U.S. patent application number 09/932256 was filed with the patent office on 2002-08-29 for novel one step rt-pcr methods, enzyme mixes and kits for use in practicing the same.
Invention is credited to Wurst, Helmut, Zhao, Ningyue.
Application Number | 20020119465 09/932256 |
Document ID | / |
Family ID | 23628575 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119465 |
Kind Code |
A1 |
Zhao, Ningyue ; et
al. |
August 29, 2002 |
Novel one step RT-PCR methods, enzyme mixes and kits for use in
practicing the same
Abstract
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(s) are produced.
Inventors: |
Zhao, Ningyue; (Milpitas,
CA) ; Wurst, Helmut; (Cupertino, CA) |
Correspondence
Address: |
Bret E. Field
Bozicevic, Field and Francis LLP
Suite 200
200 Middlefield Road
Menlo Park
CA
94025
US
|
Family ID: |
23628575 |
Appl. No.: |
09/932256 |
Filed: |
August 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09932256 |
Aug 16, 2001 |
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09411351 |
Oct 1, 1999 |
|
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6300073 |
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Current U.S.
Class: |
435/6.11 ;
435/199; 435/6.12; 435/7.9; 435/91.2 |
Current CPC
Class: |
Y10S 435/81 20130101;
C12N 9/1276 20130101; C12N 9/1252 20130101 |
Class at
Publication: |
435/6 ; 435/7.9;
435/91.2; 435/199 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/542; C12P 019/34; C12N 009/22 |
Claims
What is claimed is:
1. An enzyme composition comprising: a mutant thermostable DNA
polymerase; and a mutant reverse transcriptase.
2. The enzyme composition according to claim 1, wherein said mutant
DNA polymerase is a mutant Taq polymerase.
3. The enzyme composition according to claim 2, wherein said mutant
Taq polymerase is a deletion mutant.
4. The enzyme composition according to claim 3, wherein said
deletion mutant is an N-terminal deletion mutant.
5. The enzyme composition according to claim 1, wherein said mutant
reverse transcriptase is a mutant of moloney murine leukemia virus
reverse transcriptase.
6. The enzyme composition according to claim 5, wherein said mutant
is a point mutation mutant.
7. The enzyme composition according to claim 6, wherein said mutant
exhibits substantially the same RT activity as wild type moloney
murine leukemia virus reverse transcriptase.
8. The enzyme composition according to claim 1, wherein said enzyme
composition further comprises an antibody specific for said mutant
thermostable DNA polymerase.
9. An enzyme composition comprising: an N-terminal deletion mutant
of Taq polymerase; a point mutation mutant of moloney murine
leukemia virus reverse transcriptase; and an antibody specific for
said N-terminal deletion mutant of Taq polymerase.
10. The enzyme composition according to claim 9, wherein the ratio
of said mutant Taq polymerase mutant to said reverse transcriptase
mutant ranges from about 0.8 to 6.5.
11. The enzyme composition according to claim 9, wherein the amount
of said antibody in said composition ranges from about 0.9 to 1.1
.mu.g.
12. A kit for use in a one step nucleic acid amplification
procedure, said kit comprising: (a) a mutant thermostable DNA
polymerase; (b) a mutant reverse transcriptase; and (b) at least
one of the following components: (i) dNTPs; and (ii) buffer.
13. The kit according to claim 12, wherein said kit further
includes a thermostabilizing agent.
14. The kit according to claim 12, wherein said kit further
includes a glycine based osmolyte.
15. The kit according to claim 12, wherein said kit further
includes at least one nucleic acid.
16. The kit according to claim 12, wherein said kit further
includes an RNase inhibitor.
17. A method for producing an amplified amount of DNA from a
template RNA, said method comprising: (a) preparing an aqueous
reaction mixture comprising: (i) said RNA template; (ii) a mutant
thermostable DNA polymerase; (iii) a mutant reverse transcriptase;
(iv) dNTPs (v) buffer reagents; and (vi) at least one nucleic acid
primer; (b) subjecting said reaction mixture at a first set of
reverse transcription reaction conditions suitable for reverse
transcription of said RNA template into cDNA; and (c) subjecting
said reaction mixture at a second set of PCR conditions suitable
for amplification of said cDNA; whereby an amplified amount of DNA
is produced from a template RNA.
18. The method according to claim 17, wherein said reaction mixture
further includes an antibody specific for said mutant thermostable
polymerase.
19. The method according to claim 17, wherein said reaction mixture
further comprises a glycine based osmolyte.
20. The method according to claim 17, wherein said reaction mixture
further comprises a thermostabilizing reagent.
21. The method according to claim 17, wherein mutant thermostable
polymerase is an N-terminal deletion mutant of Taq polymerase.
22. The method according to claim 17, wherein said mutant reverse
transcriptase is a point mutation mutant of moloney murine leukemia
virus reverse transcriptase.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn.119 (e), this application is a
continuation of U.S. patent application Ser. No. 09/411,351, filed
Oct. 1, 1999, the disclosure of which is herein incorporated by
reference.
INTRODUCTION
[0002] 1. Technical Field
[0003] The field of this invention is nucleic acid amplification,
and particularly one-step RT-PCR.
[0004] 2. Background of the Invention
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] Relevant Literature
[0011] 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_whatsn-
ew/archive/nz.sub.--1.sub.--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
[0012] 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
[0013] 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.
[0014] 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.
[0015] 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.
[0016] Enzyme Compositions
[0017] 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.degree. 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.degree. C., with poly(A)/oligo(dT) as a
substrate.
[0018] Thermostable Polymerase
[0019] 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 .mu.l of diluted enzyme fractions with 5
.mu.g of activated calf thymus DNA (Worthington, Freehold, N.J.) in
a buffer containing 25 mM TAPS-KOH pH 9.3, 50 mM KCL, 5 mM
MgCl.sub.2, 1.4 mM .beta.-mercaptoethanol, 200 .mu.M each dNTP and
.alpha.-.sup.32P dCTP (30-80 cpm/pmol) for 10 min at 72.degree. C.
in a total volume of 50 .mu.l. The reaction is terminated by
addition of 10 .mu.l of 60 mM EDTA, and the products are
precipitated by the addition of 60 .mu.l of 20% trichloroacetic
acid and incubation on ice for 15 min. The acid-insoluble product
is then separated from the acid soluble nucleotides by filtration
through GF/C filters. One unit represents conversion of 10 nmol of
nucleotides in 30 min at 72.degree. C.) By thermostable is meant
that the enzyme maintains its polymerase activity at temperatures
at least in excess of 55.degree. C. and up to about 72.degree. C.
or higher. The thermostable polymerase is further characterized by
having a higher Mg.sup.2+ optimum as compared to wild type Taq
polymerase (Barnes, W. M., Gene (1992) 112:29-35.
[0020] Generally, the thermostable polymerase has a molecular
weight that is less than the molecular weight of naturally
occurring or wild type Thermus aquaticus polymerase. The molecular
weight of polymerases finding use in the subject compositions
typically ranges from about 60 to 70 kDal, usually from about 62 to
68 kDal, and more usually from about 64 to 68 kdal, as measured by
SDS-PAGE electrophoresis. The thermostable polymerase typically has
an amino acid sequence in which the C-terminal portion is
substantially identical to the carboxy domain of the naturally
occurring Thermus aquaticus DNA polymerase as reported in Lawyer et
al., J. Biol. Chem (1989) 264:6427 and having a Genbank accession
no J04639, particularly amino acid residues 289 to 832 of the
naturally occurring Thermus aquaticus DNA polymerase. By
substantially identical or the same is meant that the C-terminal
portion of subject enzyme, which is from about 530 to 550 amino
acids in length, usually from about 540 to 550 amino acids in
length and more usually 540 to 545 amino acids in length, where in
many instances it is 543 amino acids in length, has an amino acid
sequence that has a sequence identity of at least about 90%,
usually at least about 95% and more usually at least about 99%,
with residues 289 to 832 of the amino acid sequence of naturally
occurring Thermus aquaticus polymerase, as measured using the BLAST
algorithm, as described in Altschul et al., (1990) J. Mol. Biol.
215: 403-410 (using the published default settings). In many
embodiments of the subject invention, the C-terminal 543 amino acid
residues, e.g. 10 to 553, 17 to 560, etc, depending on the
particular embodiment of the invention, of the polymerase are
identical to residues 289 to 832 of wild type Thermus aquaticus
polymerase. Where the amino acid sequence of the C-terminal domain
of the polymerase does differ from residues 289 to 832 of the
naturally occurring sequence, the difference is not one that
significantly provides for a significantly reduced polymerase
activity or specificity as compared that observed for the wild type
enzyme, where any reduced polymerase activity will not exceed a
4-fold reduction, and usually will not exceed a 2 to 3 fold
reduction.
[0021] Adjacent to the C-terminal domain described above is the
N-terminal region of the enzyme. The N-terminal region at least
comprises a sequence of nine amino acid residues that has less than
50% but at least 40% amino acid sequence identity with residues 280
to 288 of naturally occurring Thermus aquaticus polymerase, as
measured using the BLAST algorithm described above, where the
number of amino acid residues in the N-terminal domain that are
identical with residues 280 to 288 is usually four.
[0022] Generally, the sequence of this nine residue domain is:
MRGHEX.sub.1GLX.sub.2
[0023] wherein X.sub.1 and X.sub.2 are hydrophilic residues, more
specifically, polar uncharged hydrophilic residues. X.sub.1 is
usually either threonine or serine, and in many preferred
embodiments is serine. X.sub.2 is usually either asparagine or
glutamine, and in many preferred embodiments is glutamine.
[0024] A preferred thermostable enzyme is further described in PCT
Application No. PCT/US99/13305, the disclosure of which is herein
incorporated by reference.
[0025] Reverse Transcriptase
[0026] Also present in the subject enzyme compositions is a mutant
reverse transcriptase. The mutant reverse transcriptase is
typically a mutant moloney murine leukemia virus reverse
transcriptase (i.e. a mutant MMLV-RT), where the mutant preferably
retains substantially all of the reverse transcriptase activity of
the wild type MMLV-RT, where by substantially all is meant at least
about 80%, usually at least about 90% and more usually at least
about 95%, where in many embodiments the mutant reverse
transcriptase retains about 100% of the wild type mmlv-rt reverse
transcriptase activity, as determined by the activity assay
disclosed in Blain & Goff, J. Biol. Chem. (1993)
268:23585-23592.
[0027] In many embodiments, the mutant reverse transcriptase is a
point mutation mutant, in which a single residue of the wild type
reverse transcriptase has been changed. Of particular interest in
many embodiments is the R657S point mutant of MMLV-RT, where this
particular enzyme is disclosed in Blain & Goff, J. Virol.
(1995) 69:4440-4452 and Blain & Goff, J. Biol. Chem. (1993)
268:23585-23592.
[0028] Optional Components
[0029] In addition to the polymerase and reverse transcriptase
components of the subject enzyme compositions, the subject enzyme
compositions may also include a number of additional
components.
[0030] One component of interest that is included in many preferred
embodiments of the enzyme composition is a water-soluble
temperature sensitive inhibitor of the thermostable DNA polymerase.
Inhibitors of interest are those that bind to and inactivate the
polymerase at temperature T.sub.1 which is generally below about
85.degree. C. For most practical purposes, T.sub.1 is below about
55.degree. C. Advantageously, however, the water-soluble
temperature sensitive inhibitor dissociates from the DNA polymerase
and becomes ineffective to inactivate the DNA polymerase at
temperature T.sub.2 which is generally above about 40.degree. C.
Preferably, T.sub.2 is at least 5.degree. C. above T.sub.1. In many
embodiments, T.sub.1 is generally from about 40.degree. C. to about
55.degree. C. and T.sub.2 is generally from about 75.degree. to
about 95.degree. C. The inhibitor can be any biological or chemical
molecule which will complex with the thermostable DNA polymerase to
effect the noted temperature-dependent responses in the polymerase.
Generally, the combined molecule (or complex) of DNA polymerase and
temperature sensitive inhibitor is water-soluble. The inhibitor can
be DNA polymerase-binding proteins which bind and release the DNA
polymerase in response to temperature. Particularly useful
inhibitors are antibodies (monoclonal or polyclonal) specific to
the DNA polymerase which have the noted binding and releasing
properties. The term "antibodies" includes the biological molecules
one skilled in the art would normally understand that term to
include, but in addition, it includes genetically prepared
equivalents thereof, and chemically or genetically prepared
fragments of antibodies (such as Fab fragments). The antibodies
(and fragments thereof), can be used singly or in mixtures in the
practice of this invention. Of particular interest in many
embodiments are monoclonal antibodies. The monoclonal antibodies
generally have an affinity for the thermostable DNA polymerase as
defined by having an association constant of at least about
1.times.10.sup.7 molar.sup.-1. Preferably, the antibody is of
either the IgG or IgM class. Most preferably, it is of the IgG
class. Specific monoclonal antibodies of interest include the mouse
monoclonal antibodies TP1, TP2, TP3, TP4, TP5, TP6, TP7, TP8, TP9,
and TP14, where these and other inhibitors of interest are further
disclosed in U.S. Pat. No. 5,338,671, the disclosure of which is
herein incorporated by reference.
[0031] Other components that may be present in the enzyme
composition include: proofreading enzymes, polymerase inhibitory
oligonucleotides or analogues thereof, etc.
[0032] The enzyme composition may be present as a liquid (aqueous
composition), where the composition may be frozen for storage
stability. Storage stable compositions will typically comprise the
enzyme in combination with a buffer medium. Buffer mediums of
interest typically comprise: buffering agents, e.g. Tris, Tricine,
HEPES, phosphate, etc.; solvents, e.g. water, glycerol, etc.;
salts, e.g. KCl, NaCl, (NH.sub.4).sub.2SO.sub.4, etc.; reducing
agents, e.g. .beta.-mercaptoethanol, DTT, DTE, etc.; chelating
agents, e.g. EDTA, CDTA, etc.; detergents, e.g. Triton X100; Tween
20, Thesit, NP40, etc.; and the like. Alternatively, the
composition may be present as a substantially non-aqueous dried,
storage stable composition, e.g. a freeze dried composition, to
which water is added prior to use.
[0033] Kits
[0034] As summarized above, also provided are kits for use in
preparing amplified amounts of DNA from a template RNA(s). The
subject kits are characterized by at least including a mutant
thermostable polymerase and a mutant reverse transcriptase, as
described above, as well as at least one of dNTPs and a buffer
composition (or the dried precursor reagents thereof, either
prepared or present in its constituent components, where one or
more of the components may be premixed or all of the components may
be separate). In many embodiments, the subject kits will include
both of these additional components, i.e. the kits will include the
polymerase and reverse transcriptase enzymes, which may be present
in a composition as described above or separate, as well as dNTPs
and a buffer or components thereof.
[0035] By dNTPs is meant a mixture of deoxyribonucleoside
triphosphates (dNTPs). Usually the kit will comprise four different
types of dNTPs corresponding to the four naturally occurring bases,
i.e. dATP, dTTP, dCTP and dGTP. The total amount of dNTPs present
in the kit ranges, in many embodiments, from about 1.0 to 1000
.mu.M, usually from about 1.0 to 500 .mu.M and more usually from
about 1.0 to 100 .mu.M, where the relative amounts of each of the
specific types of dNTPs may be the same or different. See e.g. U.S.
patent application Ser. No. 08/960,718, the disclosure of which is
herein incorporated by reference.
[0036] The aqueous PCR buffer medium that is present in the subject
kits includes a source of monovalent ions, a source of divalent
cations and a buffering agent. Any convenient source of monovalent
ions, such as KCl, K-acetate, NH.sub.4-acetate, K-glutamate,
NH.sub.4Cl, ammonium sulfate, and the like may be employed, where
the amount of monovalent ion source present in the buffer will
typically be present in an amount sufficient to provide for a
conductivity in a range from about 500 to 20,000, usually from
about 1000 to 10,000, and more usually from about 3,000 to 6,000
micro-ohms. The divalent cation may be magnesium, manganese, zinc
and the like, where the cation will typically be magnesium. Any
convenient source of magnesium cation may be employed, including
MgCl.sub.2, Mg-acetate, and the like. The amount of Mg.sup.2+
present in the buffer is one that is elevated as compared to that
employed in wild type Taq polymerase systems, and is one that is
close to the optimum concentration for MMLV-RT, where the Mg2+
concentration may range from 0.5 to 10 mM, but will preferably
range from about 2 to 5 mM. Representative buffering agents or
salts that may be present in the buffer include Tris, Tricine,
HEPES, MOPS and the like, where the amount of buffering agent will
typically range from about 5 to 150 mM, usually from about 10 to
100 mM, and more usually from about 20 to 50 mM, where in certain
preferred embodiments the buffering agent will be present in an
amount sufficient to provide a pH ranging from about 6.0 to 9.5.
Other agents which may be present in the buffer medium include
chelating agents, such as EDTA, EGTA and the like and non-ionic
detergents, such as Tween 20, Triton X100, NP40, and the like. As
mentioned above, the aqueous buffer medium may be present in the
subject kits as a fluid or frozen aqueous composition, as dried
buffer precursors that may be separate or combined, e.g. as a
freeze dried composition.
[0037] The subject kits may further include a number of optional
components. Optional ingredients that may be present include: a
thermostabilizing agent; a glycine based osmolyte, one or more
nucleic acids, e.g. oligonucleotides, an RNase inhibitor, and the
like. Each of these additional optional components is now described
in greater detail.
[0038] The first optional component mentioned above is a
thermostabilizing agent. The thermostabilizing agent should
decrease the rate of denaturation of the reverse transcriptase to
allow cDNA synthesis at elevated temperatures, where representative
agents include: sugars, e.g. trehaloses, sucrose, raffinose, etc.;
polymerase, e.g. PEG, Dextran, polysaccharides, etc.; and the like,
where in many embodiments, trehalose is preferred. When included in
the subject kits, the amount of thermostabilizing agent will
typically range from about 0.9 to 15 mmol, usually from about 0.9
to 3.0 mmol and more usually from about 1.5 to 3.0 mmol.
[0039] Another optional component mentioned above is the glycine
based osmolyte. Glycine-based osmolytes suitable for use in the
present invention include trimethylglycine (BETAINE.TM.), glycine,
sarcosine and dimethylglycine. Glycine based osmolytes and their
use in amplification reactions are further described in U.S. Pat.
No. 5,545,539, the disclosure of which is herein incorporated by
reference.
[0040] The kits may further include an RNase inhibitor. Suitable
RNase inhibitors of interest include: human placental RNase
inhibitor, recombinant RNase inhibitor, etc., where recombinant
RNase inhibitor is of particular interest in many embodiments.
[0041] The kits may further include one or more nucleic acids,
where the nucleic acids will generally be oligonucleotides that
find use in the reverse transcription or amplification reactions,
described in greater detail below. As such, nucleic acids that may
be present include oligodTs, random primers and PCR primers. When
present, the length of the dT primer will typically range from 12
to 30 nts. In certain embodiments, the oligo dT primer may be
further modified to include an arbitrary anchor sequence, where the
arbitrary anchor sequence or portion of the primer will typically
range from 15 to 25 nt in length. Also present may be one or more
sets of PCR primers, where such primers may be control primers etc.
In certain embodiments, the primers may be a set of gene specific
primers, as described in U.S. patent application Ser. No.
08/859,998, the disclosure of which is herein incorporated by
reference.
[0042] Other optional components that may be included in the
subject kits include: one or more control sets of total RNA, e.g.
mouse total RNA, water, and the like.
[0043] The various reagent components of the kits may be present in
separated containers, or may all be (or in part be) precombined
into a reagent mixture for combination with template DNA.
[0044] Finally, in many embodiments of the subject kits, the kits
will further include instructions for practicing methods of
producing amplified amounts of DNA from a template RNA(s), as
described in greater detail below, where these instructions may be
present on one or more of: a package insert, the packaging, reagent
containers and the like.
[0045] Utility
[0046] The above described enzyme compositions and/or kits find use
in methods of producing an amplified amount of DNA from a template
RNA(s), i.e. producing one or more amplified amounts of DNA from
one or more template RNAs. In particular, the above described
enzyme compositions and/or kits find use in the one step RT-PCR
reactions of the subject invention, as described in greater detail
below.
[0047] In the subject one-step RT-PCR reactions, an amplified
amount of DNA is produced from one or more, usually a plurality of,
RNAs in a single reaction container without the sequential addition
of reagents to the reaction container. Specifically, the one step
RT-PCR methods of the subject invention include the following
steps: (a) preparing a reaction mixture; (b) subjecting the
prepared reaction mixture to a first set of reverse transcription
reaction conditions; and (c) subjecting the reaction mixture to a
second set of PCR conditions. Each of these steps is now described
separately in greater detail.
[0048] The reaction mixture is prepared by combining at least the
following components: (a) a mutant thermostable DNA polymerase, as
described above; (b) a mutant reverse transcriptase, as described
above; (c) one or more RNA templates; (d) dNTPs; (e) a quantity of
reaction buffer; (f) reverse transcription primer, e.g. oligo dT;
and (g) PCR primers. Other components that may be introduced into
the prepared reaction mixture include: (a) a polymerase inhibitor;
(b) a thermostabilizing reagent; (c) a glycine based osmolyte; (d)
an RNase inhibitor; (e) control RNA and primers; and (f) water. The
components are combined in a suitable container, e.g. a thin walled
PCR reaction tube.
[0049] The following guidelines are based on the preparation of a
50 .mu.l total volume reaction mixture. As such, the below specific
amounts should be varied proportionally where different amounts of
total reaction mixture are prepared, where such calculations are
well within those of skill in the art. In preparing the reaction
mixture, the amount of template RNA (e.g. total RNA) that is
employed is typically at least about 10 pg, usually at least about
1 ng and more usually at least about 10 ng, where the amount of
template RNA may be as great as 1 .mu.g or greater, but typically
does not exceed about 1 .mu.g and usually does not exceed about 500
ng. As indicated above, the template RNA may be a single type of
RNA, such that the template RNA is a homogenous sample, but is
generally a heterogenous sample of two or more, usually at least
about 50 or more and more usually at least about 100, 1000, or 5000
or more different RNAs (which differ from each other in terms of
sequence). As such, the template RNA may be total RNA or mRNA, or a
fraction thereof, derived from a physiological sample of
interest.
[0050] The amount of mutant thermostable DNA polymerase that is
included in the reaction mixture may vary, but typically ranges
from about 5 to 30 U, usually from about 20 to 30 U. Likewise,
while the amount of mutant reverse transcriptase may vary, the
amount of this enzyme typically ranges from about 4 to 50 U,
usually from about 5 to 15 U. In general, the total amount of these
two enzymes included in the reaction mixture ranges from about 15
to 80 U, usually from about 25 to 45 U. The enzymes may be added to
the reaction mixture separately or together as an enzyme
composition, as described above.
[0051] Also included in the reaction mixture are dNTPs, i.e. an
amount of each of dATP, dTTP, dCTP and dGTP. The total amount of
dNTPs included in the reaction mixture ranges, in many embodiments,
from about 20 to 80 nmols, usually from about 30 to 50 nmols and
more usually from about 35 to 40 nmols, where the relative amounts
of each of the specific types of dNTPs may be the same or
different. See e.g. U.S. patent application Ser. No. 08/960,718,
the disclosure of which is herein incorporated by reference.
[0052] Also included in the reaction mixture is a quantity of
reaction buffer, where suitable reaction buffers are described
supra. The amount of reaction buffer used to prepare the subject
reaction mixtures typically ranges from about 4 to 5.5 .mu.l,
usually from about 4.5 to 5.0 .mu.l.
[0053] As mentioned above, the above amounts are provided for a 50
.mu.l RT PCR reaction, and may be adjusted to any other reaction
volume. Such adjustments are well within the abilities of those of
skill in the art.
[0054] Also included in the reaction mixture is a reverse
transcription primer, e.g. oligo dT, where such primers are
described above. The amount of reverse transcription primer that is
included typically ranges from about 10 to 30 pmol, usually from
about 15 to 20 pmol. Random (e.g. hexameric) primers may also be
included as primers for RT.
[0055] The reaction mixture also includes PCR primers. The
oligonucleotide PCR primers from which the reaction mixture is
prepared are of sufficient length to provide for hybridization to
complementary template DNA under annealing conditions (described in
greater detail below) but will be of insufficient length to form
stable hybrids with non-complementary template DNA. The primers
will generally be at least 10 bp in length, usually at least 15 bp
in length and more usually at least 16 bp in length and may be as
long as 30 bp in length or longer, where the length of the primers
will generally range from 18 to 50 bp in length, usually from about
20 to 35 bp in length. The template DNA may be contacted with a
single primer or a set of two primers, depending on whether linear
or exponential amplification of the template DNA is desired. Where
a single primer is employed, the primer will typically be
complementary to one of the 3' ends of the template DNA and when
two primers are employed, the primers will typically be
complementary to the two 3' ends of the double stranded template
DNA.
[0056] As mentioned above, a number of additional optional
components may be included in the reaction mixture. One such
component is a polymerase inhibitor, e.g. an polymerase specific
antibody, as described above. When present, the amount of the
antibody typically ranges from about 0.2 to 2.2 .mu.g, usually from
about 0.9 to 1.1 .mu.g. The reaction mixture may further include a
thermostabilizing reagent, e.g. trehalose. When present, the amount
of this reagent typically ranges from about 10 to 30 .mu.mol,
usually from about 20 to 30 .mu.mol. Also present may be a glycine
based osmolyte, e.g. betaine. When present, the amount of this
reagent ranges from about 25 to 75 .mu.mol, usually from about 40
to 50 .mu.mol. Also present may be an RNase inhibitor, e.g.
recombinant RNase inhibitor. When present, the amount of this
reagent typically ranges from about 4 to 25 U, usually from about
10 to 20 U, where U is defined as 1 Unit of inhibitor being equal
to the amount of protein required to inhibit the activity of 5 ng
RNase A by 50%.
[0057] In preparing the reaction mixture, the various constituent
components may be combined in many different orders. For example,
the buffer may be combined with primer, polymerase and then
template DNA, or all of the various constituent components may be
combined at the same time to produce the reaction mixture. In many
preferred embodiments, the enzymes are introduced into the reaction
mixture last.
[0058] Following preparation of the reaction mixture, the reaction
mixture is first subject to a set of conditions sufficient for
reverse transcription of the RNA template present in the reaction
mixture to occur, i.e. the reaction mixture is subjected to cDNA
synthesis conditions. This first set of conditions is characterized
by maintaining the reaction mixture at a substantially constant
temperature for a period of time sufficient for cDNA synthesis to
occur. The temperature at which the reaction mixture is maintained
during this portion of the subject methods generally ranges from
about 37 to 55, usually from about 45 to 52 and more usually from
about 48 to 50.degree. C. The duration of this step of the subject
methods typically ranges from about 15 to 90 min, usually from
about 30 to 60 min and more usually from about 50 to 60 min.
[0059] The next step of the subject methods is to subject the
reaction mixture, which now includes cDNAs which are the result of
the reverse transcription of the first step, to PCR conditions for
a period of time sufficient for a desired amount of amplified DNA
to be produced. The polymerase chain reaction (PCR) is well known
in the art, being described in U.S. Pat. Nos. 4,683,202; 4,683,195;
4,800,159; 4,965,188 and 5,512,462, the disclosures of which are
herein incorporated by reference. In subjecting the cDNA comprising
reaction mixture to PCR conditions during this step of the subject
methods, the reaction mixture is subjected to a plurality of
reaction cycles, where each reaction cycle comprises: (1) a
denaturation step, (2) an annealing step, and (3) a polymerization
step. The number of reaction cycles will vary depending on the
application being performed, but will usually be at least 15, more
usually at least 20 and may be as high as 60 or higher, where the
number of different cycles will typically range from about 20 to
40.
[0060] The denaturation step comprises heating the reaction mixture
to an elevated temperature and maintaining the mixture at the
elevated temperature for a period of time sufficient for any double
stranded or hybridized nucleic acid present in the reaction mixture
to dissociate. For denaturation, the temperature of the reaction
mixture will usually be raised to, and maintained at, a temperature
ranging from about 85 to 100, usually from about 90 to 98 and more
usually from about 93 to 96.degree. C. for a period of time ranging
from about 3 to 120 sec, usually from about 5 to 60 sec.
[0061] Following denaturation, the reaction mixture will be
subjected to conditions sufficient for primer annealing to template
DNA present in the mixture. The temperature to which the reaction
mixture is lowered to achieve these conditions will usually be
chosen to provide optimal efficiency and specificity, and will
generally range from about 50 to 75, usually from about 55 to
70.degree. C. Annealing conditions will be maintained for a period
of time ranging from about 15 sec to 60 sec.
[0062] Following annealing of primer to template DNA or during
annealing of primer to template DNA, the reaction mixture will be
subjected to conditions sufficient to provide for polymerization of
nucleotides to the primer ends in manner such that the primer is
extended in a 5' to 3' direction using the DNA to which it is
hybridized as a template, i.e. conditions sufficient for enzymatic
production of primer extension product. To achieve polymerization
conditions, the temperature of the reaction mixture will typically
be raised to or maintained at a temperature ranging from about 65
to 75, usually from about 67 to 73.degree. C. and maintained for a
period of time ranging from about 15 sec to 20 min, usually from
about 30 sec to 5 min.
[0063] The above steps of subjecting the reaction mixture to
reverse transcription reaction conditions and PCR conditions be
performed using an automated device, typically known as a thermal
cycler. Thermal cyclers that may be employed for practicing the
subject methods are described in U.S. Pat. Nos 5,612,473;
5,602,756; 5,538,871; and 5,475,610, the disclosures of which are
herein incorporated by reference.
[0064] The subject methods are characterized in that they are
extremely efficient. As such, the subject methods can be used to
prepare a large amount of amplified DNA from a small amount of
template RNA. For example, the subject methods can be used to
prepare from about 0.2 to 3.0, usually from about 0.8 to 1.5 .mu.g
amplified DNA from an initial amount of 1 ng to 1 .mu.g, usually
100 ng to 500 ng of total RNA template in from about 25 to 40
cycles. The subject methods are also highly sensitive, being able
to generate amplified DNA from exceedingly small amounts of
template RNA, where by exceedingly small is meant less than about 1
.mu.g, usually less than about 100 ng and more usually less than
about 1 ng, where the methods generally require at least about 10
pg template RNA.
[0065] The subject one step RT-PCR methods find use in any
application where the production of enzymatically produced primer
extension product from template RNA is desired, such as the
generation of libraries of cDNA from small amounts of mRNA, the
generation of gene expression profiles of from or more distinct
physiological samples, e.g. as required in gene expression analysis
assays, and the like.
[0066] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0067] I. Protocol for Performing One Step RT-PCR
[0068] The following is a representative protocol for performing a
one step RT-PCR reaction according to the subject methods.
[0069] 1. Quickly thaw each tube in the kit and place on ice. Carry
out all additions on ice.
[0070] 2. Spin each tube briefly in a microcentrifuge and put back
on ice.
[0071] 3. In a sterile, RNAse free PCR thin wall tube, add the
following reagents and mix well by pipetting up and down.
1 x ul RNAse free dH2O 1 ul 20 uM oligo dT y ul Total RNA(1 ug-1
ng) 1 ul 45 uM primers 5 ul 10 .times. One step RT-PCR buffer 1 ul
50 .times. dNTP 0.5 ul RNAse inhibitor(40 U/ul) 25 ul 1.2 M
Trehalose 10 ul 5 M betaine 1 ul 50 .times. RT-PCR enzyme mix 50 ul
Total volume
[0072] 4. Program the Thermocycler for the following:
[0073] 50.degree. C. 1 hr, 1 cycle
[0074] 94.degree. C. 5 min, 1 cycle
[0075] 94.degree. C. 30 sec, 65.degree. C. 68.degree. C. 1-3 min*,
25-35 cycles
[0076] 68.degree. C. 3 min, 1 cycle
[0077] 4.degree. C. soaking
[0078] *1-1.5 min/kb
[0079] II. Efficiency of One Step RT-PCR of Subject Invention
[0080] The protocol described in I above was used to amplify the
following specific transcripts of varying abundance from an initial
1 .mu.g of human placental total RNA.
2 Relative # PCR Lane Transcript abundance cycles 1 EGFR3 low 40 2
p53 low 30 3 IFN-8-R med 25 4 ILGF-1 low 40 5 PDGFB low 25 6 IFN-8
low 30 7 b-actin high 30 8 IL-8 low/med 30 9 G3PDH high 25
[0081] It was observed that the protocol of I above provided high
yields of all fragments, regardless of initial transcript
abundance.
[0082] Assay of Sensitivity of the Subject One Step RT-PCR
[0083] The target transcript was reverse transcribed at 50.degree.
C. for 1 hr and amplified using 40 PCR cycles, according to the
protocol of I above. RT-PCR products were then analyzed via
agarose/EtBr gel electrophoresis. In one experiment, serial 10-fold
dilutions of a synthetic RNA (synthesized in vitro using T7 RNA
polymerase) served as a template: 5.times.10.sup.5 molecules of
synthetic RNA; 5.times.10.sup.4 molecules; 5.times.10.sup.3
molecules; 500 molecules; 50 molecules; 5 molecules; 1 molecule; no
template. Amplified product was clearly visible where only 50
template molecules were present. In a second experiment, different
amounts of mouse liver total RNA were used as a template using the
protocol of I above to amplify the .beta.-actin transcript: 1 .mu.g
of total RNA; 100 ng; 10 ng; 1 ng; 100 pg; 10 pg; 1 pg; no
template. Amplified product was detected from as little as 10 pg of
total RNA template.
[0084] It is evident from the above results and discussion that
novel enzyme compositions and reagent kits are provided, as well as
novel methods for performing a one-step RT-PCR reaction mixture
using these compositions and/or kits. Advantages of the subject
invention over the prior art methods of RT-PCR include: ability to
perform the entire RT-PCR reaction in a single container without
the addition of additional reagents during the process, which
feature reduces the possibility of contamination of the sample;
greater efficiency as compared to other prior art methods of
RT-PCR; high sensitivity; and the like. As such, the subject
invention represents a significant contribution to the art.
[0085] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0086] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
* * * * *
References