USE OF TAQ POLYMERASE MUTANT ENZYMES FOR NUCLEIC ACID AMPLIFICATION IN THE PRESENCE OF PCR INHIBITORS

Abstract

The present invention generally relates to detection of a target nucleic acid in standard PCR, real-time PCR, RT PCR, and real-time RT PCR. One aspect of the invention provides mutant DNA polymerase enzymes that are resistant to PCR inhibitors, such as dye, blood, and soil. Another aspect of the invention provides for methods of real-time PCR assays using mutant DNA polymerase enzymes resistant to PCR inhibitors with samples containing dye, blood, and/or soil. Another aspect of the invention provides for methods of standard PCR assays using mutant DNA polymerase enzymes resistant to PCR inhibitors with samples containing blood and/or soil.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of PCT/US07/78571, filed Sep. 14, 2007, which claims priority from U.S. Provisional Application Ser. No. 60/825,692 filed on Sep. 14, 2006, each of which is incorporated herein by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form, filed electronically via EFS-Web, comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to nucleic acid detection in standard and real-time PCR and RT PCR.

BACKGROUND

Standard and real time polymerase chain reaction (PCR) assays are limited by inhibitors found in many common sample types as well as components of the PCR assays themselves. Examples of PCR inhibitors include those found in commercial dyes used in PCR as well as those found in blood and soil.

Commonly used fluorescent dyes, such as SYBR Green, can significantly inhibit the Taq polymerase (GenBank Accession No. J04639; SEQ ID NO: 4) at concentrations higher than about 0.25-1×. This dye inhibition of Taq polymerase imposes limitations on the sensitivity and product specificity and may cause dye-dependant false negative results (see e.g., Monis et al. (2005) Anal Biochem 340, 24-34; Stubner (2002) J Microbiol Methods 50, 155-64; Nath et al. (2000) J Biochem Biophys Methods 42, 15-29; Gundry et al. (2003) Clin. Chem. 2003; 49:396-406).

The success and sensitivity of DNA detection in important clinical, diagnostic and forensic applications of PCR of blood specimens is limited by the presence of blood inhibitors of Taq polymerase, such as the heme, IgG fractions, and other blood components. Plain Taq enzyme can be completely inhibited between 0.004% and 0.2% blood (vol/vol) (see e.g., Al-Soud et al. (2000) J. Clin. Microbiol. 38, 4463-70; Al-Soud et al. (2000) J. Clin. Microbiol. 38, 345-50; Al-Soud et al. (1998) Environ. Microbiol. 64, 3748-53). In order to overcome this inhibition, high cost and additional labor-demanding methods are currently used to purify DNA from blood prior to PCR. Nevertheless, this inhibition is still a serious concern with many PCR-based human blood tests, since even after purifying DNA from the blood, traces of the PCR inhibitors can generate as high as 14% false negative results, as published for hepatitis B blood tests (Kramvis et al. (1996) J Clin Microbiology 34, 2731-2733).

Sensitive and precise PCR detection of microorganisms in soil is necessary, for example, in specific agricultural purposes, infectious disease control, and bioterrorism related pathogen tests. Direct extraction of total DNA from soil samples results in a co-extraction of humic acid, known as the most potent soil inhibitor to PCR analysis. Humic substances represent a mixture of partially characterized polyphenols that are produced during the decomposition of organic matter. Other inhibitory components include fulvic acid, polysaccharides and metal ions that can be present in varying concentrations in the soil samples (see e.g., Tsai et al. (1992) Environ. Microbiol. 58, 2292-2295; Watson et al (2000) Can. J. Microbiol. 46, 633-642; Yeates et al. (1998) Biol. Proced. Online 1, 40-47; LaMontagne et al. (2001) Journal of Microbiological Methods 49, 255-264).

A general technical problem with the soil samples is the high variation in the concentrations of various inhibitory substances, depending on the soil source, which may generate inconsistent results. This fact significantly complicates the development of standard DNA purification protocol for processing the samples before PCR. Unlike blood, crude soil extracts exhibit relatively little or no fluorescence quenching effect in real-time PCR.

U.S. patent application Ser. No. 11/005,559 (incorporated herein by reference in its entirety) describes blood-resistant mutants of Taq DNA polymerase, which can be used to amplify the DNA target directly in blood in standard, non-real-time PCR. Unlike existing protocols, this method requires no DNA purification steps prior to PCR, thus reducing the time and cost of important clinical blood tests. Today's PCR analyses, especially clinical and forensic analyses, increasingly utilize real-time PCR protocols, which allow accurate quantitation of the data. The blood, however, has a strong quenching effect on the fluorescence detected in real-time PCR, and such problem has previously been solved by using higher fluorescent dye concentrations.

Thus, there exists a need for inhibitor-resistant DNA polymerases and methods of their use for gene detection in standard and real-time PCR, both in the presence and absence of blood and soil in the samples, and for real-time assays, the presence of dye.

SUMMARY

Disclosed herein are simple methods for real-time PCR analyses of samples utilizing mutant polymerase enzymes that are dye-resistant; dye- and blood-resistant; dye- and soil-resistant; and or dye-, blood- and soil-resistant.

One aspect provides a method of amplifying a target nucleic acid in a real-time polymerase chain reaction (PCR). The method can include forming an assay mixture comprising a sample, where the sample comprises a target nucleic acid; a plurality of primers specific for the target nucleic acid; a buffer suitable for real-time PCR; at least one dye; and at least one polymerase having dye-resistant activity. The method can include amplifying the target nucleic acid in the assay mixture in a real-time PCR.

In some embodiments, the target nucleic acid is a DNA molecule. In some embodiments, the target nucleic acid is an RNA molecule. In some embodiments, the real-time PCR is a real-time reverse transcriptase (RT) PCR. In some embodiments, the target nucleic acid is an RNA and the real-time PCR is a real-time reverse transcriptase (RT) PCR. In some embodiments, the target nucleic acid is an RNA; the real-time PCR is a real-time reverse transcriptase (RT) PCR; and the assay mixture further comprises a reverse-transcriptase

In some embodiments, the at least one polymerase is a dye-resistant and blood-resistant polymerase. In some embodiments, the at least one polymerase is a dye-resistant and soil-resistant polymerase. In some embodiments, the at least one polymerase is a dye-resistant, blood-resistant, and soil-resistant polymerase.

In some embodiments, the at least one polymerase has a polypeptide sequence comprising SEQ ID NO: 1. In some embodiments, the at least one polymerase has a polypeptide sequence at least 95% identical to SEQ ID NO: 1 and polymerase activity. In some embodiments, the at least one polymerase has a polypeptide sequence at least 95% identical to SEQ ID NO: 1; comprises at least one amino acid substitution at an amino acid residue position selected from the group consisting of positions 626, 707, and 708; and has polymerase activity.

In some embodiments, the at least one polymerase has a polypeptide sequence comprising SEQ ID NO: 2. In some embodiments, the at least one polymerase has a polypeptide sequence at least 95% identical to SEQ ID NO: 1 and dye-resistant polymerase activity. In some embodiments, the at least one polymerase having a polypeptide sequence at least 95% identical to SEQ ID NO: 2 comprises at least one amino acid substitution at an amino acid residue position selected from the group consisting of 626, 707, and 708.

In some embodiments, the sample further comprises whole blood or a blood component. In some embodiments, the blood component is blood plasma or blood serum. In some embodiments, the whole blood is at least about 1% up to about 25% of a total volume of the PCR assay mixture. In some embodiments, the blood component is at least about 1% up to about 25% of a total volume of the PCR assay mixture.

In some embodiments, the sample further comprises soil or soil extract. In some embodiments, the soil is at least about 1% up to about 50% of a total volume of the PCR assay mixture. In some embodiments, the soil extract is at least about 1% up to about 90% of a total volume of the PCR assay mixture. In some embodiments, the soil comprises a humic acid and the soil is present in the assay mixture at a soil equivalent amount that provides up to about 25 ng of humic acid per 50 uL reaction volume. In some embodiments, the soil extract comprises a humic acid and the soil extract is present in the assay mixture at a soil extract equivalent amount that provides up to about 25 ng of humic acid per 50 uL reaction volumeor soil extract

In some embodiments, the at least one dye is a fluorescent dye selected from the group consisting of SYBR Green, Ethidium Bromide, PICO, TOTO, YOYO or LC Green. In some embodiments, the at least one dye is SYBR Green. In some embodiments, the dye is present in the PCR assay mixture at least about 0.5× up to about 256×, where × is a manufacturer unit for concentration for use in PCR.

In some embodiments, the assay mixture further comprises a PCR enhancer. In some embodiments, the PCR enhancer comprises betaine.

Another aspect provides an isolated polypeptide having dye-, soil-, or blood-resistant polymerase activity, or a combination thereof. In some embodiments, the isolated polypeptide comprises a polypeptide sequence of SEQ ID NO: 2. In some embodiments, the isolated polypeptide comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 2 and having dye-, soil-, or blood-resistant polymerase activity, or a combination thereof. In some embodiments, the isolated polypeptide comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 2 comprising at least one amino acid substitution at an amino acid residue position selected from the group consisting of 626, 707, and 708, and having dye-, soil-, or blood-resistant polymerase activity, or a combination thereof.

Another aspect provides a method of amplifying a target nucleic acid in a polymerase chain reaction (PCR) comprising forming an assay mixture comprising a sample comprising a target nucleic acid, wherein the sample optionally contains whole blood or a blood component selected from blood plasma or blood serum, or soil or soil extract; primers specific for the target nucleic acid; a buffer; at least one polymerase described above; and amplifying the target nucleic acid in the assay mixture in a PCR.

Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a series of images and graphs depicting results of PCR amplification of a 0.6 kbp Bacillus cereus target DNA with wild type KlenTaq and mutant KlenTaq 10 polymerases conducted in the presence of 7 twofold decreasing concentrations of SYBR Green. FIG. 1A is gel electrophoresis analysis. FIG. 1B is melting curve analysis for wild type KlenTaq. FIG. 1C is melting curve analysis for KlenTaq-10. Further details regarding methodology are presented in Example 1.

FIG. 2 is a series of images and graphs depicting results of PCR amplification of a 0.6 kbp Bacillus cereus target DNA with mutant FLAC-22 and a commercial enzyme, Fast Start, conducted in the presence of 7 twofold decreasing concentrations of SYBR Green. FIG. 2A is gel electrophoresis analysis. FIG. 2B is melting curve analysis for FLAC-22. FIG. 2C is melting curve analysis for Fast Start. Further details regarding methodology are presented in Example 3.

FIG. 3 is a series of images and graphs depicting results of PCR amplification of an exogenous 0.6 kbp Bacillus cereus target DNA with wild type KlenTaq and mutant KlenTaq10 polymerases in the presence of 5 and 10% blood in addition to very high concentrations of SYBR with subsequent decreasing amounts of SYBR. FIG. 3A is gel electrophoresis analysis. FIG. 3B is melting curve analysis for wild type KlenTaq with 10% blood. FIG. 3C is melting curve analysis for wild type KlenTaq with 5% blood. FIG. 3D is melting curve analysis for KlenTaq-10 with 10% blood. FIG. 3E is melting curve analysis for KlenTaq-10 with 5% blood. Further details regarding methodology are presented in Example 3.

FIG. 4 is a series of images and graphs depicting results of PCR amplification of an exogenous 0.32 kbp 16 S microbial target DNA with FLAC-22 and Fast Start Taq polymerases in the presence of constant 5% blood (excluding the positive control) and decreasing amounts SYBR green. FIG. 4A is gel electrophoresis analysis. FIG. 4B is a melting curve analysis for FLAC-22. FIG. 4C is a melting curve analysis for Fast Start. Further details regarding methodology are presented in Example 4.

FIG. 5 is a series of images and graphs depicting results of PCR amplification with FLAC-22, KlenTaq-10, Fast Start, Jump Start, and AmpliTaq Gold Taq polymerases in the presence of 6 two-fold decreasing concentrations of SYBR green. FIG. 5A is a gel electrophoresis analysis of FLAC-22 and KlenTaq-10. FIG. 5B is a gel electrophoresis analysis of Fast Start and Jump Start. FIG. 5C is a gel electrophoresis analysis of AmpliTaq Gold. FIG. 5D is a melting curve analysis of FLAC-22. FIG. 5E is a melting curve analysis of KlenTaq-10. FIG. 5F is a melting curve analysis of Fast Start. FIG. 5G is a melting curve analysis of Jump Start. FIG. 5H is a melting curve analysis of AmpliTaq Gold. Further details regarding methodology are presented in Example 5.

FIG. 6 is a series of graphs illustrating a successful real-time PCR detection of an anthrax genome target (1.3 kb Bacillus anthracis) at 5-fold dilutions in 5% human blood, with FLAC-22 mutant enzyme, using 32×SYBR Green concentration to compensate for the quenching effect of blood on the fluorescent dye. FIG. 6A is a quantitation graph showing fluorsescence as a function of PCR cycle. FIG. 6B is a standard curve graph showing the log of fluorescence quantity as a function of PCR cycle. FIG. 6C is a melting curve graph. Further details regarding methodology are presented in Example 6.

FIG. 7 is a series of graphs and images depicting the high resistance of Klentaq-10 to PCR inhibitors in soil. Various amounts of a crude soil extract were added to the reaction mixture and a test target from a soil-born bacteria, Bacillus Cereus, was amplified successfully with the mutant enzyme, while two commercial wild-type Taq enzymes failed. FIG. 7A shows fluorescence as a function of cycle for Fast Start. FIG. 7B shows fluorescence as a function of cycle for Jump Start. FIG. 7C shows fluorescence as a function of cycle for KlenTaq-10. FIG. 7D is a gel electrophoresis analysis of Fast Start and Jump Start. FIG. 7E is a gel electrophoresis analysis of KlenTaq-10. Further details regarding methodology are presented in Example 7.

FIG. 8 is a series of images and graphs depicting the soil inhibition resistance feature of the two novel mutant enzymes, Klentaq-10 and FLAC-22. A real-time PCR assay of a human (CCR5 gene) target was performed in the presence of a crude soil extract mixed with the DNA template. FIG. 8A is a gel electrophoresis analysis of wild type KlenTaq and KlenTaq-10. FIG. 8B is a melting curve graph for wild type KlenTaq. FIG. 8C is a melting curve graph for wild type KlenTaq-10. FIG. 8D is a gel electrophoresis analysis of FLAC-22 and Fast Start. FIG. 8E is a melting curve graph for FLAC-22. FIG. 8F is a melting curve graph for Fast Start. The two mutant enzymes outperformed the respective wild-type enzymes, Klentaq and Taq, in tolerating higher soil concentrations. The SYBR Green concentration was titrated to optimize the fluorescent signal for real-time detection. Further details regarding methodology are presented in Example 8.

FIG. 9 is a series of agarose gel images depicting results of FLAC-22 and KT-10 (SEQ ID NO: 1) in real-time RT-PCR in crude sample. 100 units of MMLV RT were mixed with 1 unit FLAC-22 and 1 unit KT-10 (SEQ ID NO: 1). The negative controls contained only FLAC-22 and KT-10 (SEQ ID NO: 1) mix and MMLV-RT was omitted (FIG. 9C). One commercial RT-PCR kit was also included as a comparison (FIG. 9B). Seven pg of influenza virus RNA was spiked in plasma, serum, or whole blood. One-step RT-PCR reactions (50 μl) containing 8% of the blood, serum, or plasma samples mixed with viral RNA were performed in PTC-200 Thermal Cycler to amplify a 244 bp influenza virus RNA target (FIG. 9A). The PCR products were resolved in 1.5% agarose gel. Further details regarding methodology are presented in Example 10.

FIG. 10 is a series of graphs depicting performance of FLAC-22 and KT-10 (SEQ ID NO: 1) in real-time RT-PCR TaqMan assay in crude sample. Purified influenza virus RNA was 10-fold diluted 7 times and spiked in human serum. FIG. 10A depicts one-step RT-PCR reactions (25 μl) containing different concentrations of RNA and constant concentrations of human serum (5%), performed with an enzyme mix of 100 units of MMLV RT and 1 unit of FLAC-22 and 2.5 units of KT-10 (SEQ ID NO: 1). FIG. 10B depicts parallel reactions (to those of FIG. 10A) with purified viral RNA alone included as comparison and positive controls. The negative controls contained no RNA template. A 244 bp influenza virus target was amplified in the presence of a 200 μM TaqMan probe. The reactions were performed in an Opticon-2 real-time PCR cycler and the fluorescence signal was detected after the annealing step. Further details regarding methodology are presented in Example 11.

DETAILED DESCRIPTION

The present invention is based at least in part upon the discovery of mutant DNA polymerase enzymes that are resistant to PCR inhibitors. In various embodiments, mutant DNA polymerase enzymes are resistant to PCR inhibitors including those present in, for example, blood, soil, and/or high concentrations of dyes (e.g., fluorescent dyes used in real-time PCR). Some embodiments of the mutant DNA polymerase enzymes exhibit all three features; therefore they are capable of amplifying nucleic acid molecules in the presence of elevated concentrations of any of these inhibitory substances, or combinations thereof. Provided herein are methods for standard and real-time PCR analyses of blood- and soil-containing samples utilizing mutant polymerase enzymes that are blood-, soil- and/or dye-resistant.

As shown herein, various commercial DNA polymerase enzymes fail to work in blood-containing PCR assays, due to the dual inhibition of the reaction by the blood and the high fluorescence dye concentration required to overcome the strong fluorescence quenching effect of the blood. In the case of crude soil-containing samples, even without such a pronounced quenching effect on the dye, the commercial enzymes also fail, mostly due to potent soil-derived PCR inhibitors. Among the benefits of various aspects of the invention is the ability to accommodate a large quantity of blood and/or soil in a real-time PCR assay, thereby increasing the probability of detecting low copy number of a target nucleic acid. Moreover, the mutant DNA polymerase enzymes described herein can allow, in many cases, eliminating nucleic acid purification steps prior to PCR, which are required in the protocols of all currently available commercial PCR enzymes. In clinical applications, this can result in detection at early onset while reducing the number of false negative diagnoses.

Thus, the dye-, blood-, and/or soil-resistant mutant DNA polymerase enzymes described herein signify a valuable tool for an improved, low-cost, fast, and sensitive PCR detection (including real-time PCR detection) of nucleic acids in general, as well as clinical and forensic applications in specimens with and without crude samples comprising blood or soil extracts.

One aspect of the invention is the use of inhibition-resistant mutant DNA polymerase enzymes in real-time PCR assays with crude samples containing, for example, blood and dye, soil and dye, or combination of blood and soil plus dye. Generally, the presence of blood components (e.g., heme) in a PCR reaction mixture significantly quenches dye fluorescence, thus requiring increased dye concentrations. In PCR assays with conventional DNA polymerases, both blood and increased dye concentration are inhibitory. The compositions and methods described herein provide for the use of inhibition resistant mutant polymerase enzymes in real-time PCR applications with blood- or soil-containing samples with dye in PCR reaction mixtures.

Another aspect of the invention is the use of inhibition-resistant mutant DNA polymerase enzymes in standard (i.e., non-real time) PCR assays with crude samples containing, for example, blood, soil, or a combination of blood and soil.

Dye

One aspect of the invention provides a method for improving detection of a target nucleic acid in real-time PCR (qPCR) by using polymerase mutants (e.g., mutant Taq DNA polymerase or mutant Klentaq DNA polymerase) to overcome inhibitory effects of dyes (e.g., fluorescent dyes) used in qPCR.

A resistant polymerase can be used to amplify a DNA target in a real-time PCR of a DNA target in the presence of an inhibitory dye. A resistant polymerase can be used to amplify an RNA target in a real-time reverse transcriptase (RT) PCR of an RNA target in the presence of an inhibitory dye.

Dye-resistance can be readily determined by assays known in the art and described herein (see e.g., FIGS. 1, 2, and 8).

Various embodiments of the mutant polymerase enzymes described herein can tolerate increased concentrations of dyes. Such increased concentrations include, but are not limited to, up to about 0.5×, 1×, 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 5.5×, 6×, 6.5×, 7×, 7.5×, 8×, 8.5×, 9×, 9.5×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, 55×, 60×, 65×, 70×, 80×, 90×, 100×, 150×, 200×, 250×, or even higher (see e.g., FIGS. 1A, 1B, 2A, 2B, 2C, and 8) over the dye concentration conventionally used in the assay. As an example, X can be the standard manufacturers unit for dye concentration provided in a commercial product (e.g., SYBR Green, Molecular Probes, Eugene, Oreg.). For example, for SYBR Green, X corresponds to a concentration of about 5 to about 10 μM.

Dye tolerance of a mutant polymerase enzyme (e.g., mutant Taq DNA polymerase) can provide, for example, higher amplification rate, higher fluorescent signal, and/or increased efficiency as compared to standard concentrations of dyes. Dye-tolerant mutant enzymes can improve the detection of the amplification target, especially when having low copy number of the nucleic acid target. As an additional benefit, higher dye inhibition resistance can permit enough dye (e.g., SYBR dye) in real time reactions to overcome the background fluorescence or quenching effect of PCR inhibitors, thus permitting detection of a target nucleic acid amongst inhibitors (e.g., components in blood and soil).

Dyes for use in the methods described herein include, but are not limited to, SYBR Green (Molecular Probes, Eugene, Oreg.), LC Green (Idaho Technology, Salt Lake City, Utah), PicoGreen (Molecular Probes, Eugene, Oreg.,), TOTO (Molecular Probes, Eugene, Oreg.), YOYO (Molecular Probes, Eugene, Oreg.) and SYTO9 (Molecular Probes, Eugene, Oreg.).

With their tolerance to high dye concentrations, the mutant polymerases described herein can outperform other conventional polymerase enzymes, including top commercial PCR enzymes, with commercially available dyes used in qPCR including, but not limited to, SYBR Green (see e.g., FIGS. 1A, 2A, 2B, and 8)), LC Green (Idaho Technology, Salt Lake City, Utah), PICO, TOTO (Molecular Probes, Eugene, Oreg.), YOYO (Molecular Probes, Eugene, Oreg.), SYTO (Molecular Probes, Eugene, Oreg.), and ethidium bromide. Some of these dyes are even more inhibitory than SYBR Green to a conventional Taq enzyme in PCR.

Blood

In some embodiments, resistant polymerases described herein can amplify a target nucleic acid in the presence of blood or blood components. A resistant polymerase can be used to amplify a DNA target in a real-time PCR of a DNA target in the presence of blood or blood components. A resistant polymerase can be used to amplify an RNA target in a real-time reverse transcriptase (RT) PCR of an RNA target in the presence of blood or blood components.

Whole blood generally comprises plasma, serum, and blood cells. Blood components include, but are not limited to, red blood cells, white blood cells (e.g., leukocytes or platelets, i.e., thrombocytes), plasma, serum, hemoglobin, water, proteins, glucose, amino acids, fatty acids, mineral ions, hormones, carbon dioxide, urea, and lactic acid. Resistant polymerases described herein can be used in PCR to amplify a nucleic acid target in the presence of one or more such blood components.

Blood plasma is generally understood as a liquid suspension in which blood cells are circulated. Thus, blood plasma can include one or more of water, proteins, glucose, amino acids, fatty acids, mineral ions, hormones, carbon dioxide, urea, lactic acid, platelets (i.e., thrombocytes), and blood cells. In a human subject, blood plasma represents about 55% of whole blood, or about 2.7 to 3 liters in an average human subject. Blood plasma contains about 92% water, 8% blood plasma proteins, and trace amounts of other materials. Blood plasma can contain serum albumin, blood-clotting factors, immunoglobulins, lipoproteins, other proteins, and electrolytes (e.g., sodium and chloride). A crude sample comprising blood plasma can also contain blood cells. Resistant polymerases described herein can be used in PCR to amplify a nucleic acid target in the presence of blood plasma.

Blood serum is generally understood as plasma from which clotting proteins have been removed, leaving mostly albumin and immunoglobulins. Resistant polymerases described herein can be used in PCR to amplify a nucleic acid target in the presence of blood serum.

A blood-resistant polymerase, as that term is used herein, can generally display amplification activity in PCR assays (e.g., real-time PCR or real-time RT PCR) containing from about 1% to about 25% whole blood in the reaction mixture (vol/vol). For example, whole blood can comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of a total volume of a PCR assay mixture comprising a blood-resistant polymerase described herein. In contrast, the full-length Taq enzyme (SEQ ID NO: 4) is usually completely inhibited in a blood concentration range of about 0.004% to about 0.2% whole blood in the reaction mixture (vol/vol).

A resistant polymerase, as described herein, can generally display amplification activity in PCR assays (e.g., real-time PCR or real-time RT PCR) containing from about 1% to about 25% of a blood component in the reaction mixture (vol/vol). For example, a blood component can comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of a total volume of a PCR assay mixture comprising a blood-resistant polymerase described herein.

A resistant polymerase, as described herein, can generally display amplification activity in PCR assays (e.g., real-time PCR or real-time RT PCR) containing from about 1% to about 25% blood plasma in the reaction mixture (vol/vol). For example, blood plasma can comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of a total volume of a PCR assay mixture comprising a blood-resistant polymerase described herein.

A resistant polymerase, as described herein, can generally display amplification activity in PCR assays (e.g., real-time PCR or real-time RT PCR) containing from about 1% to about 25% blood serum in the reaction mixture (vol/vol). For example, blood serum can comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of a total volume of a PCR assay mixture comprising a blood-resistant polymerase described herein.

Blood-resistance can be readily determined by assays described herein and know in the art (see e.g., US Pat App Pub No 2006/0084074).

Soil

Various embodiments of the mutant DNA polymerases described herein are resistant to inhibitors found in soil and soil extracts.

In some embodiments, resistant polymerases described herein can amplify a target nucleic acid in the presence of an inhibitor found in soil or soil extract. A resistant polymerase can be used to amplify a DNA target in a real-time PCR of a DNA target in the presence of an inhibitor found in soil or soil extract. A resistant polymerase can be used to amplify an RNA target in a real-time reverse transcriptase (RT) PCR of an RNA target in the presence of an inhibitor found in soil or soil extract.

Soil inhibitors and soil extract inhibitors include, but are not limited to, humic acid, fulvic acid, polysaccarides, and metal ions. A soil-resistant polymerase, as that term is used herein, can generally display amplification activity in PCR assays containing from about 1% to about 90% soil or soil extract in the reaction mixture (vol/vol). For example, soil extract can comprise up to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of a total volume of a PCR assay mixture comprising a soil-resistant DNA polymerase described herein. For example, soil extract can comprise up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of a total volume of a PCR assay mixture comprising a soil-resistant DNA polymerase described herein. The amount of soil or soil extract in the assay mixture can depend upon the levels of inhibitory substances in the soil or soil extract. Generally, the mutant DNA polymerases described herein can tolerate at least an order of magnitude greater concentration of these inhibitory substances, as compared to conventional DNA polymerases. Assays to determine the level of inhibitory substances in a sample are known in the art. Soil-resistance can be readily determined by assays described herein.

Direct extraction of total DNA from soil samples results in a co-extraction of humic acid, known as the most potent soil inhibitor to PCR analysis. Humic substances represent a mixture of partially characterized polyphenols that are produced during the decomposition of organic matter. Conventional DNA polymerase enzymes are inhibited at about 1 ng of humic acid per 50 uL reaction volume. Various embodiments of the mutant DNA polymerases described herein are resistant to soil or soil extract that contains, for example, various levels of humic acid. Preferably, the volume of soil or soil extract used in the PCR assay mixture is the soil or soil extract equivalent that provides up to about 25 ng of humic acid per 50 uL reaction volume. Assays to determine the amount of humic acid is a sample are known in the art. Preferably, the volume of soil or soil extract used in the PCR assay mixture is the soil or soil extract equivalent that provides up to about 20 ng of humic acid per 50 uL reaction volume, more preferably up to about 10 ng of humic acid per 50 uL reaction volume.

Combination

Preferably, the mutant polymerase enzymes are resistant to both blood and dye (e.g., fluorescent dye) inhibition; soil and dye inhibition; or blood, soil and dye inhibition, as might occur in, for example, a PCR reaction containing dye and blood and/or soil. Even more preferably, a synergy is provided in which the combination of the mutant enzymes, blood, and fluorescent dyes enable amplification and optical detection heretofore not possible. A preferred double inhibition resistant phenotype (i.e., blood and dye resistance) of a mutant enzyme makes possible the real-time detection of samples containing blood. Furthermore, in the presence of blood, the blood- and dye-resistant mutant polymerases can tolerate higher initial concentration of dye (e.g., tolerance of about 64× to about 256× for SYBR Green dye).

In some embodiments, resistant polymerases described herein can amplify a target nucleic acid in the presence of an inhibitory dye and an inhibitor found in soil or soil extract; an inhibitory dye and blood or a blood component; or an inhibitory dye, an inhibitor found in soil or soil extract, and blood or a blood component. A resistant polymerase can be used to amplify a DNA target in a real-time PCR of a DNA target in the presence of an inhibitory dye and an inhibitor found in soil or soil extract; an inhibitory dye and blood or a blood component; or an inhibitory dye, an inhibitor found in soil or soil extract, and blood or a blood component. A resistant polymerase can be used to amplify an RNA target in a real-time reverse transcriptase (RT) PCR of an RNA target in the presence of an inhibitory dye and an inhibitor found in soil or soil extract; an inhibitory dye and blood or a blood component; or an inhibitory dye, an inhibitor found in soil or soil extract, and blood or a blood component.

Interference between the blood and the fluorescent dye is demonstrated herein to at least partially eradicate the detection of the amplified product when using normal (i.e., low) dye concentrations (e.g., 1× or lower for SYBR Green). Adding high dye concentration to the reaction (allowed through the use of dye-resistant mutant polymerases described herein) can help to overcome the quenching effect of blood components (e.g., heme) on the dye fluorescence.

PCR

In some embodiments, a resistant polymerase can be employed in a real-time PCR amplification of a DNA target.

The DNA polymerases resistant to PCR inhibitors described herein can be used in a variety of polymerase reactions known to the art (see e.g., Dorak (2006) Real-Time PCR, Taylor & Francis, ISBN 041537734X; Bustin, ed. (2004) A-Z of Quantitative PCR, International University Line, ISBN 0963681788). For example, the resistant polymerases can be employed in PCR reactions, primer extension reactions, etc. The use of the mutant polymerase enzymes described herein generally does not require any, or substantial, changes in the typical protocol, other than, for example, adding higher amounts of fluorescent dye. Thus, methods described herein can be applied to improve the nucleic acid detection in any standard real-time PCR with purified template nucleic acids and primers.

In some embodiments, a resistant polymerase can be employed in a real-time reverse transcriptase (RT) PCR amplification of an RNA target. It is noted that reverse transcriptase (RT) PCR is not to be confused with real-time polymerase chain reaction (Q-PCR), which is sometimes (incorrectly) abbreviated as RT-PCR in the art. In RT-PCR, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional PCR. Like with standard PCR, conventional RT-PCR protocols require extensive purification steps prior to amplification to purify RNA from inhibitors and ribonucleases, which can destroy the RNA template. Both the inhibition and degradation of RNA are major concerns in important clinical and diagnostics tests, which may lead to false-negative results.

Applications of RT-PCR include, but are not limited to, detection of RNA virus pathogens; analysis of mRNA expression patterns of certain genes related to various diseases; semiquantitative determination of abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression; and cloning of eukaryotic genes in prokaryotes.

Resistant polymerases described herein can be used in a variety of RT-PCR protocols known to the art (see e.g., King and O'Connel (2002) RT-PCR Protocols, 1^st Ed., Human Press, ISBN-10 0896038750). The use of the mutant polymerase enzymes described herein generally does not require any, or substantial, changes in the typical protocol, other than, for example, adding higher amounts of a fluorescent dye. Thus, methods described herein can be applied to improve target detection in any standard RT PCR.

The buffer for use in the various PCR assay mixtures described herein is generally a physiologically compatible buffer that is compatible with the function of enzyme activities and enables cells and/or biological macromolecules to retain their normal physiological and biochemical functions. Typically, a physiologically compatible buffer will include a buffering agent (e.g., TRIS, MES, PO₄, HEPES, etc.), a chelating agent (e.g., EDTA, EGTA, or the like), a salt (e.g., ammonium sulfate, NaCl, KCl, MgCl.sub.2, CaCl.sub.2, NaOAc, KOAc, Mg(OAc).sub.2, etc.) and optionally a stabilizing agent (e.g., sucrose, glycerine, Tween20, etc.).

Various PCR additives and enhancers can be employed with the methods described herein. For example, betaine can be added to the PCR assay, to further aid in overcoming the inhibition by blood and/or soil. Betaine can be included at final concentration about 1 M to about 2M. Generally, betaine alone is insufficient to overcome the inhibition of, for example, dye, blood, and/or soil when used with conventional DNA polymerases.

Mutant Polymerases

Examples of mutant enzymes useful to the invention include, but are not limited to, KlenTaq-10 (SEQ ID NO: 1) (as described in US Pat App Pub No 2006/0084074, specifically incorporated herein by reference in its entirety) and the full-length Taq enzyme mutant, FLAC-22 (SEQ ID NO: 2), as well as variant polypeptides of these reference sequences, as described below.

For the following discussion, wild type Taq numbering is used in this descriptive text so as to make clear the relationship between the polypeptides. For truncated polymerase polypeptides (e.g., Klentaq-10 of SEQ ID NO: 1; Klentaq-1 of SEQ ID NO: 3), position number 1 as notated in the Sequence Listing of SEQ ID NO: 1 or SEQ ID NO: 3 corresponds to position number 279 as notated in the full-length Taq of SEQ ID NO: 4. Similarly, position number 2 of SEQ ID NO:1 or SEQ ID NO: 3 corresponds to position number 280 of SEQ ID NO: 4.

KlenTaq-1 (SEQ ID NO: 3) has an N-terminal deletion at 278 of a full-length Taq (SEQ ID NO: 4), with Met/Gly replacing amino acids 279 and 280 (per wild type Taq numbering). It is noted that Klentaq-1 is a “wild-type” Klentaq given that it is directly derived from wild-type Taq by truncation.

KlenTaq-10 (SEQ ID NO: 1) has an N-terminal deletion at 278 of a full-length wild type Taq (SEQ ID NO: 4) and substitutions at E626K, I707L, and E708K (per wild type Taq numbering).

FLAC22 (SEQ ID NO: 2) is a full-length Taq (SEQ ID NO: 4) with substitutions at E626K, 1707L, and E708N (per wild type Taq numbering).

Also included in the scope of the invention are variant polypeptides (or encoding polynucleotides) with at least 80% sequence identity to the above-described sequences, so long as such variants retain dye-resistant polymerase activity; dye- and blood-resistant polymerase activity; dye- and soil-resistant polymerase activity; or dye-, blood-, and soil-resistant polymerase activity. For example, variant polypeptides (or encoding polynucleotides) with dye-resistant polymerase activity; dye- and blood-resistant polymerase activity; dye- and soil-resistant polymerase activity; or dye-, blood-, and soil-resistant polymerase activity can have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity to sequences disclosed herein. Preferably, variant polypeptides (or encoding polynucleotides) with dye-resistant polymerase activity; dye- and blood-resistant polymerase activity; dye- and soil-resistant polymerase activity; or dye-, blood-, and soil-resistant polymerase activity have at least 95% sequence identity to sequences disclosed herein. More preferably, variant polypeptides (or encoding polynucleotides) with dye-; dye- and blood-; dye- and soil-; or dye-, blood- and soil-resistant polymerase activity have at least 99% sequence identity to sequences disclosed herein. The species of SEQ ID NO: 1 and SEQ ID NO: 2 are representative of the genus of variant polypeptides of each of these respective sequences because all variants must possess the specified catalytic activity (e.g., dye-, blood-, and/or soil-resistant polymerase activity) and must have the percent identity required above to the reference sequence.

Design, generation, and testing of the variant polypeptides having the above required percent identities to the sequences of the mutant DNA polymerases and retaining a required resistant phenotype is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of polypeptide variants having, for example, at least 95-99% identity to the sequences of mutant DNA polymerases described herein and screen such for phenotypes including, dye-resistance, blood-resistance, and/or soil-resistance according to methods routine in the art. Generally, conservative substitutions can be made at any position so long as the required activity is retained. Amino acid positions known to be associated with the phenotypes described herein include, but are not limited to 626, 707, and 708 (per wild type Taq numbering) (see Example 9). For example, preferred substitutions at 708 include K and L for truncated and full length mutant Taq polymerases; N, Q, and I for full length mutant Taq polymerases; and W in truncated mutant Taq polymerases. Preferable substitutions include E708R and E708L.

Amino acid sequence identity percent (%) is understood as the percentage of amino acid residues that are identical with amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity=X/Y100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

Mutant DNA polymerases described herein can be produced according to methods known in the art. For example, oligonucleotides providing the specific amino acid changes to a mutant DNA polymerase described can be prepared by standard synthetic techniques (e.g., an automated DNA synthesizer) and used as PCR primers in site-directed mutagenesis. Standard procedures of expression of mutant DNA polymerase polypeptides from encoding DNA sequences can then be performed. Alternatively, the mutant DNA polymerase polypeptides can be directly synthesized according to methods known in the art.

Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. It shall be understood that any method described in an example may or may not have been actually performed, or any composition described in an example may or may not have been actually been formed, regardless of verb tense used.

Example 1

The following primer oligonucleotides were used in real-time PCR reaction to amplify a 0.6 kbp Bacillus cereus specific target from 20 pg input genomic DNA: forward 5′-AGG GTC ATT GGA AAC TGG G-3′ (SEQ ID NO 5), and reverse 5′-CGT GTT GTA GCC CAG GTC ATA-3′ (SEQ ID NO 6). The final concentration of each primer was 0.2 uM used in a common master mix. The amount of each enzyme used was 2.5 units per 50 ul reaction. Wild type KlenTaq and mutant KlenTaq-10 were challenged with a series of seven two-fold SYBR green fluorescent dye dilutions starting with 16× concentration. After 40 cycles the products were analyzed both in 2% agarose gel electrophoresis (see e.g., FIG. 1A) and temperature dissociation profile (see e.g., FIG. 1B).

Results show that KlenTaq-10 was capable of amplifying a 0.6 kbp Bacillus cereus target DNA in the presence of 4×SYBR, while the wild type failed at concentrations higher than 1× (see e.g., FIG. 1). The post PCR melting curve analysis confirms the fluorescent detection and the specificity of the product.

Example 2

The following primer oligonucleotides were used in real-time PCR reaction to amplify a 0.6 kbp Bacillus cereus specific target from 20 pg input genomic DNA: forward 5′-AGG GTC ATT GGA AAC TGG G-3′ (SEQ ID NO 5), and reverse 5′-CGT GTT GTA GCC CAG GTC ATA-3′ (SEQ ID NO 6). The final concentration of each primer was 0.2 uM used in a common master mix. The amount of each enzyme used was 2.5 units per 50 ul reaction. FLAC-22 (SEQ ID NO: 2) and Fast Start Tags were challenged with a series of seven two-fold SYBR green fluorescent dye dilutions starting with 6.4× concentration. After 40 cycles the products were analyzed both in 2% agarose gel electrophoresis (see e.g., FIG. 2A) and temperature dissociation profile (see e.g., FIG. 2B).

Results showed that FLAC-22 (SEQ ID NO: 2) was capable of amplifying a 0.6 kbp Bacillus cereus target DNA in the presence of 1.6×SYBR, while Fast Start Taq failed at concentrations higher than 0.2× (see e.g., FIG. 3). Post PCR melting curve analysis confirms the fluorescent detection and the specificity of the product.

Example 3

The following primer oligonucleotides were used in real-time PCR reaction to amplify a 0.6 kbp Bacillus cereus specific target from 20 pg input genomic DNA: forward 5′-AGG GTC ATT GGA AAC TGG G-3′ (SEQ ID NO: 5), and reverse 5′-CGT GTT GTA GCC CAG GTC ATA-3′ (SEQ ID NO: 6). The final concentration of each primer was 0.2 uM used in a common master mix. The amount of each enzyme used was 2.5 units per 50 ul reaction. Wild-type KlenTaq and KlenTaq-10 were double challenged with two blood concentrations 5% and 10% of the total reaction volume and with series of six two-fold SYBR green fluorescent dye dilutions starting with 64× concentration. After 40 cycles, the products were analyzed both in 2% agarose gel electrophoresis (see e.g., FIG. 3A) and temperature dissociation profile (see e.g., FIG. 3B).

Under normal circumstances, i.e., without blood, these conditions would be very inhibitory. But the results herein show that KlenTaq 10 was capable of amplifying with at least two times higher concentration of SYBR compared to the wild type KlenTaq (see e.g., FIG. 3). The post PCR melting curve analysis confirms that in the presence of 10% blood, at least 8×SYBR green is necessary for real-time PCR detection and at least 2×SYBR for 5% blood samples. Results also show that the resistance of KlenTaq 10 toward SYBR green is increased to at least 64× compared to 4× when there is no blood in the reaction.

Example 4

The following primer oligonucleotides were used in real-time PCR reaction to amplify a 0.32 kbp 16S microbial target: forward 5′-GGA TGC AAG CGT TAT CCG GAA TG-3′ (SEQ ID NO 7), and reverse 5′-CAT TCT TGC GAA CGT ACT CCC CA-3′ (SEQ ID NO 8). The final concentration of each primer was 0.2 uM used in a common master mix. The amount of each enzyme used was 2.5 units per 50 ul reaction. FLAC-22 (SEQ ID NO: 2) and Fast Start Tags were double challenged with 5% blood and with a series of four two-fold SYBR green fluorescent dye dilutions and no SYBR control starting with 32× concentration. A positive control, a reaction containing neither blood nor SYBR, was also included. After 40 cycles, the products were analyzed both in 2% agarose gel electrophoresis (see e.g., FIG. 4A) and temperature dissociation profile (see e.g., FIG. 4B).

Without blood, these SYBR concentrations would be very inhibitory. But the results herein show that FLAC-22 (SEQ ID NO: 2) was capable of amplifying with all SYBR concentrations whereas Fast Start was inhibited by both blood and SYBR and was able to amplify only the positive control, containing neither blood nor dye (see e.g., FIG. 4). The post PCR melting curve analysis demonstrates that in the presence of 5% blood, at least 4×SYBR green is necessary for real-time PCR detection for FLAC-22 (SEQ ID NO: 2) while the Fast Start samples did do not show the correct melting temperature of the product at all. Once again, the resistance of FLAC-22 (SEQ ID NO: 2), toward SYBR green has increased to at least 32× compared to 2× without blood in the reaction.

Example 5

The following primer oligonucleotides were used in real-time PCR reaction to amplify a 0.25 kbp Lambda specific target: forward 5′-GGG CGG CGA CCT CGC GGG TTT TCG C-3′ (SEQ ID NO 9), and reverse 5′-CTG AAT GGT ACG GAT ACT CGC ACC G-3′ (SEQ ID NO 10). The final concentration of each primer was 0.2 uM used in a common master mix. The amount of each enzyme used was 2.5 units per 50 ul reaction. FLAC-22 (SEQ ID NO: 2), KlenTaq-10, Fast Start, JumpStart and AmpliTaq Gold polymerases were challenged with a series of seven two-fold SYBR green fluorescent dye dilutions starting with 4× concentration. After 35 cycles the products were analyzed both in 2% agarose gel electrophoresis (see e.g., FIG. 5A) and temperature dissociation profile (see e.g., FIG. 5B).

Results showed that FLAC-22 (SEQ ID NO: 2) was capable of amplifying a 0.25 kbp lambda target from added purified DNA template in the presence of 2×SYBR; and KlenTaq-10 was able to amplify with at least 4×SYBR; but the commercial Taq polymerases failed at concentrations higher than 0.5× (see e.g., FIG. 5). The post PCR melting curve analysis confirms the fluorescent detection and the specificity of the product.

Example 6

Whole human blood was pre-mixed with Bacillus anthracis DNA and subjected directly to PCR. Klentaq-10 specifically amplified the target in 5% blood, as indicated by the melting curve profile. In addition, the quantitation curves were highly proportional to the input amount of DNA so that the standards followed the general curve. High concentration of SYBR Green, 32×, was used to compensate for the quenching effect of blood on the fluorescent dye.

Example 7

Two commercial Taq enzymes, Fast Start Taq (Roche) and Jump Start Tag (Sigma), and KT-10 mutant enzyme (SEQ ID NO: 1) were used to amplify an endogenous 600 bp target of Bacillus Cereus from a crude soil extract. The reactions contained four dilutions of the soil extract: 16%, 8%, 4% and 2% (see e.g., FIG. 7D-E, lanes 1-4, respectively). Reactions in lane 5 were positive controls, containing 5 ng purified B. cereus DNA without soil extract. PCR was performed in real-time cycler Opticon-2, and the amplified products were analyzed both by SYBR Green dye fluorescence (top panels) and gel electrophoresis (bottom panels). The pink curves correspond to the control reactions and the yellow, blue, green, and red ones reflect the reactions with increasing soil concentration. This example demonstrates the soil-resistance feature of the mutant Klentaq-10 enzyme, outperforming the two commercial Tags, which require DNA purification from soil prior to PCR.

Example 8

This example demonstrates that in addition to the blood- and SYBR-resistance, the two novel mutant enzymes also possess high tolerance to PCR inhibitors present in soil. A 630 bp CCR5 gene target was amplified from 4 ng of human DNA mixed with crude soil extract and various amounts of SYBR. Klentaq-10 mutant enzyme was compared to it's wild type precursor, and FLAC-22 (SEQ ID NO: 2) was compared to the wild type Fast Start Taq polymerase (Roche). SYBR Green fluorescent dye was titrated to optimize the fluorescent signal for real time detection. PCR was performed in real-time cycler Opticon2, and the amplified products were analyzed both by gel electrophoresis (bottom panels) and SYBR Green dye melting curve profile (top panels).

The soil resistant mutant enzymes showed specific melting curves with very low background signal. In contrast, the wild type Klentaq and Fast Start Taq showed a melting curve at very low SYBR concentration so the signal might get lost in the background in the presence of more soil. The results demonstrate that the two new mutant enzymes can perform easily in real time PCR of crude soil samples containing PCR inhibitors, without requiring pre-PCR DNA purification steps.

Example 9

Single amino acid changes of the wild type Taq Glu708 were implicated in both selected qualities of the mutants, blood and soil-resistance. Functional analysis revealed that the same amino acid substitutions in that position were optimal both for the blood and soil resistance features.

Saturated mutagenesis was performed at codon 708 of Taq to test functionally the full spectrum of all possible substitutions at this critical position. Among all 708 mutant variants, Klentaq-10 (SEQ ID NO: 1) and FLAC-22 (SEQ ID NO: 2) proved superior in all aspects of inhibitors resistance to PCR inhibitors.

The analysis of the 708 changes showed certain specificity for KlenTaq and Taq DNA polymerase. K and L substitutions were efficient in both enzymes (mutants Klentaq10 and 12, and the corresponding FLAC-10 and FLAC-12), while N, Q, I (FLAC-22, 3 and 4) were only functional in Taq, and W substitution was only good in KlenTaq (Klentaq-7).

The two phenotypes were not tightly coupled, as the KlenTaq 7 (E708W) and Klentaq-12 mutants (G708Trp and Glu708Leu) exhibited relatively high resistance to blood but not to soil inhibitors, while another KlenTaq mutant (Klentaq 11, E708R) was predominantly soil resistant. As per the SYBR resistance, next to Klentaq-10 and FLAC-22 (SEQ ID NO: 2) in order of performance were Klentaq-11 (E708R) and Klentaq-12(E708L).

The importance of residue 708 was confirmed by the fact that two of the possible 20 substitutions, P and C, inactivated both the KlenTaq and Taq enzyme.

Example 10

The following example illustrates real-time RT PCR amplification of crude samples containing blood components utilizing FLAC-22 (SEQ ID NO: 2) and KT-10 (SEQ ID NO: 1).

100 units of MMLV RT were mixed with 1 unit FLAC-22 (SEQ ID NO: 2) and 1 unit KT-10 (SEQ ID NO: 1). The negative controls only contained FLAC-22 (SEQ ID NO: 2) and KT-10 mix and MMLV-RT was omitted. One commercial RT-PCR kit was also included as comparison. Seven pg of influenza virus RNA was spiked in plasma, serum, or whole blood. RT-PCR reactions also contained an enhancer composition containing 0.64 M Trehalose, 0.12 M L-carnitine, 0.4% NP-40 and 10 u heparin per ml serum, plasma, or whole blood (PEC-Plus). One-step RT-PCR reactions (50 μl) containing viral RNA (lane 1) or viral RNA mixed with 8% of plasma (lane 2), serum (lane 3), or blood (lane 4) were performed in PTC-200 Thermal Cycler to amplify a 244 bp influenza virus RNA target. The PCR products were resolved in 1.5% agarose gel.

Results showed a 244 bp influenza virus gene was succesfully amplified from purified RNA and mimic crude samples containing 8% plasma, serum, and whole blood using an FLAC-22 (SEQ ID NO: 2), KT-10 (SEQ ID NO: 1) and MMLV-RT mixture (see e.g., FIG. 9). Succesful amplification of RNA targets was also obtained where FLAC-22 (SEQ ID NO: 2) and KT-10 (SEQ ID NO: 1) were blended with other reverse transcriptase (data not shown).

Results further showed that conventional commercial RT-PCR kits were only able to amplify this target from purified RNA and failed in crude samples due to the presence of potent inhibitors of the Taq DNA polymerase and the reverse transcriptase in such specimens. The negative controls, omitting the reverse transcriptase, produced no specific products. These data show that RT-PCR products were amplifications based on the RNA template and not from cDNA contamination or another source of DNA.

Thus, as demonstrated above, FLAC-22 (SEQ ID NO: 2) and KT-10 (SEQ ID NO: 1) work in RT-PCR for RNA target in crude samples in the presence of reverse transcriptases.

Example 11

The following example illustrates real-time RT PCR TaqMan assay in crude samples containing blood components utilizing FLAC-22 (SEQ ID NO: 2) and KT-10 (SEQ ID NO: 1).

Purified influenza virus RNA was 10-fold diluted 7 times and spiked in human serum. One-step RT-PCR reactions (25 μl) containing different concentrations of RNA and constant concentrations of human serum (5%), were performed with an enzyme mix of 100 units of MMLV RT and 1 unit of FLAC-22 (SEQ ID NO: 2) and 2.5 units of KT-10 (SEQ ID NO: 1). RT-PCR reactions also contained an enhancer composition containing 0.64 M Trehalose, 0.12 M L-carnitine, 0.4% NP-40 and 10 u heparin per ml serum, plasma, or whole blood (PEC-Plus). Parallel reactions with purified viral RNA alone were included as comparison and positive controls. The negative controls contained no RNA template. A 244 bp influenza virus target was amplified in the presence of a 200 μM TaqMan probe. The reactions were performed in an Opticon-2 real-time PCR cycler and the fluorescence signal was detected after the annealing step.

Results showed that the 244 bp influenza virus gene was successfully detected from mimic clinical samples containing 5% human serum, using a combination of FLAC-22 (SEQ ID NO: 2), KT-10 (SEQ ID NO: 1), and MMLV-RT. The fluorescent signal in crude samples was relatively lower than these in purified RNA, however, the sensitivity was not compromised as compared to the results with purified RNA. In addition to serum, these results were confirmed with plasma and whole blood (data not shown). The optimal concentrations of TaqMan was 200-400 μM for 5-20% plasma and serum, and 400-8000 for 5-10% whole blood.

Thus, as demonstrated above, FLAC-22 (SEQ ID NO: 2) and KT-10 (SEQ ID NO: 1) work in RT-PCR TaqMan assays for RNA target in crude samples in the presence of reverse transcriptases.

Claims

1. A method of amplifying a target nucleic acid in a real-time polymerase chain reaction (PCR) comprising:

forming an assay mixture comprising: a sample comprising a target nucleic acid; primers specific for the target nucleic acid; a buffer; at least one dye; and at least one polymerase, wherein the at least one polymerase is a dye-resistant polymerase; and

amplifying the target nucleic acid in the assay mixture in a real-time PCR.

2. The method of claim 1 wherein the target nucleic acid is a DNA molecule.

3. The method of claim 1 wherein:

the target nucleic acid is an RNA molecule;

the real-time PCR is a real-time reverse transcriptase (RT) PCR; and

the assay mixture further comprises a reverse-transcriptase.

4. The method of claim 1 wherein the at least one polymerase is (i) a dye-resistant and blood-resistant polymerase; (ii) a dye-resistant and soil-resistant polymerase; or (iii) a dye-resistant, blood-resistant, and soil-resistant polymerase.

5. The method of claim 1 wherein the at least one polymerase has a polypeptide sequence comprising SEQ ID NO: 1 or at least 95% identical to SEQ ID NO: 1 and dye-resistant polymerase activity.

6. The method of claim 5 wherein the at least one polymerase having a polypeptide sequence at least 95% identical to SEQ ID NO: 1 comprises at least one amino acid substitution at an amino acid residue position selected from the group consisting of positions 626, 707, and 708.

7. The method of claim 1 wherein the at least one polymerase has a polypeptide sequence comprising SEQ ID NO: 2 or at least 95% identical to SEQ ID NO: 1 and dye-resistant polymerase activity.

8. The method of claim 7 wherein the at least one polymerase having a polypeptide sequence at least 95% identical to SEQ ID NO: 2 comprises at least one amino acid substitution at an amino acid residue position selected from the group consisting of 626, 707, and 708.

9. The method of claim 1 wherein the sample further comprises whole blood or a blood component.

10. The method of claim 9 wherein the blood component is selected from the group consisting of blood plasma and blood serum.

11. The method of claim 9 wherein the whole blood or blood component is at least about 1% up to about 25% of a total volume of the PCR assay mixture.

12. The method of claim 1 wherein the sample further comprises soil or soil extract.

13. The method of claim 12 wherein the soil or soil extract is at least about 1% up to about 90% of a total volume of the PCR assay mixture.

14. The method of claim 12 wherein the soil or soil extract comprises a humic acid and the soil or soil extract is present in the assay mixture at a soil or soil extract equivalent amount that provides up to about 25 ng of humic acid per 50 uL reaction volume.

15. The method of claim 1 wherein the at least one dye is a fluorescent dye selected from the group consisting of SYBR Green, Ethidium Bromide, PICO, TOTO, YOYO or LC Green.

16. The method of claim 1 wherein the dye is present in the PCR assay mixture at least about 0.5× up to about 256×, where × is a manufacturer unit for concentration for use in PCR.

17. The method of claim 1 wherein the assay mixture further comprises a PCR enhancer.

18. An isolated polypeptide comprising

a polypeptide sequence of SEQ ID NO: 2;

a polypeptide sequence at least 95% identical to SEQ ID NO: 2 and having dye-, soil-, or blood-resistant polymerase activity, or a combination thereof;

a polypeptide sequence at least 95% identical to SEQ ID NO: 2 comprising at least one amino acid substitution at an amino acid residue position selected from the group consisting of 626, 707, and 708, and having dye-, soil-, or blood-resistant polymerase activity, or a combination thereof.

20. A method of amplifying a target nucleic acid in a polymerase chain reaction (PCR) comprising:

forming an assay mixture comprising: a sample comprising a target nucleic acid; wherein the sample optionally contains: whole blood or a blood component selected from blood plasma or blood serum; or soil or soil extract; primers specific for the target nucleic acid; a buffer; at least one polymerase comprising the isolated polypeptide of claim 19; and

amplifying the target nucleic acid in the assay mixture in a PCR.