Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-01
2001-10-09
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S183000, C435S810000
Reexamination Certificate
active
06300073
ABSTRACT:
TECHNICAL FIELD
The field of this invention is nucleic acid amplification, and particularly one-step RT-PCR.
BACKGROUND OF THE INVENTION
Reverse transcription (RT) and the polymerase chain reaction (PCR) are critical to many molecular biology and related applications, particularly gene expression analysis applications. In these applications, reverse transcription is used to prepare template DNA from an initial RNA sample, e.g. mRNA, which template DNA is then amplified using PCR to produce a sufficient amount of amplified product for the application of interest. The RT and PCR steps of DNA amplification can be carried out as a two step or one step process.
In two step processes, the first step involves synthesis of first strand cDNA with a reverse transcriptase, e.g. MMLV-RT, following by a second PCR step. In certain protocols, these steps are carried out in separate reaction tubes. In these two tube protocols, following reverse transcription of the initial RNA template in the first tube, an aliquot of the resultant product is then placed into the second PCR tube and subjected to PCR amplification.
In a second type of two-step process, both RT and PCR are carried out in the same tube using a compatible RT and PCR buffer. In certain embodiments of single tube protocols, reverse transcription is carried out first, followed by addition of PCR reagents to the reaction tube and subsequent PCR.
In an effort to further expedite and simplify RT-PCR procedures, a variety of one step RT-PCR protocols have been developed. See e.g. the Relevant Literature section, supra. However, there is still room for improvement of these methods in a number of areas, including sensitivity, efficiency, and the like.
Accordingly, there is continued interest in the development of additional one step RT-PCR protocols, where a highly efficient and sensitive protocol is of particular interest.
Relevant Literature
See Blain & Goff, J. Biol. Chem. (1993) 5: 23585-23592; Blain & Goff, J. Virol. (1995) 69:4440-4452; Sellner et al., J. Virol. Method. (1994) 49:47-58; PCR, ESSENTIAL TECHNIQUES (ed. J. F. Burke, J. Wiley & Sons, New York)(1996) pp61-63; 80-81; SuperScript One-Step RT-PCR System description on the world-wide web at http://www.lifetech.com/world_whatsnew/archive
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3.html; Access RT-PCR System and Access RT-PCR Introductory System described on the world wide web at http://www.promega.com/tbs/tb220/tb220.html; and AdvanTaq & AdvanTaq Plus PCR kits and User Manual available at www.clontech.com at least as early as Sep. 15, 1999.
SUMMARY OF THE INVENTION
Enzyme compositions, kits comprising the same and methods for their use in one-step RT-PCR are provided. The subject enzyme compositions at least include a mutant thermostable DNA polymerase and a mutant reverse transcriptase. In preferred embodiments, the mutant thermostable DNA polymerase is an N-terminal deletion mutant of Taq polymerase and the mutant reverse transcriptase is a point mutation mutant of MMLV-RT. The subject kits, in addition to the above described mutant thermostable DNA polymerase and mutant reverse transcriptase, include at least one of, and usually both of dNTPs and a buffer composition, where the subject kits may further include additional reagents, including nucleic acids, a thermostabilizing agent, a glycine based osmolyte and the like. In practicing the subject methods, a reaction mix that at least includes template RNA, the above described mutant polymerase and reverse transcriptase, dNTPs, buffer, and nucleic acid primers is prepared. The resultant reaction mixture is then subjected to a first set of reverse transcription conditions and then a second set of PCR conditions, whereby amplified amounts of DNA from a template RNA(s).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Enzyme compositions, kits comprising the same and methods for their use in one-step RT-PCR are provided. The subject enzyme compositions at least include a mutant thermostable DNA polymerase and a mutant reverse transcriptase. In preferred embodiments, the mutant thermostable DNA polymerase is an N-terminal deletion mutant of Taq polymerase and the mutant reverse transcriptase is a point mutation mutant of MMLV-RT. The subject kits, in addition to the above described mutant thermostable DNA polymerase and mutant reverse transcriptase, at least include one of, and usually both of, dNTPs and a buffer composition, where the subject kits may further include additional reagents, including nucleic acids, a thermostabilizing agent, a glycine based osmolyte and the like. In practicing the subject methods, a reaction mix that at least includes template RNA, the above described mutant polymerase and reverse transcriptase, dNTPs, buffer, and nucleic acid primers is prepared. The resultant reaction mixture is maintained at a first set of reverse transcription conditions and then a second set of PCR conditions, whereby amplified amounts of DNA from a template RNA are produced. In further describing the subject invention, the subject enzyme compositions will be described first, followed by a discussion of the subject kits and a review of the methods of amplifying a template RNA into DNA according to the subject invention.
Before the subject invention is further described, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
Enzyme Compositions
As summarized above, the enzyme compositions of the subject invention are characterized by having at least one mutant thermostable polymerase and at least one mutant reverse transcriptase. Where the enzyme compositions include more than one thermostable polymerase, the number of different thermostable polymerases will generally be less than five, usually less than three and in many embodiments will be two. In such embodiments, the amounts of the two or more different polymerases are generally unequal. In addition, the amounts of the thermostable polymerase(s) and the reverse transcriptase(s) in the enzyme composition differ, where the ratio of polymerase to reverse transcriptase activity typically ranges from about 0.8 to 6.5, usually from about 1.6 to 6.5 and more usually from about 1.6 to 4.0 U polymerase/U RT, where 1 Unit of polymerase is defined as the amount of enzyme that will incorporate 10 nmoles of dNTPs into acid insoluble material per 30 minutes in a 10 minute incubation at 72° C. under the assay conditions and 1 Unit of RT is defined as the amount of enzyme that will incorporate 1 nmole of dTMP into acid insoluble material in 10 minutes at 37° C., with poly(A)/oligo(dT) as a substrate.
Thermostable Polymerase
The thermostable DNA polymerase of the enzyme compositions of the subject invention is characterized by having substantial polymerase activity, specifically DNA dependent DNA polymerase activity, but substantially no nuclease activity. Since the enzyme has substantial polymerase activity, it is capable of catalyzing the synthesis of DNA from deoxynucleotide triphosphates using a DNA strand as a template. Since the subject polymerase lacks nuclease activity, it is incapable of catalyzing the hydrolysis of the phosphodiester bonds of DNA polymers. By substantial polymerase activity is meant that the polymerase activity of the enzyme is at least about 80,000 units/mg protein. (Polymerase activity is determined by incubating 5 ml of diluted enzyme fractions with 5
Wurst Helmut
Zhao Ningyue
Bozicevic, Field & Francis
Clontech Laboratories, Inc.
Field Bret E.
Sisson Bradley L.
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