Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-03
2002-02-12
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C530S300000, C530S327000, C536S023100, C536S023200
Reexamination Certificate
active
06346379
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to thermostable DNA polymerases which have enhanced efficiency for incorporating nucleoside triphosphates labeled with fluorescein family dyes. The present invention provides means for isolating and producing such altered polymerases. The enzymes of the invention are useful for many applications in molecular biology and are particularly advantageous for nucleic acid sequencing.
BACKGROUND OF THE INVENTION
Incorporation of nucleoside triphosphates (dNTPs) labeled with fluorescent dyes is important for many in vitro DNA synthesis applications. For example, dye-terminator DNA sequencing reactions require the incorporation of fluorescent dideoxynucleotide analogues for termination and labeling. In addition, in vitro synthesis of labeled products may involve incorporation of fluorescent nucleotides or nucleotide analogues. For example, fluorescently labeled DNA has been used in hybridization assays using microarrays of immobilized probes (Cronin et al., 1996
, Human Mutation
7:244).
To assure fidelity of DNA replication, DNA polymerases have a very strong bias for incorporation of their normal substrates, referred to herein as conventional deoxynucleoside triphosphates (dNTPs), and against incorporation of unconventional dNTPs including dNTPs and dNTP analogues labeled with fluorescent dyes. In the cell, this property attenuates the incorporation of abnormal bases such as dUTP in a growing DNA strand. In vitro, this characteristic is particularly evident where both conventional and unconventional fluorescently-labeled nucleoside triphosphates are present, such as in DNA sequencing reactions using a version of the dideoxy chain termination method that utilizes dye-terminators (Lee et al., 1992
, Nuc. Acids. Res
. 20:2471 which is incorporated herein by reference).
Commercially available DNA cycle sequencing kits for dye-terminator methods use chain terminator ddNTPs labeled with fluorescent dyes of the rhodamine family. However, rhodamine dyes are zwitterionic in charge and nucleoside triphosphates labeled with these dyes migrate anomalously in the electrophoretic gels used to separate the sequencing products for detection. This property of rhodamine family dyes necessitates making modifications in the standard sequencing protocol which include the use of dITP and an additional processing step before electrophoresis.
In contrast, negatively charged fluorescent dyes such as fluorescein family dyes allow 1) better separation between the labeled nucleoside triphosphates and labeled primer extension products, and 2) better electrophoretic migration of the labeled sequencing products than neutral or positively charged fluorescent dyes. Thus, the use of fluorescein family dyes avoids the need for additional processing steps required with the use of rhodamine family dyes. However, available dyes of the fluorescein family are not ideal for use in current commercially available DNA cycle sequencing formats because ddNTPs labeled with these dyes are not efficiently incorporated into sequencing products using these formats. Consequently, there is a need for commercially available thermostable DNA polymerases that can efficiently incorporate both conventional and fluorescein-labeled nucleotides. The present invention serves to meet that need. Further, an unexpected property of the mutant enzymes of this invention is the increased rate of primer extension relative to the corresponding wild-type enzyme. Another unexpected property is the increased uniformity of incorporation of the various terminator nucleotides in automated DNA sequence analysis.
SUMMARY OF THE INVENTION
The present invention provides template-dependent thermostable DNA polymerase enzymes having reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes compared to previously characterized enzymes. These enzymes incorporate nucleotides, including deoxynucleotides (dNTPs) and base analogues such as dideoxynucleotides (ddNTPs), that are labeled with fluorescein family dyes more efficiently than conventional thermostable enzymes. Genes encoding these enzymes are also provided by the present invention, as are recombinant expression vectors for providing large amounts of purified enzymes.
By the present invention, a region of criticality within thermostable DNA polymerases is identified which affects the polymerase's ability to incorporate nucleotides labeled with fluorescein family dyes, while retaining the ability to incorporate faithfully natural nucleotides. This region of criticality, or Critical Motif, can be introduced into genes for thermostable DNA polymerases by recombinant DNA methods such as site-specific mutagenesis to provide the advantages of the invention.
Thus, in one aspect, the invention provides recombinant thermostable DNA polymerase enzymes which are characterized in that the enzymes have been mutated to produce the Critical Motif and have reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes, in comparison to the corresponding wild-type enzyme.
In this aspect, the invention provides recombinant thermostable DNA polymerase enzymes which are characterized in that a) in its native form said polymerase comprises the amino acid sequence (given in one-letter code) LSXXLX(V/I)PXXE (SEQ ID NO: 1), where X is any amino acid; b) the X at position 4 in said sequence is mutated in comparison to said native sequence, except that X is not mutated to E; and c) said thermostable DNA polymerase has reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes in comparison to the native form of said enzyme. In the three-letter code, this amino acid sequence is represented as LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 1), whereby “Xaa” at positions 3, 4, 6, 9, and 10 of this sequence are any amino acid residue, and “Xaa” at position 7 of this sequence is Val or Ile.
In another embodiment, the recombinant thermostable DNA polymerases are characterized in that a) the native form of the polymerase comprises the amino acid sequence LS(Q/G)XL(S/A)IPYEE (SEQ ID NO: 2), where X is any amino acid; b) the X at position 4 in said sequence is mutated in comparison to said native sequence, except that X is not mutated to E; and c) said thermostable DNA polymerase has reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes in comparison to the native form of said enzyme. In the three-letter code, this amino acid sequence is represented as LeuSerXaaXaaLeuXaalleProTyrGluGlu (SEQ ID NO: 2), whereby “Xaa” at position 3 is Gln or Gly, “Xaa” at position 4 is any amino acid, and “Xaa” at position 6 is Ser or Ala. In a preferred embodiment, the amino acid sequence is LSQXLAIPYEE (SEQ ID NO:3), where X is any amino acid. In the three-letter code, this amino acid sequence is represented as LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby “Xaa” at position 4 is any amino acid. In a more preferred embodiment, the “Xaa” at position 4 is Lys.
In yet another embodiment, the recombinant thermostable DNA polymerases are characterized in that a) the native form of the polymerase comprises the amino acid sequence LSVXLG(V/I)PVKE (SEQ ID NO: 4); b) the X at position 4 in said sequence is mutated in comparison to said native sequence, except that X is not mutated to E; and c) said thermostable DNA polymerase has reduced discrimination against incorporation of nucleotides labeled with fluorescein family dyes in comparison to the native form of said enzyme. In the three-letter code, this amino acid sequence is represented as LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4), whereby “Xaa” at position 4 is any amino acid and “Xaa” at position 7 is Val or Ile. In a preferred embodiment, the amino acid sequence is LSVXLGVPVKE (SEQ ID NO: 5) where X at position 4 is any amino acid. In the three-letter code, this amino acid sequence is represented as LeuSerValXaaLeuGlyValProValLysGlu (SEQ ID NO: 5), whereby “Xaa” at position 4 is any amino acid.
Gelfand David Harrow
Kalman Lisa Vivian
Myers Thomas W.
Reichert Fred Lawrence
Sigua Christopher Lim
F. Hoffman-La Roche AG
Jones W. Gary
Souaya Jehanne
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