Compositions and methods for reverse transcriptase-polymerase chain reaction (RT-PCR)

Abstract

The present invention is directed to compositions and methods useful for the amplification of nucleic acid molecules by reverse transcriptase-polymerase chain reaction (RT-PCR). Specifically, the invention provides compositions and methods for the amplification of nucleic acid molecules in a simplified one- or two-step RT-PCR procedure using combinations of reverse transcriptase and thermostable DNA polymerase enzymes in conjunction with sulfur-containing molecules or acetate-containing molecules (or combinations of such sulfur-containing molecules and acetate-containing molecules), and optionally bovine serum albumin. The invention thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules. The invention also is useful in the rapid production and amplification of cDNAs which may be used for a variety of industrial, medical and forensic purposes.

Claims

1. A composition suitable for carrying out a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) comprising a Moloney murine leukemic virus (M-MLV) reverse transcriptase (RT), one or more DNA polymerases, a non-naturally occurring buffering agent, and bovine serum albumin, wherein said one or more DNA polymerases is selected from the group consisting of Tne, Tma, Taq, Pfu, Tth, Tfi, Pwo, and Tfl; or wherein said one or more DNA polymerases comprises a first DNA polymerase having 3 exonuclease activity and a second DNA polymerase having substantially reduced 3 exonuclease activity; and wherein the unit ratio of said RT to said DNA polymerase(s) is greater than about 3:2; and wherein said bovine serum albumin is at a concentration of about 2 to 20 g/ml.

2. The composition of claim 1, wherein said bovine serum albumin is at a concentration which relieves RT inhibition of nucleic acid amplification.

3. The composition of claim 1, further comprising one or more sulfur-containing molecules, one or more acetate-containing molecules, and/or one or more potassium-containing molecules.

4. The composition of claim 3, wherein said one or more acetate-containing molecules is at a concentration of about 1 mM to about 500 mM.

5. The composition of claim 3, wherein said one or more sulfur-containing molecules is at a concentration of at least 18 mM sulphur.

6. The composition of claim 1, wherein (a) said DNA polymerase having 3 exonuclease activity is selected from the group consisting of Pfu, Pwo, Tne, Tma, Kod, and mutants, variants and derivatives thereof; and/or (b) said DNA polymerase having substantially reduced 3 exonuclease activity is selected from the group consisting of Taq, Tfl, Tth, and mutants, variants and derivatives thereof.

7. The composition of claim 3, wherein a source of said one or more sulfur-containing molecules is selected from: (a) a sulfur-containing buffer; or (b) a sulfur-containing salt.

8. The composition of claim 3, wherein a source of said one or more acetate-containing molecules is selected from: (a) an acetate-containing salt; or (b) an acetate-containing buffer.

9. The composition of claim 1, further comprising one or more nucleotides which are deoxyribonucleoside triphosphates or derivatives thereof, or dideoxyribonucleoside triphosphates or derivatives thereof.

10. The composition of claim 1, wherein said reverse transcriptase is substantially reduced in RNase H activity.

11. The composition of claim 1, wherein said reverse transcriptase is a mutant Moloney murine leukemic virus (M-MLV) reverse transcriptase.

12. The composition of claim 1, further comprising one or more oligonucleotide primers.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photograph of an ethidium bromide (EtdBr)-stained gel demonstrating the inhibition of RT-PCR by reverse transcriptase. Lanes 1, 10, 11 and 20 contain 100 bp DNA sizing ladders.

(2) FIG. 2A is photograph of an EtdBr-stained gel demonstrating the protective role of bovine serum albumin (BSA) in RT-PCR of -actin mRNA from 100 pg of HeLa total mRNA template. Lane 1 contains a DNA sizing ladder, and the arrow indicates the 1026 bp -actin target sequence.

(3) FIG. 2B is a photograph of an EtdBr-stained gel demonstrating the protective role of BSA in RT-PCR of CAT mRNA from 10.sup.5 copies of CAT mRNA template. Lane 1 contains a DNA sizing ladder, and the arrow indicates the 653 bp CAT target sequence.

(4) FIG. 3 is a photograph of an EtdBr-stained gel demonstrating the performance of various RTs in sulfate ion- or BSA-containing buffers. Lane 1: 100 bp DNA sizing ladder, Lanes 2-5: 5 units/reaction of AMV-RT; Lanes 6-9: 5 units/reaction of M-MLV-RT; Lanes 10-13: 5 units/reaction of M-MLV-RT (RNase H.sup.). Arrow indicates 500 bp CAT target sequence.

(5) FIG. 4 is a photograph of an EtdBr-stained gel demonstrating the effect of reverse transcription reaction temperature on RT-PCR product formation. Duplicate samples of 100 ng (lanes 1-6) or 10 ng (lanes 7-12) of total HeLa RNA were reverse transcribed at the indicated temperatures, and a 606 bp DNA polymerase III subunit target sequence (lanes 1-6) or a 253 bp -actin target sequence (lanes 7-12) were amplified by PCR. M: DNA sizing markers.

(6) FIG. 5 is a photograph of an EtdBr-stained gel demonstrating improved yield of RT-PCR products obtained by conducting the RT reaction at higher temperatures. Duplicate samples of 10 ng of tobacco total RNA (lanes 1-6) or of 100 ng of total HeLa RNA (lanes 7-12) were reverse transcribed at 45 C. (lanes 1, 2, 7 and 8), 50 C. (lanes 3, 4, 9 and 10) or 55 C. (lanes 5, 6, 11 and 12), and a 500 bp GADPH target sequence (arrowhead; lanes 1-6) or a 1475 bp DNA polymerase III subunit target sequence (arrow, lanes 7-12) were amplified by PCR. M: DNA sizing markers; L: 100 bp nucleic acid sizing ladder.

(7) FIG. 6 is a photograph of an EtdBr-stained gel demonstrating the sensitivity and efficiency of the invention in RT-PCR of large (2-3 kb) products. A 2.78 kb tuberous sclerosis II fragment was amplified from varying amounts of total HeLa RNA that was reverse transcribed using the compositions of the invention without (lanes 1-6) or with (lanes 7-12) the addition of 1 l of ELONGASE Enzyme Mix, or using a RT kit from Supplier A (lanes 13-18). Lanes 1, 2, 7, 8, 13 and 14: 1 ng of HeLa RNA; Lanes 3, 4, 9, 10, 15 and 16: 10 ng of HeLa RNA; Lanes 5, 6, 11, 12, 17 and 18: 100 ng of HeLa RNA; M: DNA sizing markers.

(8) FIG. 7 is a photograph of an EtdBr-stained gel demonstrating the sensitivity and efficiency of the invention in RT-PCR of long (>3 kb) products. 100 ng of total HeLa RNA was reverse transcribed using the compositions of the invention with the addition of 1 l of LONGASE Enzyme Mix, and fragments of the adenomatous polyposis coli gene that were 7.3 kb (lanes 1, 2), 7.7 kb (lanes 3, 4) or 8.9 kb (lanes 5, 6) in size were amplified by PCR. M: DNA sizing markers (HindIII digest of DNA).

DETAILED DESCRIPTION OF THE INVENTION

Overview

(9) The present invention is directed to compositions and methods for use in reverse transcriptase-polymerase chain reaction (RT-PCR) production and analysis of nucleic acids. In particular, the invention provides compositions comprising a variety of components in various combinations. Such components include one or more sulfur-containing molecules or one or more acetate-containing molecules (or combinations of one or more sulfur-containing molecules and one or more acetate-containing molecules), one or more enzymes having reverse transcriptase activity, one or more DNA polymerases, one or more primers, one or more nucleotides and a suitable buffer. These compositions may be used in the methods of the invention to produce, analyze, quantitate and otherwise manipulate nucleic acid molecules using a one- or two-step RT-PCR procedure.

(10) Compositions

(11) The buffer in the compositions of the invention provide appropriate pH and ionic conditions for the enzymes having reverse transcriptase activity and DNA polymerase enzymes. The nucleotides used in the compositions (e.g., deoxyribonucleoside triphosphates (dNTPs)), and the primer nucleic acid molecules provide the substrates for synthesis or amplification of nucleic acid molecules in accordance with the invention. The compositions of the invention may also include inhibition-relieving reagents to assist in overcoming inhibition in RT-PCR reactions.

(12) Buffer and Ionic Conditions

(13) The buffer and ionic conditions of the present compositions have been optimized to relieve RT-mediated inhibition of RT-PCR. Preferred compositions of the invention comprise one or more sulfur-containing molecules, which provide sulfur in ionic form such as sulfate ions, sulfite ions, sulfonate ions (e.g., p-toluenesulfonic acid) and the like. Additional preferred compositions of the invention comprise one or more acetate-containing molecules, or combinations of one or more sulfur-containing molecules and one or more acetate-containing molecules.

(14) The sulfur-containing molecules should be formulated into the compositions to preferably provide a concentration of sulfur in the solution of at least 18 mM, more preferably a concentration of at least 19 mM and most preferably a concentration of at least 20 mM. Particularly preferred concentration ranges for sulfur in the present compositions include about 18 mM to about 500 mM, about 18 mM to about 150 mM, about 18 mM to about 100 mM, about 18 mM to about 75 mM, about 18 mM to about 50 mM or about 18 mM to about 40 mM, and most preferably about 18 mM to about 50 mM.

(15) The sulfur-containing molecules are preferably formulated into the present compositions in the form of one or more salts or buffers. Examples of suitable sulfur-containing salts according to the invention include, but are not limited to, ammonium sulfate, magnesium sulfate, manganese sulfate, potassium sulfate, sodium sulfate and the like. Most preferred are ammonium sulfate, magnesium sulfate and manganese sulfate. Examples of suitable sulfur-containing buffers according to the invention include, but are not limited to, TRIS-sulfate and other sulfuric acid-based buffers, as well as sulfonic acid-based buffers such as AMPSO (3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid), BES (N,N-bis[2-hydroxyethyl]-2-aminomethanesulfonic acid), MOPS (3-N-morpholino)-propanesulfonic acid), MOPSO (3-N-morpholino)-2-hydroxypropanesulfonic acid, TES (2-{[tris-(hydroxymethyl)-methyl]amino}ethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid), HEPPS (N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid), HEPPSO (N-2-hydroxyethylpiperazine-N-2-hydroxypropanesulfonic acid), TAPS (TES (3-{[tris-(hydroxymethyl)methyl]amino}propanesulfonic acid, CHES (2-(N-cyclo-hexylamino)ethanesulfonic acid), MES (2-N-morpholino)ethanesulfonic acid, PIPES (piperazine-N,N-bis-2-ethanesulfonic acid), POPSO (piperazin-N,N-bis[2-hydroxy]propanesulfonic acid), TAPS(N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid), TAPSO (3-[N-tris{hydroxymethyl}methylamino]-2-hydroxypropanesulfonic acid), ACES (N-2-acetamide-2-aminoethane sulfonic acid), DIPSO (3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), CAPSO (3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid) and CAPS (3-[cyclohexylamino]propanesulfonic acid). Other sulfur-containing ionic salts and buffers, and other sulfur-containing molecules, suitable for use in the compositions of the invention will be apparent to one of ordinary skill in the art.

(16) The acetate-containing molecules should be formulated into the compositions to preferably provide a concentration of acetate ion in the solution of about 1 mM to about 500 mM. Particularly preferred concentration ranges for acetate ion in the present compositions include about 1 mM to about 500 mM, about 5 mM to about 250 mM, about 10 mM to about 200 mM, about 25 mM to about 150 mM, about 50 mM to about 100 mM, or about 60 mM.

(17) The acetate-containing molecules are preferably formulated into the present compositions in the form of one or more salts or buffers. Examples of suitable acetate-containing salts according to the invention include, but are not limited to, ammonium acetate, magnesium acetate, manganese acetate, potassium acetate, sodium acetate and the like. Examples of suitable acetate-containing buffers according to the invention include, but are not limited to, TRIS-acetate (tris[{hydroxymethyl}]aminomethane] acetate), ADA (N-[2-acetamido]-2-iminodiacetic acid), and imidazole acetate (2-hydroxy-3-[4-imidazolyl]-propionic acid).

(18) In accordance with the invention, one or more potassium-containing molecules may be formulated into the present compositions to substitute for, and thereby reduce the concentration requirement for, sulfur. In this aspect of the invention, the addition of both sulfur-containing molecules and potassium-containing molecules decreases the concentration requirement for sulfur by about 13-75%, preferably by about 25-50%. For example, when potassium-containing molecules are formulated into the present compositions, the concentration of sulfur-containing molecules may be reduced from about 18 mM to about 2-14 mM, or preferably to about 4-9 mM. It will be understood, of course, that the one or more potassium ions may also be used in the above-described compositions of the invention that contain one or more acetate-containing molecules instead of, or in addition to, the one or more sulfur-containing molecules.

(19) The potassium-containing molecules should be formulated into the compositions at a preferred concentration of at least 2 mM, preferably at least 5 mM, still more preferably at least 10 mM, and most preferably at least 20 mM. Particularly preferred concentration ranges of potassium-containing molecules in the present compositions include about 2 mM to about 500 mM, about 2 mM to about 200 mM, about 2 mM to about 100 mM, about 2 mM to about 75 mM, about 2 mM to about 50 mM, about 2 mM to about 40 mM, about 2 mM to about 30 mM, about 2 mM to about 20 mM and about 2 mM to about 10 mM.

(20) The potassium-containing molecules are preferably formulated into the present compositions in the form of one or more salts or buffers. Examples of suitable potassium salts according to the invention include, but are not limited to, potassium sulfate, potassium sulfite, potassium chloride, potassium nitrate and the like. Most preferred are potassium chloride, potassium sulfate and potassium acetate. Preferred potassium buffers according to the invention include, but are not limited to, potassium phosphate (monobasic), potassium phosphate (dibasic) and the like. Other potassium salts and buffers, and other potassium-containing molecules, suitable for use in the present compositions will be apparent to one of ordinary skill in the art.

(21) Molecules and buffers containing sulfur, acetate or potassium that are suitable for use in the present compositions are available commercially from a wide variety of sources, e.g., from Sigma (St. Louis, Mo.).

(22) Reverse Transcriptase Enzymes

(23) The compositions of the present invention also comprise enzymes having reverse transcriptase activity. According to the present invention, the enzymes having reverse transcriptase activity are Moloney Murine Leukemia Virus (M-MLV) reverse transcriptases. Preferred enzymes for use in the invention include those that are substantially reduced in RNase H activity. By an enzyme substantially reduced in RNase H activity is meant that the enzyme has less than about 20%, more preferably less than about 15%, 10% or 5%, and most preferably less than about 2%, of the RNase H activity of a wildtype or RNase H.sup.+ enzyme such as wildtype M-MLV reverse transciptase. The RNase H activity may be determined by a variety of assays, such as those described, for example, in U.S. Pat. No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988) and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), the disclosures of all of which are fully incorporated herein by reference.

(24) Particularly preferred enzymes for use in the invention include, but are not limited to, M-MLV reverse transcriptase (RNase H or substantially reduced in RNase H activity), and RSV reverse transcriptase (RNase H or substantially reduced in RNase H activity). Enzymes having reverse transcriptase activity are commercially available (for example, SUPERSCRIPT I reverse transcriptase or SUPERSCRIPT II reverse transcriptase and M-MLV, available from Life Technologies, Inc.; Rockville, Md.).

(25) DNA Polymerases

(26) The compositions of the invention also comprise one or more DNA polymerases, which are preferably thermostable DNA polymerases. Those DNA polymerases may be isolated from natural or recombinant sources, by techniques that are well-known in the art (See WO 92/06200, U.S. Pat. Nos. 5,455,170 and 5,466,591, WO 96/10640 and U.S. patent application Ser. No. 08/370,190, filed Jan. 9, 1995, the disclosures of all of which are incorporated herein by reference), from a variety of thermophilic bacteria that are available commercially (for example, from American Type Culture Collection, Rockville, Md.) or may be obtained by recombinant DNA techniques (see, e.g., WO 96/10640 and U.S. patent application Ser. No. 08/370,190, filed Jan. 9, 1995). Suitable for use as sources of thermostable polymerases or the genes thereof for expression in recombinant systems are the thermophilic bacteria Thermus thermophilus, Thermococcus litoralis, Pyrococcus furious, Pyrococcus woosii and other species of the Pyrococcus genus, Bacillus sterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum, Thermus favus, Thermus ruber, Thermus brockianus, Thermotoga neapolitana, Thermotoga maritima and other species of the Thermotoga genus, and Methanobacterium thermoautrotophicum, and mutants, variants or derivatives thereof. It is to be understood, however, that thermostable DNA polymerases from other organisms may also be used in the present invention without departing from the scope or preferred embodiments thereof. As an alternative to isolation, thermostable DNA polymerases are available commercially from, for example, Life Technologies, Inc. (Rockville, Md.), New England BioLabs (Beverly, Mass.), Finnzymes Oy (Espoo, Finland), Stratagene (La Jolla, Calif.), Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and Perkin Elmer Cetus (Norwalk, Conn.).

(27) Particularly preferred thermostable DNA polymerases for use in the compositions and methods of the present invention include, but are not limited to, Taq, Tne, Tma, Tli/VENT, DEEPVENT, Pfu, Pwo, Tfi or Tth DNA polymerases, or mutants or derivatives thereof. Taq DNA polymerase is commercially available, for example from Life Technologies, Inc. (Rockville, Md.), or may be isolated from its natural source, the thermophilic bacterium Thermus aquaticus, as described previously (U.S. Pat. Nos. 4,889,818 and 4,965,188). The DNA polymerase may be isolated from its natural source, the thermophilic bacterium Thermotoga neapolitana (See WO 96/10640 and U.S. patent application Ser. No. 08/370,190, filed Jan. 9, 1995), and Tma DNA polymerase from its natural source, the thermophilic bacterium Thermotoga maritima (See U.S. Pat. No. 5,374,553, the disclosure of which is incorporated herein by reference). Methods for producing mutants and derivatives of thermophilic DNA polymerases, particularly of Tne and Tma polymerases, are disclosed in co-pending U.S. patent application Ser. No. 08/689,807 of Deb K. Chatterjee, and in co-pending U.S. patent application Ser. No. 08/689,818 of Deb K. Chatterjee and A. John Hughes, both filed Sep. 6, 1996, which are incorporated by reference herein in their entirety. Tfi, Tli/VENT and DEEPVENT are available commercially (e.g., from New England BioLabs; Beverly, Mass.), or may be produced as described (Bej, A. K., and Mahbubani, M. H., in: PCR Technology: Current Innovations, Griffin, H. G., and Griffin, A. M., eds., CRC Press, pp. 219-237 (1994) for Tli/VENT; Flaman, J.-M., et al., Nucl. Acids Res. 22(15):3259-3260 (1994) for DEEPVENT). Thermostable DNA polymerases are preferably added to the present compositions at a final concentration in solution of about 0.1-200 units per milliliter, about 0.1-50 units per milliliter, about 0.1-40 units per milliliter, about 0.1-36 units per milliliter, about 0.1-34 units per milliliter, about 0.1-32 units per milliliter, about 0.1-30 units per milliliter, or about 0.1-20 units per milliliter, and most preferably at a concentration of about 20 units per milliliter.

(28) In preferred compositions of the invention, the concentration of DNA polymerases is determined as a ratio of the concentration of the enzymes having reversetranscriptase activity. Thus, in particularly preferred compositions the unit ratio of the reverse transcriptase enzymes to the DNA polymerase enzymes ranges from about 0.2:2 to about 500:2, preferably from about 0.5:2 to about 250:2 and most preferably a ratio of greater than 3:2. Of course, other suitable ratios of unit activities of reverse transcriptase enzymes to DNA polymerases suitable for use in the invention will be apparent to one of ordinary skill in the art.

(29) Inhibition-Relieving Reagents

(30) In accordance with the methods of the invention, one or more additional inhibition-relieving agents may optionally be added to the present compositions to assist in overcoming the inhibition of RT-PCR reactions by RTs such as M-MLV RT. Preferred inhibition-relieving agents for use in the present compositions include peptides, polypeptides and proteins such as (but not limited to) human serum albumin, bovine serum albumin, ovalbumin, Albumax, casein, gelatin, collagen, globulin, lysozyme, transferrin, myoglobin, hemoglobin, -lactalbumin, fumarase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), amyloglucosidase, carbonic anhydrase, -lactoglobulin, aprotinin, soybean trypsin inhibitor, trypsinogen, phosphorylase b, myosin, actin, -galactosidase, catalase, tryptic soy digests, tryptose, lectins and the like, or fragments or derivatives thereof. Particularly preferred for use in the compositions and methods of the invention are bovine serum albumin, human serum albumin, Albumax and casein. Peptides, polypeptides or proteins are preferably added to the compositions to give a final concentration in the solution of about 0.01 to about 100 g/ml, preferably about 0.1 to about 100 g/ml, more preferably about 1 to about 50 g/ml and most preferably about 2 to about 20 g/ml.

(31) dNTPs

(32) The compositions of the invention further comprise one or more nucleotides (e.g., deoxynucleoside triphosphates (dNTPs)). The nucleotide components of the present compositions serve as the building blocks for newly synthesized nucleic acids, being incorporated therein by the action of the reverse transcriptases or DNA polymerases. Examples of nucleotides suitable for use in the present compositions include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, -thio-dATP, -thio-dTTP, -thio-dGTP, -thio-dCTP or derivatives thereof all of which are available commercially from sources including Life Technologies, Inc. (Rockville, Md.), New England BioLabs (Beverly, Mass.) and Sigma Chemical Company (Saint Louis, Mo.). The dNTPs may be unlabeled, or they may be detectably labeled by coupling them by methods known in the art with radioisotopes (e.g., .sup.3H, .sup.14C, .sup.32P or .sup.35S), vitamins (e.&, biotin), fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels, dioxigenin and the like. Labeled dNTPs may also be obtained commercially, for example from Life Technologies, Inc. (Rockville, Md.) or Sigma Chemical Company (Saint Louis, Mo.). In the present compositions, the dNTPs are added to give a working concentration of each dNTP of about 10-1000 micromolar, about 10-500 micromolar, about 10-250 micromolar, or about 10-100 micromolar, and most preferably a concentration of about 100 micromolar.

(33) Primers

(34) In addition to nucleotides, the present compositions comprise one or more primers which facilitate the synthesis of a first DNA molecule complementary to all or a portion of an RNA template (e.g., a single-stranded cDNA molecule). Such primers may also be used to synthesize a DNA molecule complementary to all or a portion of the first DNA molecule, thereby forming a double-stranded cDNA molecule. Additionally, these primers may be used in amplifying nucleic acid molecules in accordance with the invention. Such primers include, but are not limited to, target-specific primers (which are preferably gene-specific primers), oligo(dT) primers, random primers or arbitrary primers. Additional primers that may be used for amplification of the DNA molecules according to the methods of the invention will be apparent to one of ordinary skill in the art.

(35) Methods of RT-PCR

(36) In the RT-PCR reaction, the reaction mixtures are incubated at a temperature sufficient to synthesize a DNA molecule complementary to all or portion of the RNA template. Such conditions typically range from about 20 C. to 75 C., more preferably from about 35 C. to 60 C. and most preferably from about 45 C. to about 55 C. After the reverse transcription reaction, the reaction is incubated at a temperature sufficient to amplify the synthesized DNA molecule. Preferably the amplification is accomplished via one or more polymerase chain reactions (PCRs). Preferred conditions for amplification comprise thermocycling, which may comprise alternating heating and cooling of the mixture sufficient to amplify the DNA molecule and which most preferably comprises alternating from a first temperature range of from about 90 C. to about 100 C., to a second temperature range of from about 45 C. to about 75 C., more preferably from about 50 C. to about 75 C. or from about 55 C. to about 75 C., and most preferably from about 65-C to about 75 C. According to the invention, the thermocycling may be performed any number of times, preferably from about 5 to about 80 times, more preferably greater than about 10 times and most preferably greater than about 20 times

(37) The compositions and methods of the present invention may also be used for the production, analysis and quantitation of large nucleic acid molecules (e.g., by long PCR or long RT-PCR), preferably nucleic acid molecules that are larger than about 4-8 kilobases in size, more preferably larger than about 5-7 kilobases in size, and most preferably nucleic acid molecules that are larger than about 7 kilobases in size. In this aspect of the invention, combinations of DNA polymerases, preferably mixtures of one or more DNA polymerases lacking 3-5 exonuclease activity (i.e., a 3 exo.sup. polymerase) with one or more DNA polymerases having 3-5 exonuclease activity (i.e., a 3 exo.sup.+ polymerase), may be added to the compositions of the invention (see U.S. Pat. No. 5,436,149; see also co-pending U.S. patent application Ser. No. 08/801,720, of Ayoub Rashtchian and Joseph Solus, filed Feb. 14, 1997, and the co-pending U.S. patent application of Ayoub Rashtchian and Joseph Solus entitled Stable Compositions for Nucleic Acid Amplification and Sequencing, filed Mar. 27, 1998, the disclosures of all of which are incorporated herein in their entireties). Preferred 3 exo.sup. and 3 exo.sup.+ polymerases for use in this aspect of the invention are thermostable 3 exo.sup. and 3 exo.sup.+ polymerases. Particularly preferred 3 exo.sup.+ polymerases include, but are not limited to, Taq, Tne(exo.sup.), Tma(exo.sup.), VENT(exo.sup.), DEEP VENT(exo.sup.), Pfu (exo.sup.) and Pwo(exo.sup.) polymerases, or mutants, variants or derivatives thereof which are preferably added to the compositions of the invention at a concentration in the solution of about 0.1-200 units per milliliter, about 0.1-50 units per milliliter, about 0.1-40 units per milliliter, about 0.1-36 units per milliliter, about 0.1-34 units per milliliter, about 0.1-32 units per milliliter, about 0.1-30 units per milliliter, or about 0.1-20 units per milliliter, and most preferably at a concentration of about 20 units per milliliter. Particularly preferred 3 exo.sup.+ polymerases include, but are not limited to, VENT, Pfu, Pwo, Tne, Kod and Tma, and most preferably DEEPVENT, DNA polymerases, which should be added to the mixtures in sufficient quantity to give a final working concentration of about 0.0002-200 units per milliliter, about 0.002-100 units per milliliter, about 0.002-20 units per milliliter, about 0.002-2.0 units per milliliter, about 0.002-1.6 units per milliliter, about 0.002-0.8 units per milliliter, about 0.002-0.4 units per milliliter, or about 0.002-0.2 units per milliliter, most preferably at concentrations of about 0.40 units per milliliter. These thermostable DNA polymerases are available commercially, for example from Life Technologies, Inc. (Rockville, Md.), New England BioLabs (Beverly, Mass.), Finnzymes Oy (Espoo, Finland), Stratagene (La Jolla, Calif.), Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and Perkin-Elmer Cetus (Norwalk, Conn.). The mixtures of the compositions of the invention and the 3 exo and 3 exo.sup.+ polymerases may be used in the methods of the invention to result in enhanced sensitivity of detection, and yield, of large RT-PCR products.

(38) It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

Example 1: Inhibition of RT-PCR by RT

(39) To examine inhibition of RT-PCR amplification by RT, CAT RNA was used as a template. RT-PCR reactions were conducted in a 50 l final volume in PCR buffer (20 mM Tris-HCl, 50 mM KCl) or ELONGASE buffer (60 mM Tris-SO.sub.4 (pH 9.1), 18 mM (NH.sub.4).sub.2SO.sub.4; total sulfur concentration approximately 23 mM) containing 20 units of M-MLV(H.sup.) RT, 1 mM dithiothiothreitol (DTT), 0.2 mM each of sense and antisense primers, 0.2 mM dNTPs, 1.25 mM MgCl.sub.2, 2 units of Taq and various amounts of CAT mRNAs (0, 10, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6 or 10.sup.7 copies per reaction). RT-PCR conditions were one cycle of 45 C. for 30 minutes and 94 C. for 2 minutes, followed by 40 cycles of 94 C. for 15 seconds/60 C. 30 seconds and then 72 C. for 5 minutes.

(40) Upon analysis of the amplification products by 1.5% agarose gel electrophoresis (FIG. 1), significant amplification of the 500 bp target sequence was observed in reactions carried out in ELONGASE buffer using 10.sup.6 or 10.sup.7 copies of CAT mRNA template (FIG. 1, lanes 8 and 9). In contrast, no significant PCR product was observed for reactions carried out in standard PCR buffer at all template concentrations (FIG. 1, lanes 18, 19). These results indicate that the presence of RT in PCR reaction mixtures inhibits the amplification reaction, but that this inhibition is at least partially relieved using ELONGASE buffer.

Example 2: Role of Sulfur in Relief of RT Inhibition in PCR Amplification

(41) To determine if the sulfate ion in ELONGASE buffer might be a key component for the relief of RT inhibition observed in Example 1, various reaction parameters such as pH, ionic conditions and buffer composition were studied in detail. RT-PCR reactions were carried out in a 50 l final volume containing 1 mM DTT, 0.2 mM dNTPs, 1.5 mM MgSO.sub.4, 0.2 mM each of sense and antisense human -actin primers, 1 pg of total HeLa RNA template, 2 units of Taq DNA polymerase and 10 units M-MLV(H.sup.) RT in buffered salt solutions comprising various ionic and buffer conditions. RT-PCR conditions were 30 minutes at 45 C. and 2 minutes at 94 C., followed by 40 cycles of 94 C. for 15 seconds/55 C. for 30 seconds/68 C. for 90 seconds, and one final extension for 5 minutes at 72 C. The ability to detect an amplified 1,026-bp RT-PCR product from 1 pg of total HeLa RNA was used as an assessment of the amplification success under the specific reaction conditions.

(42) Upon analysis of 1,026-bp b-actin RT-PCR products in 1% agarose gel electrophoresis, the results shown in Tables 1-7 were obtained.

(43) Table 1: Compositions comprising Tris-HCl (pH 8.5.9.3) demonstrated a sensitivity of about 1 pg total HeLa RNA when 18 mM (NH.sub.4).sub.2SO.sub.4 was present. However, this increased sensitivity was obtained over a broader pH range (pH 7.8-9.3) when Tris-SO.sub.4, buffer was used in place of Tris-HCl.

(44) TABLE-US-00001 TABLE 1 Optimal pH in Tris-HCl and Tris-SO.sub.4 Buffers. pH 7.5 7.8 8.0 8.3 8.5 8.8 9.0 9.3 9.5 Tris-HCl, 60 mM.sup.1 + + + + + + + + + (NH.sub.4).sub.2SO.sub.4, 18 mM + + + + + + + + + Sensitivity (Yield + ++ + of PCR Product) Tris-SO.sub.4, 60 mM + + + + + + + + + (NH.sub.4).sub.2SO.sub.4, 18 mM + + + + + + + + + Total Sulfur Concentration 41 38 35 32 30 26 23 22 21 (mM).sup.2 Sensitivity (Yield + + + + + ++ + of PCR Product) .sup.1The pH in the Tris-HCl reaction mixture was adjusted using HCl. Therefore, no sulfur was contributed via pH adjustment, and the sulfur concentration for each pH tested was approximately 18 mM. .sup.2The pH in the Tris-SO.sub.4 reaction mixture was adjusted using sulfuric acid. Therefore, the addition of sulfuric acid increased the sulfur concentration to approximately that shown.

(45) Table 2: In order to detect the 1,026-bp RT-PCR product in compositions comprising Tris-HCl buffer, the inclusion of 20 mM (NH.sub.4).sub.2SO.sub.4 in the compositions was essential. However, if Tris-SO.sub.4 buffer (20-80 mM, pH 8.0-9.0) was used in place of Tris-HCl, the inclusion of (NH.sub.4).sub.2SO.sub.4 was not required.

(46) TABLE-US-00002 TABLE 2 Requirement for Sulfur and Potassium. pH 9.0 8.5 8.0 Tris-HCl, 60 mM + + + + + + + + + + + + + + + + + + (NH.sub.4).sub.2SO.sub.4, mM.sup.1 0 10 20 40 60 80 0 10 20 40 60 80 0 10 20 40 60 80 Sensitivity (Yield of PCR Product) + Tris-SO.sub.4, mM 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 KCl, 40 mM + + + + + + + + + + + + + + + Total Sulfur Concentration (mM).sup.2 20 21 23 25 26 22 26 30 34 38 24 29 35 41 46 Sensitivity (Yield of PCR Product) + + + + + + .sup.1The pH in the Tris-HCl reaction mixture was adjusted using HCl. Therefore, no sulfur was contributed via pH adjustment, and the sulfur concentration for each pH tested was approximately 18 mM. .sup.2The pH in the Tris-SO.sub.4 reaction mixture was adjusted using sulfuric acid. Therefore, the addition of sulfuric acid increased the sulfur concentration to approximately that shown.

(47) Table 3: Requirement for sulfur for sensitive detection of 1,026 bp RT-PCR product was also demonstrated by use of taurine (NH.sub.2CH.sub.2SO.sub.3H), which contains sulfur ion. Taurine relieved the RT-mediated inhibition of RT-PCR about as well as did ammonium sulfate.

(48) TABLE-US-00003 TABLE 3 Requirement for Sulfur. Tris-HCl, 60 mM + + + + + + + + + + (pH 9.0) [Taurine], mM 0 10 20 40 60 0 0 0 0 0 [Ammonium 0 0 0 0 0 0 10 20 40 60 Sulfate], mM Sensitivity (yield + ++ +++ +++ ++ +++ of PCR product)

(49) Table 4: The increased detection sensitivity of the Tris-SO.sub.4 buffer system shown in Table 2 (60 mM, pH 8.5-9.0) could be further enhanced by the addition of 20-40 mM KCl, indicating that potassium-containing molecules not only were suitable substitutes for sulfur-containing molecules in the present compositions, but may also enhance the sensitivity of the RT-PCR reaction in their own right.

(50) Table 5: Similar detection sensitivity and further enhancement by the addition of KCl was obtained in the Tris-taurine buffer system.

(51) TABLE-US-00004 TABLE 4 Role of Potassium in Suboptimal Concentrations of Sulfur. pH 9.0 8.5 8.0 Tris-SO.sub.4, 60 mM + + + + + + + + + + + + + + + + + + (NH.sub.4).sub.2SO.sub.4, mM 0 10 20 40 60 80 0 10 20 40 60 80 0 10 20 40 60 80 Sensitivity (Yield of PCR Product) ++ + + Tris-SO.sub.4, 60 mM + + + + + + + + + + + + + + + + + + KCl, mM 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Sensitivity (Yield of PCR Product) + + + +++ +++

(52) TABLE-US-00005 TABLE 5 Requirement for Sulfur and Potassium. [Tris-taurine], mM (pH 8.9) 60 60 60 60 60 60 60 60 60 60 100 200 60 60 60 60 60 [Ammonium sulfate], mM 0 10 20 40 60 0 0 0 0 0 0 0 0 0 0 0 0 [Taurine], mM 0 0 0 0 0 0 0 0 0 0 0 0 0 10 20 40 60 [KCl], mM 0 0 0 0 0 0 10 20 40 60 0 0 0 0 0 0 0 Sensitivity (yield of PCR product) +++ +++ + ++ +++ +++ + ++ + ++ +++ +++

(53) Table 6: The addition of NH.sub.4Cl in place of (NH.sub.4).sub.2SO.sub.4 in the present compositions did not relieve the RT-mediated inhibition of RT-PCR, indicating that sulfur-containing molecules are key components for the relief of RT inhibition in RT-PCR.

(54) TABLE-US-00006 TABLE 6 Requirement for Sulfur. Detection Sensitivity (Yield of PCR Product) Buffer Composition (Additive to (NH.sub.4).sub.2SO.sub.4, NH.sub.4Cl, Tris-HCl, 60 mM) 20 mM 40 mM Magnesium Sulfate, 1.5 mM + Magnesium Acetate, 1.5 mM + Magnesium Chloride, 1.5 mM +

(55) Table 7: Requirement for sulfur ion for relief of RT-mediated inhibition of PCR was less stringent in Tris-acetate buffer systems than in Tris-sulfate buffer systems. In the Tris-acetate buffer system, RT-PCR products of 1,026 bp size were able to be observed even in the absence of sulfur ions.

(56) TABLE-US-00007 TABLE 7 Requirement for Sulfur and Effect of Buffers. Tris-SO.sub.4, + + + + + 60 mM (pH 9.0) Tris-acetate, + + + + + 60 mM (pH 8.4) [(NH.sub.4).sub.2SO.sub.4], mM 0 10 20 40 80 0 10 20 40 80 Sensitivity (yield ++ +++ + ++ +++ of PCR product)

Example 3: Role of Bovine Serum Albumin (BSA) in RT-PCR

(57) To investigate other reaction components which might relieve the RT-mediated inhibition of RT-PCR, BSA was added to the present compositions. Compositions were formulated comprising increasing amounts of M-MLV(H.sup.) RT (from 10 units to 260 units) and various amount of BSA, and these compositions used in RT-PCR reactions. Reactions were conducted in a 50 l final volume containing 60 mM Tris-SO.sub.4 (pH 9.1), 18 mM (NH.sub.4).sub.2SO.sub.4, 0.2 mM dNTPs, 1.2 mM MgSO.sub.4, 0.02 mM DTT, 0.2 mM each of human -actin CAT mRNA sense and antisense primers, 2 units of Taq DNA polymerase and 100 pg of total HeLa RNA template for -actin amplification, or 10.sup.5 copies of total HeLa RNA template for CAT amplification. RT-PCR conditions were 30 minutes at 45 C. and two minutes at 94 C., followed by 40 cycles of 94 C. for 15 seconds/55 C. for 30 seconds/68 C. for 90 seconds, and then one final extension of five minutes at 72 C.

(58) Upon analysis of the 1,026-bp -actin RT-PCR products in 1% agarose gel electrophoresis (FIG. 2A). RT inhibition was not observed with 10 units of RT, but increasing amounts of RT (135 units-260 units) inhibited RT-PCR completely. However, such RT inhibition was relieved by the addition of 200-1000 ng of BSA per reaction (i.e., a final BSA concentration of about 4-20 g/ml). Similar results were obtained from the analysis of the 653-bp CAT RT-PCR products (FIG. 2B), where only a slightly higher amount of BSA (300-1000 ng per reaction i.e., a final BSA concentration of about 6-20 g/ml) was required for the relief of RT inhibition. Together, these results indicate that the incorporation of proteins such as BSA into the compositions of the invention may assist in overcoming the inhibition of RT-PCR caused by RT.

Example 4: Performance of M-MLV, AMV, M-MLV(H) for RT-PCR in Sulfur- and BSA-containing Buffer

(59) To study the performance of various RTs in RT-PCR, AMV-RT, M MLV-RT, and M-MLV(H) RT were used in the present compositions. RT-PCR reactions for M-MLV-RT and M-MLV H RT (SUPERSCRIPT II reverse transcriptase) were conducted in a 50 l final volume containing 60 mM Tris-S04 (pH 9.1), 18 mM (NH4)2S04, 0.2 mM dNTPs, 250 ng BSA, 1.0 mM DTT, 0.2 mM each of CAT sense and antisense primers, 2 units of Taq DNA polymerase, 1.5 mM MgSO.sub.4, and various amount of CAT RNA template (0, 10.sup.3, 10.sup.4 or 10.sup.5 copies per reaction). The compositions containing AMV-RT were the same except that they lacked BSA. For each reaction, 5 units each of AMV-RT, M-MLV-RT or M-MLV(H) RT were used. RT-PCR cycling conditions were 30 minutes at 45 C. and 2 minutes at 94 C., followed by 40 cycles of 94 C. for 15 seconds/60 C. for 30 seconds, and then a final extension of five minutes at 72 C.

(60) As shown in FIG. 3, upon analysis of 500-bp CAT RT-PCR products in 1.5% agarose gel electrophoresis the reactions performed with AMV-RT, M-MLV-RT, and M-MLV(H.sup.)-RT demonstrated efficient and sensitive RT-PCR product yield, with no inhibition of the RT-PCR reactions observed under any of the reaction conditions.

Example 5: RT-PCR Amplification of Long Nucleic Acid Templates

(61) Having demonstrated the simplicity and sensitivity of the present methods in RT-PCR amplification of templates up to 3.5 kb in size, the efficacy of the invention in amplifying mRNAs up to 8.9 kb was examined.

(62) Total HeLa RNA was isolated with TRIzol Reagent (Life Technologies, Inc.; Rockville, Md.) and amplified as above. To examine possible temperature effects, identical reactions were assembled and incubated at 45 C. to 55 C. in duplicate. The total HeLa RNA used varied from 1 ng to 100 ng depending on the abundance of the mRNA. After the RT incubation for 30 minutes, the reactions were incubated at 94 C. for two minutes, followed by 40 cycles of 94 C. for 15 seconds, 55 C. for 30 seconds and 68 C. for 60 seconds, followed by a final 68 C. extension for five minutes.

(63) For larger RT-PCR products, the magnesium concentration was increased to 1.8 mM from the standard 1.5 mM, and 1 l of ELONGASE Enzyme Mix (Life Technologies, Inc.; Rockville, Md.) was added to each reaction. The final 50 l reaction consisted of 1 ELONGASE buffer with 1.8 mM MgSO.sub.4 and other salts, 200 M of each dNTP, 2 g/ml BSA, 0.2 M of primers (GIBCO BRL Custom Primers; Life Technologies, Inc., Rockville, Md.), 100 ng of total HeLa RNA, 1 l of the RT/Taq enzyme mix of the invention and 1 l of ELONGASE Enzyme Mix. For the experiments using the same template of primers, a master mix of buffer, enzyme mixes, and primers or template was made to ensure consistency. The samples were incubated at 50 C. for 30 minutes and then 94 C. for two minutes. Amplification was performed with 40 cycles of 94 C. for 15 seconds, 58 C. for 30 seconds and 68 C. for six to nine minutes (one minute/kb). The RT-PCR products were resolved and visualized on 0.8 or 1.0% (w/v) agarose-TAE gels containing ethidium bromide.

(64) Temperature of the RT Reaction.

(65) SUPERSCRIPT II reverse transcriptase (RT) has improved temperature stability compared to M-MLV-RT (Gerard, G., et al., FOCUS 14:91 (1992)). To test the effect of temperature on RT-PCR products, 36 primer sets (representing different genes from human, rat, plant, and one in vitro transcript) were examined. As shown in FIG. 4, substantial product yield and specificity were observed at 45 C. for many of the fragments examined; increased incubation temperature did not result in increased yield or specificity. This result, however, was dependent upon the specific template and primer chosen (FIG. 5): for some template-primer combinations, the smearing of bands caused by mispriming that was characteristic of RT reactions conducted at 45 C. disappeared with increased temperature (FIG. 5; lanes 1-6), while in some cases a significant increase in product yield was observed when the RT reactions were conducted at elevated temperatures (FIG. 5; lanes 7-12).

(66) Long RT-PCR Products.

(67) For studies of full-length coding sequences or for amplification of long segments of RNA, the use of the compositions of the present invention supplemented with ELONGASE Enzyme Mix was tested. As shown in FIG. 6, the present system and Supplier A's kit were able to amplify a 2.8-kb product from 100 ng, but not from 10 ng, of total RNA. The addition of ELONGASE Enzyme Mix to the present system not only increased the product yield with 100 ng of total RNA, but also increased the sensitivity to allow amplification of long templates from 10 ng total RNA (FIG. 6; lanes 7-12). In contrast, Supplier A's kit was not able to amplify the 2.8-kb target from 10 ng of total RNA (FIG. 6; lanes 13-18) even though it contained a polymerase enzyme mix designed for long templates.

(68) The compositions and methods of the invention were also found to be useful in amplifying large (>3-5 kb) RT-PCR products. As shown in FIG. 7, the compositions of the present invention, when supplemented with ELONGASE Enzyme Mix, produced RT-PCR products up to 8.9 kb in size. The ladder of bands observed upon amplification with these compositions is not uncommon for long RT-PCR, and the variation in product yields may be partially due to the use of different primer sets. These results demonstrate that the addition of ELONGASE Enzyme Mix to the present compositions makes it possible to amplify long and rare mRNAs directly from total RNA preparations by the methods of the invention. Further, the thermostability of SUPERSCRIPT II RT, facilitating RT reactions at temperatures of up to 55 C., can increase the specificity and product yield for some templates and primer sets.

(69) Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

(70) All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.