Taq DNA polymerise background are R660D, E681G, and F667Y, i.e., Taq DNA
polymerise mutant (R660D, E681 G, and F667Y).
Different embodiments of DNA polymerise having mutations in the nucleotide label interaction region differ with respect to the degree of reduction in discrimination against specific fluorescently labeled nucleotides. These differences may be measured by an assay in order to determine which specific embodiments have the greatest degree of reduction in discrimination against the particular fluorescently labeled nucleotides of interest. Generally, such assays measure competition between a fluorescently labeled nucleotide and an unlabeled nucleotide for incorporation into the same site on a primed template. One example of such an assay (referred to herein as a "selectivity assay") is described in detail below in Example 2.
The mutant DNA polymerises of the invention may comprise numerous mutations in addition to discrimination reduction mutations in the nucleotide label interaction region.
These secondary mutations may be either inside or outside the nucleotide label interaction region. Secondary mutations may be selected so as to have as to confer some useful property on the mutant DNA polymerise. For example, additional mutations may be introduced to increase thermostabiiity; decrease thermostability, increase processivity, decrease processivity, decrease 3'-5' exonuclease activity, increase 3'-5' exonuclease activity, decrease 5'-3' SUBSTITUTE SHEET ( rule 26 ) exonuclease activity, increase S'-3' exonuclease activity, and increase incorporation of dideoxynucleotides. Alternatively, the secondary mutations may be essentially neutral in known effect.
Of particular interest are embodiments of the subject mutant DNA polymerise that S comprise one or more secondary mutation that reduce 3'-S' exonuclease activity. DNA
polymerises that are deficient in 3'-S' exonuclease activity have superior properties for PCR
and for chain termination polynucleotide sequencing. Mutations that reduce 3'-S' exonuclease activity in DNA polymerise are well known to person of ordinary skill in the art.
Detailed guidance on how to introduce mutations that reduce 3'-S' exonuclease activity can be found, among other places in U.S. Patent No. 4,795,699 (Tabor); U.S. Patent No.
5,541,099; U.S. Patent No. 5,489,523; and Bernad et al., Cell 59:219-288 ( 1989). Examples of such mutations in Taq DNA polymerise include G46D. For embodiments of the mutant DNA polymerises that are used for sequencing, it is preferable to include a G46D (or analogous mutations in DNA polymerises other than Taq) in addition to mutations in the nucleotide label interaction region.
Also of interest among secondary mutations in the subject DNA polymerise mutants are mutations that increase incorporation of dideoxynucleotides, i.e., reduce the ability of a DNA polymerise to discriminate against dideoxynucleotide as opposed to deoxynucleotides. Guidance on making such mutations can be found, among other places in published PCT application W096/12042 (application number PCT/LJS95/12928). Of particular interest is the mutation F667Y in Taq and analogous mutations in other DNA
polymerise. While F667Y is not pan of the nucleotide label interaction region in Taq DNA
polymerise with respect to Tet(II)~ddLTP, F667Y mutations may reduce discrimination against fluorescein-type dye labeled nucleotides (see Table 1 ). Accordingly, for use in certain 2S procedures, e.g., DNA sequencing, be desirable to combine an F667Y
mutations with one or more discrimination reducing mutations in the nucleotide label interaction region so as to reduce discrimination of the polymerise between deoxynucleotides and 2'3' dideoxynucleotides. Mutant DNA polymerise of the invention having the F667Y
mutation (or equivalent thereof) are particularly useful in Singer type DNA sequencing with fluorescently labeled 2'3' dideoxynucleotide chain terminators.
Numerous genes encoding DNA polymerises have been~isolated and sequenced. This sequence information is available on publicly accessible DNA sequence databases such as SUBSTITUTE SHEET ( rule 26 ) GENBANK. A large compilation of the amino acid sequences of DNA polymerises from a wide range of organism can be found in Braithwaite and Ito, Nucl. Acids Res.
21 (4):787-802 ( 1993). This information may be used in designing various embodiments of DNA
polymerises of the invention and polynucleotide encoding these enzymes. The publicly available sequence information may also be used to clone genes encoding DNA
polymerises through techniques such as genetic library screening with hybridization probes.
Other embodiments of the invention are polynucleotide sequences encoding the mutant DNA polymerises provided herein. Poiynucleotide sequences encoding the mutant DNA polymerise of the invention may be used for the recombinant production of the mutant DNA polymerises. Polynucleotide sequences encoding mutant DNA polymerises having reduced discrimination against fluorescently labeled nucleotide may be produced by a variety of methods. A preferred method of producing polynucleotide sequences encoding mutant DNA polymerises having reduced discrimination against fluorescently labeled nucleotides is by using site-directed mutagenesis to introduce desired discrimination reducing mutations into polynucleotides encoding the parent DNA polymerise molecules. Site-directed mutagenesis techniques are well known in the art as exemplified by U.S. Patent No.
4,711,848; U.S. Patent No. 4,873,192; U.S. Patent No. 5,071,743; U.S. patent, 5,284,760; U.S. Patent No.
5,354,670; U.S. Patent No. 5,556,747; Zoller and Smith, Nucleic Acids Res.
10:6487-6500 ( 1982), and Edelman et al DNA 2:183 ( 1983 ). Detailed protocols for site-directed mutagenesis are also given many general molecular biology textbooks such as Sambrook et al Molecular Cloning a Laboratory Manual 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor ( 1989), Ausubel et al. Current Protocols in Molecular Biology, (current edition).
Additionally, many text books on PCR (the polymerise chain reaction), such as Diefenbach and Dveksler, PCR Primer: A Laboratorx Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY ( 1995), describe methods of using PCR to introduce directed mutations. Genes encoding parent DNA polymerise may be isolated using conventional cloning techniques in conjunction with publicly-available sequence information. Alternatively, many cloned polynucleotide sequences encoding DNA polymerises have been deposited with publicly-accessible collection sites, e.g., the American type culture collection deposit accession number ATCC 40336 is a phage clone of Taq DNA polymerise.
In addition to producing the mutant DNA polymerise encoding polynucleotides of the invention by introducing directed mutations into polynucleotides encoding parent DNA

SUBSTITUTE SHEET ( rule 26 ) polymerises, it is possible (although difficult) to produce the poiynucleotides of the invention primarily by in vitro DNA synthesis techniques. In vi~ro DNA synthesis techniques are well known to those skilled in the art and examples of in vitro DNA synthesis can be found in U. S.
Patent No. 5,252,530; U.S. Patent No. 4,973,679; U.S. Patent No. 5,153,319;
U.S. Patent S No. 4,668,777; U.S. Patent No. 4,500,707; U.S. Patent No. 5,132,418; U.S.
Patent No.
4,415,732; U.S. Patent No. 4,458,066; and U.S. Patent No. 4,811,218. When producing relative polynucleotide molecules by in vitro DNA synthesis, smaller molecules are usually produced first and subsequently joined together by hybridization and ligation.
Mutant DNA
polymerise encoding poiynucleotides may also be produced by a combination of in vitro synthesis and site-directed mutagenesis of cloned genes.
Polynucleotide encoding the mutant DNA polymerise of the invention may be used for the recombinant expression of the mutant DNA polymerises. Generally, the recombinant expression of the mutant DNA polymerise is effected by introducing a mutant DNA
polymerise into an expression vector adapted for use in particular type of host cell. Thus, another aspect ofthe invention is to provide expression vectors comprising a polynucleotide encoding a mutant DNA polymerise of the invention, such that the polymerise encoding polynucleotide is functionally inserted int the expression vector. The invention also provide host cells comprising the expression vectors of the invention. Host cells for recombinant expression may be prokaryotic or eukaryotic. Example of host cells include bacterial cells, yeast cells, cultured insect cell lines, and cultured mammalian cells lines.
Preferably, the recombinant host cell system is selected so as to closely match the organism from which the mutant DNA polymerise was derived. For example, prokaryotic DNA polymerises are preferably expressed in a prokaryotic expression system. A wide range of expression vectors are well known in the art. Description of various expression vectors and how to use them can be found among other places in U.S. Patent No. 5604118; U.S. 5,583,023;
U.S. Patent No. 5,432,082; U.S. Patent No. 5,266,490; U.S. Patent No. 5,063,158; U.S.
Patent No.
4,966,841; U.S. Patent No. 4,806,472; U.S. Patent No. 4,801,537; and Goedel et al., Gene Expression Technology Methods of Enzymology, Vol. 185, Academic Press, San Diego (1989). The expression ofDNA polymerises in recombinant cell systems is a well-established technique. Examples of the recombinant expression of DNA polymerise can be found in U. S.
Patent No. 5,602,756; U.S. Patent No. 5,545,552; U.S. Patent No. 5,541,31 I;
U.S. Statutory Inventor RegistrationH1,531; U.S. Patent No. 5,500,363; U.S. Patent No.
5,489,523; U.S.

SUBSTITUTE SHEET ( rule ?6 ) Patent No. 5,455,170; U.S. Patent No. 5,352,778; U.S. Patent No. 5,322,785;
and U.S.
Patent No. 4,935,361.
Other embodiments ofthe invention include multiple DNA polymerise compositions particularly useful for polynucleotide sequencing, such compositions comprise at least two different mutant DNA polymerises of the invention, wherein ( 1 ) the first mutant DNA
polymerise is receptive with respect to a first fluorescently labeled nucleotide; (2) the second mutant DNA polymerise is receptive with respect to a second fluorescently labeled nucleotide; and (3) the first and second fluorescently labeled nucleotides differ from one another with respect to their nucleotide bases and fluorescent labels. The first and second fluorescently labeled bases may also differ with respect to one another by way of the linker, the base attachment position, or the fluorescent dye attachment site. The subject compositions are useful for catalyzing the sequencing reactions in Singer type DNA
sequencing with fluorescent dye labeled 2'3' dideoxy chain terminating nucleotides. Chain termination sequencing with fluorescently labeled terminators preferably employs at least two, and more preferably 4 different fluorescently labeled chain terminators, wherein each different base is labeled with a distinctive fluorescent label. Because of the necessary structural differences between the different fluorescently labeled chain terminators required for a sequencing reactions, i.e., nucleotide bases and fluorescent labels, there are many mutant DNA polymerises of the invention that are not receptive to all of the fluorescently labeled terminators necessary for a given sequencing reaction. Thus, there are embodiments of the subject DNA polymerises that may have undesirably high levels of discrimination against one or more of the labeled terminators used in a sequencing reaction set The subject compositions oftwo or more mutant polymerises ameliorates this problem by simultaneously employing multiple mutant DNA polymerises that are receptive to different chain labeled terittinators, thereby having at least one of the mutant polymerises "compensate" for the discrimination against a particular fluorescently labeled terminator by the other polymerises catalyzing the sequencing reactions. The ratio of the different DNA
polymerises in the composition preferably are selected so as to result in approximately equal levels of total activity for each of the different mutant DNA polymerises. Differences in specific activity between the different mutant polymerises may be taken into account when equalizing total activity ratios between the polymerises. Differences in activity levels between the various mutant DNA polymerises in the subject compositions may also be compensated for by _17_ SUBSTITUTE SHEET ( rule 26 ) adjusting the levels of the different fluorescently labeled terminators in the subject compositions. The subject multiple polymerise compositions may comprise two, three, four, or more difiE'erent mutant DNA polymerises. The mutant polymerise may or may not be derived from the same species or strain. The different mutation DNA
polymerises in the subject mutant polymerise compositions may or may not be receptive for one or more of the fluorescently labeled nucleotides in a given set fluorescently labeled dideoxynucleotides for sequencing.
The invention also includes various methods of using the mutant DNA
polymerises (or subject multiple mutant DNA polymerise compositions) of the invention. The mutant DNA polymerises of the invention may be substituted for the corresponding parent DNA
polymerises in most procedures that employ DNA polymerises. In order to more fully take advantage of the properties of the subject mutant DNA polymerises, the amount (or concentration) of labeled and unlabeled nucleotides used in the methods of the invention may be changed with respect to the amounts (or concentrations) used in the corresponding I S methods employing convention DNA polymerises. These changes in the amount of nucleotide may be optimized by routine experimentation. Methods of the invention comprise the step of extending a primed polynucleotide template with at least one fluorescently labeled nucleotide, wherein the extension is catalyzed by a mutant DNA polymerise of the invention.
Thus, the subject methods result in the formation of one or more dii~erent fluorescently labeled polynucleotides produced by primer extension. The subject methods of synthesizing a fluorescently labeled polynucleotide may be used in a variety of procedures including, but not limited to, Singer sequencing (e.g., dideoxy nucleotide chain termination), the polymerise chain reaction (PCR), polynucleotide labeling, minisequencing. The reduced discrimination against fluorescently labeled nucleotide properties of the subject mutant DNA
polymerise is particularly useful for Singer DNA sequencing reactions, including cycle sequencing. The use of the subject mutant DNA polymerises for Singer sequencing reduces the amount of fluorescently labeled chain terminating nucleotides required for a sequencing reaction an may in many case be used to increase the number of bases that may be identified in single sequencing reaction that is analyzed on an automated fluorescence-based sequencing apparatus such as an Applied Biosystems 310 or 377(Applied Biosystems Division of Perkin-Eimer, Foster City, CA. ). Detailed protocols for Singer sequencing are known to those skilled in the art and may be found, for example in Sambrook et al, Molecular Cloning, A
_18_ SUBSTITUTE SHEET ( rule 26 ) ___T ._ _-__ ~

Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY
( 1989).
The invention also provides kits for synthesizing fluorescently labeled poiynucleotides.
The kits may be adapted for performing specific polynucleotide synthesis procedures such as DNA sequencing or PCR. Kits of the invention comprise a mutant DNA polymerise of the invention and a fluorescently labeled nucleotide that exhibits reduced discrimination with respect to the mutant DNA polymerise in the kit. Kits preferably contain detailed instructions on how to perform the procedures for which the kits are adapted. Optionally, the subject kit may further comprise at least one other reagent required for performing the method the kit is adapted to perform. Examples of such additional reagents include unlabeled nucleotides, buffers, cloning vectors, restriction endonucleases, sequencing primers, and ampiif cation primers. The reagents include in the kits of the invention may be supplied in premeasured units so as to provide for greater precision and accuracy.
Other embodiments of the invention include kits comprising ( I ) the subject compositions of multiple mutant DNA polymerises, and (2) fluorescently labeled chain terminating nucleotides suitable for use with the subject compositions, i.e., each labeled chain terminator is receptive with respect to at least one of the mutant DNA
polymerises in the composition. Additional embodiments ofthe invention include kits for sequencing DNA that comprise a multiple mutant polymerise composition of the invention and at least two different fluorescently labeled chain terminating nucleotides are labeled at different bases, wherein each of the fluorescently labeled chain terminating nucleotides is receptive with respect to at least one mutant DNA polymerise in the composition.
The invention, having been described above, may be better understood by reference to the following examples. The examples are offered, for among other reasons, to illustrate specific embodiment of the invention and should not be construed as a limitation on the invention.
EXAMPLES
Example 1 Purification of Mutant Forms of Tag DNA Polvmerase Lysates ofE. coil containing recombinant constructs designed for the productiion of - l 9-SUBSTITUTE SHEET ( rule 26 ) recombinant mutant Taq DNA polymerases were made essentially as described in tDesai, U.J. and Pfaffle, P.K., Biotechniques, 19:780-784 (1995). In order to prevent the polymerase from binding to chromosomal and plasmid DNAs contaminating the lysate, 5 M NaCl was added dropwise to the heat treated, clarified lysates to bring the final NaCl concentration to 0.25 M. DNA was then precipitated from this mixture by dropwise addition of 5% polyethylimine (in 20 mM TRIS~C1, pH 8.5) to make the final concentration of PEI 0.3%. Precipitation was allowed to continue for 5 minutes on ice.
A white, cloudy precipitate was removed by centrifugation at 15,000 x g for 15 minutes at 4°C. The supernatant fluid was decanted and saved. Following centrifugation, the NaCI
concentration was reduced to 0.13 M by monitoring conductivity of the solution during the addition of TETT minus NaC 1 (20 mM TRIS~C 1, 0.1 mM EDTA. 0.05% Tween-20T"", 0.05% Triton-X100T"", 1% glycerol, pH 8.5).
Excess PEI was removed using a Bio-Rex 70 (BIO-RAD, Richmond, CA) column (2.5 x 30 cm). The column was poured and equilibrated with TETT Buffer + 0.1 M
NaC 1. The polymerase does not bind to the Bio-Rex 70 under these conditions.
To remove contaminating E. coli proteins, the Bio-Rex 70 column eluate was loaded directly onta a Heparin-Agarose (Sigma Chemical Company, St. Louis, MO) column ( 1.5 x 30 cm) which was also poured and equilibrated in TETT Buffer +
0.1 M
NaC 1. The heparinagarose column was washed with 2 column volumes of TETT +
0.1 M NaCl and Taq DNA polymerase was eluted as a sharp peak using TETT + I M
NaCl.
Elution was monitored at 280 nm.
The heparin-agarose column fractions corresponding to the peak absorbance were pooled, and concentrated to 0.15 ml using Ultrafree-15 Centrifugal Filter Devices (Millipore Corporation, MA) according to the manufacture's recommendations for centrifugation speeds and times. The concentrate was diluted to 15 ml with TETT Buffer + 5% glycerol and the sample was re-concentrated to 0.15 ml. This was repeated one more time to reduce the final NaCl concentration below I mM in the protein samples.
The concentrated polymerase samples were diluted two-fold using TETT + 5%
glycerol and an equal volume of TETT -~- 95% glycerol was added to bring the final glycerol concentration to about 50°%. Samples were stored at -20°C. Protein concentrations were determined using the "Bradford Protein Assay" (BIO-RAD, Richmond, CA). Activity was measured using a radiometric assay (described elsewhere).
Typical yields of polymerase from 2-liters of induced E. coli culture (corresponding to 30- 50 ml of heat treated, clarified lysate) ranged from 4 to 24 mg. SDS-PAGE analysis of the purified samples showed one dark band of about 94,000 molecular weight) and several minor ones after Coomassie Blue staining. The gels indicated a typical purification level of > 90% .
Example 2 Selectivity Assay An unlabeled versus dye-labeled terminator assay ("terminator" is defined as a non-extendible base such as 2',3'- ddNTPs) was used to screen mutant Taq DNA
polymerise samples for better Tet{II)~ddCTP incorporating mutant forms of this polymerise. This assay is based upon two substrates competing for the same active site at the same time during a steady state reaction in which only the polymerise concentration is limiting.
Therefore, the assay measure the polymerase's "selectivity" for the unlabeled versus the fluorescein-labeled terminator. The DNA Primer/template used in this assay format is given below:
5'->(FAM)-CCC TCG CAG CCG TCC AAC CAA CTC A
GGG AGC GTC GGC AGG TTG GTT GAG TGC CTC TTG TTT<-5' The next template position following the 3'-end of the primer is indicated above by the bold and underlined G.
The reaction consisted of 80 mM TRIS~Cl (pH 9.0 at 20 °C) 1000 nM DNA primer/tempiate [ S'-(FAM)25mer / 36 G, template ]
2 mM MgCI, 5 0 pM TET(II)~ddCTP
1 pM ddCTP
0.25 Units of enryme 40 pL reaction volume 60 °C reaction temperature Samples (2 pL) were removed from the reaction mixture at pre-determined times (typically, 20 second intervals for 0.25 Units of polymerise activity per pL) and added to ice SUBSTITUTE SHEET ( rule ?6 ) cold 50 ~tL 0.5 M EDTA (pH 8.0). Timed aliquots were mixed and held on ice for further processing.
Samples of each time point were processed to remove excess, unincorporated TET(II)~ddCTP. Typically, 1.6 pL of each quenched sample were added to 250 ~tL
of 0.8 S M LiCI plus 0.2 pg/ml E. coli tRNA. followed by 750 uL of 95% ethanol. After mixing, the nucleic acids were allowed to precipitate for 20 minutes at -20 °C. The precipitates were recovered by centrifugation using standard procedures. The supernatant fluid was discarded and pellets were dissolved in 50 pL of SO% forntamide. Gel samples were heat treated (95 °C
for 2 minutes) and 2 pL were loaded per sample lane on a 16% denaturing DNA
sequencing gel. Gels were run on an Applied Biosystems Model 373 Sequencer using GeneScan Fragment Analysis software to measure the amount of FAM fluorescence in the bands corresponding to the 25-mer primer, the 26-mer product (indicating a ddC
incorporation event) and the apparent " 27-mer" product band (indicating a TET(II)~ddC
incorporation event).
The fluorescence signal in each of the bands was summed and the percent of signal in each band was used for further calculations as a normalization to avoid Iane to lane loading differences. Energy transfer from the %-FAM moiety present on the apparent "27-mer"
product molecules to the Tet(II) moiety on the newly incorporated 3'-base was not corrected since all ratios were compared to "wild type" or Taq G46D.) The normalized fluorescent signals in the 26-mer and "apparent" 27-mer product bands were corrected for the different concentrations of the two molecules used in the reaction and the corrected values were plotted versus time. The velocity of incorporation for each substrate was determined using least square fits to the data. The ratio of ddC / TET(II)~ddC incorporation rates is equal to the selectivity bias that the sample poiymerase shows for the unlabeled versus the TET(II)-labeled nucleotides and reflects the following relationship:
vaac -vT~~.aac - (~,~ / ~1 sac ( ddC ]= (Iy / I~,i ~«.aac ~ Tet(II)~ddC
where:
vac - velocity of ddC incorporation vmunraac - velocity of Tet(II)~ddC incorporation - catalytic rate constant SUBSTITUTE SHEET ( rule 26 ) KM - nucleotide equilibrium binding constant [ ddC ] - concentration of ddCTP in the reaction [ Tet(II)~ddC ] - concentration of Tet(II}~ddCTP in the reaction In this assay format, "wild-type" Taq or (Taq G46D) showed a selectivity bias or ddC
Tet(II)~ddC number of about 85 to 1. Mutants showing lower selectivity bias ratios were submitted to further testing. The Table 2 below shows the results for a few of the mutants tested by way of a few examples:
Table 2 Tai Selectivity Number WT / Mutant G46D 85 85 / 85 or G46D; R660D 8 85 / 8 or ~

G46D; R595E 28 85 / 28 or ~

G46D; F667Y 28 85 / 28 or =

G46D; E681G 40 85 / 40 or =

G46D; D655L 40 85 / 40 or ~

Example 3 Next Nucleotide Rate Effect Assay An additional kinetic step between "ground state" nucleotide binding or initial collision and correct base pair formation and the group transfer reaction would be expected to slow the polymerise dissociation rate from an Enz~DNA complex having a 3'-dideoxynucleotide in an assay termed the "Next Nucleotide Rate Effect" (Patel et al., 1991 ).
This assay measures the steady state rate of incorporation of ddTTP (i.e., the enzyme is limiting) in the absence or presence of the next correct nucleotide. The primer template pair is shown below:
5'->(FAM)-CCC TCG CAG CCG TCC AAC CAA CTC A
GGG AGC GTC GGC AGG TTG GTT GAG TAG GTC TTG TTT<-5' The next template position is indicated by the bold, underlined A. The next template SUBSTITUTE SHEET ( rule 2G ) _«-position beyond A is G. Under steady state reaction conditions; essentially all of the available polymerise is bound,to the primer/template. When ddTTP is present alone in solution, it is incorporated following binding to its template position, A: Additional incorporation events require the polymerise to dissociate from the Enz~DNA complex and find another available S primer/template that, has not already undergone and incorporation event.
Hence the rate of incorporation under', these conditions is the dissociation rate of the polymerise from the Enz~DNA complex.' If the next correct nucleotide, dGTP or ddCTP, is also present in the reaction mixture, the dissociation rate ofthe polymerise from the Enz~DNA~ddCTP complex, for example; will be slower if there is an additional kinetic step between the group transfer reaction that incorporated the ddTTP and an attempt by the polymerise to incorporate ddCTP
in a processive mode of synthesis. This slower rate of dissociation an be detected as a slower incorporation rate of ddTTP since no chemistry can occur once ddTTP and the polymerise can no be processive-despite the presence of another correct nucleotide. As shown in Figure 2, the presence of the next correct nucleotide does indeed slow the turnover or dissociation 1 S rate of the polymerise (Taq G46D; F667Y). Figure 2 also shows that the presence of a fluorescein dye on the next correct nucleotide (in this case, Tet(II)~ddCTP), appears to accelerate the turnover rate. We interpret this to mean that the polymerise is constantly undergoing a conformational change and that it can attempt to undergo the change even in the absence of the next correct nucleotide. However, the presence of a fluorescein dye on the next correct nucleotide blocks the ability of the polymerise to undergo such a change and thereby causes an immediate dissociation of the enzyme following the group transfer step for ddTTP incorporation: Hence; the fluorescein dye appears to accelerate the polymerise dissociation rate by eliminating a kinetic step (or steps) following the group transfer reaction.
Figure 3 shows the results for a Next Nucleotide Rate Effect assay for a "multiple"
2S mutant form of Taq DNA polymerise, Taq G46D; R660D; F667Y; E681 G. In this case, the presence of Tet(II) on the next correct nucleotide is "transparent" to the mutant polymerise.
We interpret this to mean that the mutant polymerise can indeed undergo the same kinetic steps following group transfer that "wild-type" versions of this polymerise undergo. We also interpret these results to indicate that the F667Y mutation belongs in a different class than the R660D or E681G mutations since Taq G46D; F667Y still shows a "fluorescein-effect" in the "Next Nucleotide Rate Effect" assay, however, the multiple mutant, Taq G46D;
R660D;
F667Y; E681 G, does not.

SUBSTITUTE SHEET ( rule 26 ) Printed by VisuaIPatent Typical assay conditions for the Next Nucleotide Effect assay were as follows:
1000 nM primer/template DNA
80 mM TRIS~Cl(pH 9.0 @ 20° C) 2.4 mM MgCh 0.02 UnitsIpL polymerise activity 400 pM each nucleotide (when present) Samples were taken and processed in the same manner as described under "Selectivity Assay." In this case, it is possible to distinguish a ddC-incorporation event from a Tet(II)~ddC incorporation event by the migation rate of the resulting fragments in a 16% gel.
Incorporation of ddC results in a "normal" 26-mer band that migrates as expected above or slower than the 25-mer primer. Incorporation of Tet(II)~ddC results in slower migration causing the band to migrate with an apparent size equivalent to a 27- or 28-mer.
Examoie 4 Analysis of Additional Mutants Table l, provided beiaw, provides a summary of results obtained with selectivity assays performed with several different Taq mutants. The analogous site for the mutation in the enzymes E coif DNA polymerise I and phage T7 DNA polymerise are also noted. The term "FS" refers to a Taq DNA polymerise having a F667Y mutation.
References Barnes, W.M. ( 1992) The fidelity of Taq polymerise catalyzing PCR is improved by an N-terminal deletion. Gene 112: 29-35.
Brandis, J.W., Edwards, S.G. and Johnson, K.A. (1996) Slow rate of phosphodiester bond formations accounts for the strong bias that Taq DNA polymerise shows against 2',3'-dideoxynucleotide terminators. Biochemistry 3S: 2189-2200.
Desai, U.J. and PfafRe, P.K. Single-step purification of a thermostabie DNA
polymerise expressed in Escherichia coli. Biotechnignes l 9: 780-784.

SUBSTITUTE SHEET ( ruie 26 Printed by VlsualPatent Fersht, A. (1985) in "Enzyme Structure and Function," W.I-I. Freeman and Company, 2nd ed., pp. 111-112.
Johnson, K.A. (1993) Conformational coupling in DNA polymerase fidelity. Ann.
Rev. Biochem. 62: 685-7 13.
Patel, S.S., Wong, L, and Johnson, K.A. (1991) Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30: 511-525.
Eguivalents While the invention has been described and illustrated with reference to specific embodiments, those skilled in the art will recognize that modifications and variations may be made without departing from the principles of the invention as described hereinabove and set forth in the following claims.

No.Taq pollTT (Final)'Unita~ T'eT(11)ddCTP/ 7AMRAdTTP/
Mutant Equiv.Equiv.Icg/plpa Spve.ROXddCTP/ l ddTTP
p Aet.ddCTP ddCTP

(Mut I Wt ]
- I (Mut I Wt - J I (Mut I
Wt j 1 G46D ~ ~ 4.4 200 1 I ~ I 4a.61 I
t I

I I I 28.019501 ~~~~-_"4.,lae~_,~y _G46D _ I 69.6t-..~_ (LS.Iy I ~

.
I G46D fLS.21 I I 78.362401 ~
I ~ 79.7-,..~
. . ,;7~_ ' ~

I ~c _~s ' I f3 I I Fh5 I 3 I J
_ I I 9 2 0 ~~ _ 2 G46D F667W F762Y5263.6 5 1.411 1 1 I
I

(LS.t) F762Y62625.015016.0 ' S 6480 RST3E 8668842911.001 0.0 I

4 6460: E6tSL E710E4804.8 0 0.0 S 6480 EB15D w E710E48010.4600 57.7~,~.'''rrie~%"
I G46D EB151 E710E4807.0 140 20.0",.
6 G46D RSBTK RB82V4438.9 420 4T.2:..~nosiistNl 7 1 1 ~ TO;

8 Ci48D R573K ' R66884299.3 0 9 6460 LBST>' ET52T5179.5 450 47.41 t 10G46D R58TK 8682'V443nd nd 1 6460 GT54S 0849Q815f 0 0.0 1 3.0 126460 E61SK 10 E4801.8 0 0.0 toG46D R573C 8868_8429t4.oaoo ST.tt t t G48D D6SSL T7S0LS151t.3400 35.42 0.7 tSG48D 07S4K 08490815nd nd nd 16G48D R59SK 8890H460nd nd nd t 6460 K631M H926H7048.3 300 38.11 1 T

t G48D LB82G LT77'1540'5.3 200 36.41 t 6 19G4aD RsssK 8754DSt922.91s s.e . aa~aEtttl.

20G4so Asa3E N7T6v64112.ssoo 70.31 21G48D G7S4K Q849QB155.0 0 0.0 - __ _ 22G4so Rs93H 8666Essa1s.7Too 41.9 23G48D.RS95E 8890H48023.5S0 2.1 3 24G4BD A6a3V N778V54f11.4340 29.81 25G48D t7592A 8887A45Tnd nd nd G48D R680D RTSSDSt913.31sa 14.3== =;t0 ~~:~
~ ~1 2 Q46D TsIOG 8735507 t 22 1 1 7 3.0 5 T.3 28G4sD EsstG 077 '1540'7.S 1T0 - 2 - 22.7 29G4iD V664E V749514 !.2 210 22.8 3 G46D Qsl3E 6708_64781 71 4.7 0 ' S.2 31G4sD DstoA 07050475ls.s0 0.0 32G48D E820K E917883 11.6475 40.9 33G46D L8t7A L9t4L890iS.24T0 30.9 34G4sD Isa4G 17796542 356480 RiBOD F887Y 19.1179 9.4 10 3 G46D RS96D R880D 10.50 0.0 s F887Y

3TG48D D6dSL Rt60D 18.8228 12.1i 0 Fdi7Y

38Gi6D RB60D Fi67Y~E881G 13.1404 30.812 3 6480 RS93E FH6TY 9.5 0 0.0 9 G~~ ~ L~ JK 12.70 0.0 4 G4sD asa2css3 13.20 0.0 _ _ 4zGssD: psass s7st ts.zsso 34.s _ _ K73t (ysatas aeL

tvstvd -.
_ ___ mutants >
'wt' IArf ~

Table _z~_ SUBSTITUTE SHEET ( rule 26 ) R6b4- Mutants Lvsate enotvne Specific ActivityTet Selectivity ~

TE'i (II]ddCTP
/ ddCTP

(iYlutant / V'I~

Acidic-r~spar~ic 29 CS; R660D 14 10' acid 38 FS: R660D 9 10 39 R595E; FS: R660D0 nd 40 Db52; FS; R660DI2 10 41 FS; R660D; E681G~ 31 1 ~

49 CS; R660D 41 n,~

Glutamic 51 ' FS; R660E; 11 7 Acid E681G

72 FS;R660E 1 ,7 a i -Lysine 50 FS; R660K 28 Icy Histidinc 101 FS; R660H I3 1 Imino-Proline 66 FS; R660P 8 1 -t~liohatic-Alanine 68 FS; R660A 4 4 Isoleucine73 FS; R6bOZ 5 0.g***

Valinc 90 FS; R660V 10 I

55 FS; R660V; E681G1 I

L.eucine 91 FS: G660L 8 0.6"*

52 FS; R.660L; 28 1 Glycinc 47 FS; R660G: E681G18 6 78 FS: R.660G 8 2 Polar Unchar~ed-Glucaminc 53 CS; R6b0Q, 47 I

69 FS; R660Q 5 3 Setine 98 FS; R.660S 16 7 Cystcine 93 FS; R660C 14 4 Asparaoine97 FS: R660N I3 3 Thrconinc 96 FS; R660T 26 3 Mcthioninc romatic-Phcnyalaniac92 FS: R.660F 9 0.1"*

Tyiosine 95 FS; R660Y I7 1 Table 3 E6g1-Mutants v Soeciac ActivitvTet Selectivity at r eriotv a Tl_ I"(11]ddGTP /
ddCTP

(Mucarc / W'n cidic-Aspartic 71 FS; Eb8lD 9 4**
acid Lysine 75 FS:1~68IK 52 Ark Histidinc86 FS; E681H 37 7 Im~no .

ProIine 74 FS; E681P 19 9 Aliehatic-Alaninc b3 FS; E681A I3 ~6 lsoleucinc99 FS; E681I 37 27 VaIine 76 FS; EbBI V 110 10 Leucine 87 FS; Eb8lL 22 14 Glycinc ,48 FS; Eb8lG 37. 6 ' polar arged.
Unch Glutamine Serine b FS; E681 S 12 S
l Cysteinc ~88 FS; Eb8lC 20 2 Aspara~uuc89 FS; Efi8lN 40 4 Thieoninc81 FS; EbBIT 35 6 Methianine~ FS; E681M 32 47 Aro~~tic-Phenyalaninc Tyrosine 80 FS; E681Y 42 3 Tryptophan84 FS; E681W 37 I7 *Ratio means > 1 improved TET(II~ddCTP
incorporation.

enzyme.

**Ratio meanswild-type activity.
= 1 ***Ratio meansactivity worse < 1 than wild-rypc.

M > I >~N > L >
V > P > H=K=G=T=.S
> D=A=N > Y=C

''Table 3 (Continued) Tryptophan 9~t FS; R660W
D > E=S > C=A=Q=T =V> G > K=P=V=Y=W=H > I = L » F
~ 7 4 4 3 3 3 2 1 I 1 1 1 0.9 0.6 O.I
*Ratio > 1 means improved TET(IyddCTP incorporation. blast be "8~" to be "transparent to the enryme.
**Ratio = 1 means wild-type acdvir~.
***Ratio < 1 means activity worst than wild-type.
'Table 3 (Continued) SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: THE PERKIN-ELMER CORPORATION
(B) STREET: 850 Lincoln Centre Drive (C) CITY: Foster City, California (E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 94404 (ii) TITLE OF INVENTION: DNA POLYMERASES HAVING IMPROVED LABELED
NUCLEOTIDE INCORPORATION PROPERTIES
(iii) NUMBER OF SEQUENCES:4 (iv) CORRESPONDENCE ADDRESS:
John H. Woodley Sim & McBurney 330 University Avenue, 6'" Floor Toronto, Canada MSG 1R7 (v) COMPUTER READABLE FORM:
(A) COMPUTER: IBM PC compatible (B) OPERATING SYSTEM: PC-DOS/MS-DOS
(C) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (vi) CURRENT APPLICATION DATA:
(A)APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER: US 60/039,610 (B) FILING DATE: March 12, 1997 (C) CLASSIFICATION:
(viii) PATENT AGENT INFORMATION
(A) NAME: John H. Woodley (B) REFERENCE NUMBER: JHW 5565-47 (2) INFORMATION FOR SEQ ID NO. 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides (B) TYPE: nucleic acid (C) STRANDENESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1:
ccctcgcagc cgtccaacca actca 25 (2) INFORMATION FOR SEQ ID NO. 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 nucleotides (B) TYPE: nucleic acid (C) STRANDENESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:
gggagcgtcg gcaggttggt tgagtgcctc ttgttt 36 (2) INFORMATION FOR SEQ ID NO. 3 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides (B) TYPE: nucleic acid (C) STRANDENESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 3:
ccctcgcagc cgtccaacca actca 25 (2) INFORMATION FOR SEQ ID NO. 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 nucleotides (B) TYPE: nucleic acid (C) STRANDENESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4:
gggagcgtcg gcaggttggt tgagtaggtc ttgttt 36

Claims (12)

WHAT IS CLAIMED IS:

1. A DNA polymerase having at least one mutation in the nucleotide label interaction region, said nucleotide label interaction region being selected from the group consisting of (i) the O-helix, (ii) the K helix, and (iii) the inter O-P
helical loop, wherein the mutation is at an amino acid residue position selected from the group consisting of R595, D655 and E681, wherein the position of said amino acid residue within the DNA
polymerase being defined with respect to Taq DNA polymerase and wherein the DNA
polymerase has reduced discrimination for a fluorescein-type dye labeled nucleotide as compared with the naturally occurring DNA polymerase.

2. The DNA polymerase according to claim 1, wherein the DNA polymerase is Taq DNA polymerase.

3. The DNA polymerase according to 2, wherein the mutation is selected from the group consisting of D655L, E681G, and R595E.

4. The DNA polymerase according to claim 3, comprising a mutation set belonging to the group consisting of (G46D, R595E, R660D, F667Y), and (G46D, R660D, F667Y, E681G), and (G46D, F667Y, E681G).

5. The DNA polymerase according to claim 1, wherein the DNA polymerase is a thermostable DNA polymerase.

6. A polynucleotide encoding a DNA polymerase according to claim 1.

7. An expression vector having a promoter, wherein the vector comprises a polynucleotide according to claim 6 in functional combination with the promoter.

8. A host cell comprising an expression vector according to claim 7.

9, A method of synthesizing a fluorescently labeled polynucleotide, said method comprising extending a primed polynucleotide template with at least one fluorescently labeled nucleotide, wherein the extension is catalyzed by a mutant DNA
polymerase according to claim 1.

10. A method according to claim 9, wherein the primed template is a primed template in a chain termination sequencing reaction.

11. A method according to claim 9, wherein the primed template is a primed template in a polymerase chain reaction.

12. A kit for fluorescently labeling a polynucleotide, the kit comprising a DNA
polymerase according to claim 1 and a fluorescently labeled nucleotide.