U.S. patent application number 14/579985 was filed with the patent office on 2016-06-09 for recombinant reverse transcriptases.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to LIANGJING CHEN, GARY LATHAM, ROBERT SETTERQUIST.
Application Number | 20160160194 14/579985 |
Document ID | / |
Family ID | 35096525 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160160194 |
Kind Code |
A1 |
CHEN; LIANGJING ; et
al. |
June 9, 2016 |
RECOMBINANT REVERSE TRANSCRIPTASES
Abstract
The present invention relates to a gene that encodes a
hyperactive reverse transcriptase having DNA polymerase activity
and substantially reduced RNase H activity, vectors containing the
gene and host cells transformed with the invention. The present
invention also includes a method of producing the hyperactive
reverse transcriptase, producing cDNA from mRNA using the reverse
transcriptase of the invention, kits and assay templates made using
the hyperactive reverse transcriptase.
Inventors: |
CHEN; LIANGJING; (AUSTIN,
TX) ; SETTERQUIST; ROBERT; (AUSTIN, TX) ;
LATHAM; GARY; (AUSTIN, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
CARLSBAD |
CA |
US |
|
|
Family ID: |
35096525 |
Appl. No.: |
14/579985 |
Filed: |
December 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13730108 |
Dec 28, 2012 |
8956841 |
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14579985 |
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12568364 |
Sep 28, 2009 |
8361754 |
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13730108 |
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10827498 |
Apr 19, 2004 |
7595179 |
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12568364 |
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Current U.S.
Class: |
435/194 ;
536/23.2 |
Current CPC
Class: |
C12N 9/1276 20130101;
C12Y 207/07049 20130101 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Claims
1.-35. (canceled)
36. An isolated protein comprising DNA polymerase activity and
substantially reduced RNase H activity comprising one or more
mutations in the processivity domain and/or in the nucleotide
selection domain.
37. The protein of claim 36, wherein the protein produces a yield
of greater than about 1, 5, 7, 10, 12, 15, 25 .mu.g of an aRNA from
100 ng of template RNA in a single amplification reaction.
38. The protein of claim 36, wherein the protein produces an aRNA
yield of greater than about 20% as compared to an equivalent
wild-type Reverse Transcriptase enzyme.
39. The protein of claim 36, wherein the protein produces a yield
of greater than about 5 or 10 .mu.g of an aRNA from 10 pg of
template RNA after a two-round amplification reaction.
40. The protein of claim 36, wherein the protein produces a cDNA
greater than about 6, 9 or even 11 kilobases in a single cDNA
synthesis reaction.
41. The protein of claim 36, wherein the protein produces a cDNA
greater than about 6 to about 15 kilobases in a single cDNA
synthesis reaction.
42. The protein of claim 36, wherein the protein produces a cDNA
greater than about 15 kilobases in a single cDNA synthesis
reaction.
43. The protein of claim 36, wherein the DNA polymerase activity is
greater than about 200 Units per microgram.
44. The protein of claim 36, wherein the DNA polymerase activity is
between about 0.1 and 300 Units per microgram.
45. The protein of claim 36, wherein the RNase H activity is
between about 0.1 and about 25 percent of the wild-type RNase H
activity.
46.-61. (canceled)
62. An isolated and purified nucleic acid comprising a hyperactive
reverse transcriptase with a mutation in the processivity domain
and/or in the nucleotide selection domain.
63. The nucleic acid of claim 62, wherein the hyperactive reverse
transcriptase comprises a mutation that corresponds to and includes
an H638G mutation of the MMLV-RT.
64. The nucleic acid of claim 62, wherein the hyperactive reverse
transcriptase comprises a hyperactive reverse transcriptase further
comprising an F155Y mutation.
65. The nucleic acid of claim 62, wherein the hyperactive reverse
transcriptase comprises a hyperactive reverse transcriptase further
comprising an F155Y mutation and an H638G mutation.
66. The nucleic acid of claim 62, wherein the nucleic acid of SEQ
ID No.: 1 further comprises a nucleic acid segment encoding a
leader sequence.
67. The nucleic acid of claim 62, wherein the nucleic acid of SEQ
ID NO.: 1 further comprises a nucleic acid segment encoding a
protein segment other than the hyperactive reverse
transcriptase.
68.-85. (canceled)
86. A kit for nucleic acid synthesis, comprising, in a suitable
container: a hyperactive reverse transcriptase; and a reaction
solution for the reverse transcriptase.
87. The kit of claim 86, further comprising an insert that
comprises information for using the reverse transcriptase.
88. The kit of claim 86, wherein the reaction solution comprises a
reverse transcriptase reaction buffer.
89. The kit of claim 86, further comprising a primer.
90. The kit of claim 86, wherein the reaction solution comprises a
reverse transcriptase buffer.
91. The kit of claim 86, wherein the reaction solution comprises a
PCR buffer.
92. The kit of claim 86, further comprising a mix of
nucleotides.
93. The kit of claim 86, further comprising containers comprising
individual nucleotides.
94. The kit of claim 86, wherein the reaction solution comprises a
buffer for in vitro transcription.
95. The kit of claim 86, further comprising a template purification
column.
96. The kit of claim 86, further comprising magnetic particles
suitable for nucleic acid purification.
97.-128. (canceled)
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to recombinant Reverse
Transcriptase (RT) enzymes with modified activity, and more
particularly, to selectively mutated RTs with enhanced RNA
directed, DNA polymerase activity that produces longer cDNAs,
higher aRNA yields.
BACKGROUND OF THE INVENTION
[0002] Without limiting the scope of the invention, its background
is described in connection with the use of RT enzymes for use in a
wide variety of assays used by molecular biologists, as an
example.
[0003] Heretofore, in this field, the RNA directed, DNA polymerase
activity responsible for the synthesis of complementary DNA (cDNA)
from an RNA template has been accomplished using reverse
transcriptase enzymes, whether purified or recombinant One such use
for RTs if for transcription-based amplification systems, e.g.,
amplifying RNA and DNA target sequences.
[0004] Transcription-based amplification methods find use in a wide
variety of settings, e.g., routine clinical laboratory use in
diagnostic tests such as direct detection of pathogens. Another
such use for RTs is in the initial step for RT-PCR (polymerase
chain reaction) used to amplify an RNA target for analysis and/or
cloning. In RT-PCR, the RT is used to make an initial complementary
DNA (cDNA) copy of the RNA target, which is then amplified by
successive rounds of DNA replication.
[0005] RTs have three primary enzymatic activities: a RNA-directed,
DNA polymerase activity; a DNA-directed, DNA polymerase activity;
and an RNase H activity. The RNase H activity specifically degrades
RNA found in an RNA:DNA duplex. An initial goal of many molecular
biologists was to identify an RT that had no detectable RNase H
activity while still maintaining DNA polymerase activity. An RT
having no RNase H activity would finds particular use because
degradation of the RNA strand of RNA:DNA intermediates by RNase H
causes unwanted degradation of the template reducing cDNA yields.
U.S. Pat. Nos. 5,244,797, 5,540,776, 5,668,005, 6,063,608,
6,589,768 and 6,610,522, disclose one such mutant RT, wherein a
gross deletion mutant with no detectable RNase H activity is
taught.
[0006] U.S. Pat. No. 5,998,195 teaches a method of reducing the
level of RNase activity in an RT preparation by using an expression
vector or plasmid containing a cloned version of the gene for an
MMLV-RT which, when used to transform a suitable host cell such as
E. coli, leads to the expression of the gene and the generation of
a gene product having the DNA- and RNA-directed DNA polymerase
activities and RNase H activity associated with retroviral reverse
transcriptases. A host cell with a reduced level of ribonuclease
activity as compared to wild-type strains is used to provide a
source of RT that has endogenous levels of RNase activity below
that of previous recombinant preparations.
[0007] Yet others have purified RT, e.g., Goff et al., U.S. Pat.
No. 4,943,531 (1990) and Kotewicz et al., U.S. Pat. No. 5,017,492,
which have described methods for the purification of reverse
transcriptase derived from Moloney Murine Leukemia Virus (MMLV-RT)
and expressed in E. coli. These expression constructs and isolation
and purification methods form the basis for the majority of
commercial reverse transcriptase preparations.
SUMMARY OF THE INVENTION
[0008] The present invention relates to a gene that encodes a
hyperactive reverse transcriptase having enhanced DNA polymerase
activity. Increased DNA polymerase activity is achieved by one or
more point mutations in the DNA processivity domain of the RT.
Using the mutant RTs of the present invention greatly enhanced
yields of aRNA may be achieved from template amounts in picogram
amounts. In addition to enhanced amplification, the hyperactive RTs
were found to consistently produce extra-long cDNAs, that is,
messages exceeding 9 kb.
[0009] The present invention may also include one or more mutations
to the nucleotide selection domain, which is located near the amino
terminus of the RT. The present invention may also include one or
more mutations in the processivity domain, which facilitates the
formation of longer cDNA products. Mutants in the processivity
domain of RT also exhibit substantially reduced RNase H activity
(e.g., between about 0.1, 0.5, 1.0, 2.5, 5.0, 10 to about 50%
percent of the wild-type activity of MMLV RT). The hyperactive
mutants described herein are able to produce, enhanced
amplification of mRNA to cDNA from very small quantities of
template in both single and double rounds of amplification while
maintaining message ratio fidelity.
[0010] More particularly, the present invention includes an
isolated hyperactive reverse transcriptase that includes one or
more point mutations in the processivity domain and/or one or more
point mutations in the nucleotide selection domain. The reverse
transcriptase may be, e.g., an AMV, M-MLV, HTLV-1, BLV, RSV, HFV,
R2 Bombyx mori or HIV reverse transcriptase. The hyperactive
reverse transcriptase is encoded by a modified nucleotide sequence
that encodes a modified amino acid sequence modified in the
processivity domain generally correspond with amino acids 497 to
671 of M-MLV reverse transcriptase. The modifications to the
nucleotide selection domain may correspond to amino acids 153 to
158 of M-MLV reverse transcriptase. As disclosed herein, the
reverse transcriptase may be used in the preparation of full-length
cDNA and may be a hyperactive reverse transcriptase that is
produced recombinantly and purified to, e.g., greater than about
90% pure.
[0011] Examples of mutations that have been developed and that show
an increase in activity over the wild-type enzyme, as described and
characterized herein include, e.g., mutations in the processivity
domain with one or more of the following mutations corresponding to
the amino acids in MMLV-RT: H638G, Y586A, D653N, D524N, D524E and
E562D. For mutations in the nucleotide selection domain these may
include one or more of the following mutations corresponding to the
amino acids in MMLV-RT: F155, D153, A154, F155, F156, C157, or
L158. In one group of specific examples that demonstrate the
structure and functional relationship between the mutations, the
mutation in the processivity domain may include one or more of the
following mutations corresponding to the amino acids in MMLV-RT:
H638G, Y586A, D653N, D524N, D524E and E562D and the mutation in the
nucleotide selection domain may include one or more of the
following mutations corresponding to the amino acids in MMLV-RT:
F155Y. The hyperactive reverse transcriptase produces a yield of
greater than about 1, 5, 7, 10, 15 or about 25 .mu.g of an aRNA
from 100 ng of template RNA in a single amplification reaction.
Alternatively, the hyperactive reverse transcriptase produces a
yield of greater than about 1, 2, 5 or even 10 .mu.g of an aRNA
from 10 pg of template RNA after a two-round amplification
reaction. The hyperactive reverse transcriptase may produces a cDNA
greater than about 6, 9 or even 11 or from between about 6 to about
15 kilobases, or greater than 15 kilobases in a single cDNA
synthesis reaction. The hyperactive reverse transcriptase has a DNA
polymerase activity of greater than about 200 Units per microgram,
e.g., between about 0.1 and 300 Units per microgram. Generally, the
reverse transcriptase of the present invention has an RNase H
activity of between about 0.1 and about 25 percent of the wild-type
RNase H activity of reverse transcriptases.
[0012] The present invention also includes an isolated reverse
transcriptase having substantially reduced RNase H activity that
one or more point mutations in the processivity domain. The RNase I
activity of the reverse transcriptase has between about 0.1 and 50%
of wild-type activity or between about 1 and 10% of wild-type RNase
H activity of a wild-type reverse transcriptase, e.g., an MMLV-RT.
The mutation in the processivity domain may include one or more of
the following mutations corresponding to the amino acids in
MMLV-RT: H638G, Y586A, D653N, D524N, D524E and E562D and may
further include a mutation in the nucleotide selection domain
comprises a mutation of residue F155 in MMLV-RT. The reverse
transcriptase may be isolated and purified and include one or more
mutations in the processivity domain corresponding to the amino
acids in MMLV-RT: H638G, Y586A, D653N, D524N, D524E and E562D and a
mutation in the nucleotide selection domain at F155Y.
[0013] Yet another embodiment of the present invention is an
isolated protein having DNA polymerase activity and substantially
reduced RNase H activity comprising one or more mutations in the
processivity domain and one or more mutations in the nucleotide
selection domain. The isolated protein having DNA polymerase
activity and substantially reduced RNase H activity may produces a
yield of greater than about 1, 5, 7, 10, 12, 15, 25 .mu.g of an
aRNA from 100 ng of template RNA in a single amplification
reaction. Another method for characterizing the activity of the
reverse transcriptase enzyme mutants disclosed herein is that the
reverse transcriptase protein produces an aRNA yield of greater
than about 20% as compared to an equivalent wild-type Reverse
Transcriptase enzyme. Another characteristic is that the reverse
transcriptase protein produces a yield of greater than about 1, 5
or 10 .mu.g of an aRNA from 10 pg of template RNA after a two-round
amplification reaction; a cDNA greater than about 6, 9 or even 11
kilobases in a single cDNA synthesis reaction; a cDNA greater than
about 6 to about 15 kilobases in a single cDNA synthesis reaction
or even a cDNA greater than about 15 kilobases in a single cDNA
synthesis reaction. Generally, the DNA polymerase activity is
greater than about 200 Units per microgram, e.g., between about 0.1
and 300 Units per microgram. Functionally, the mutant reverse
transcriptase will have between about 0.1 and about 25 percent of
the wild-type RNase H activity.
[0014] The present invention also includes an isolated and purified
reverse transcriptase protein comprising one or more mutations in
the nucleotide selection domain and may be selected from, e.g.,
AMV, M-MLV, HTLV-1, BLV, RSV, HFV, R2 Bombyx mori and/or HIV
reverse transcriptase. The reverse transcriptase may also be
modified at the nucleotide sequence to encode a modified amino acid
sequence in the processivity domain corresponding to amino acids
497 to 671 of M-MLV reverse transcriptase. When the nucleotide
selection domain is mutated this may be one or more point mutations
in the nucleotide selection domain corresponding to amino acids 153
to 158 of M-MLV reverse transcriptase and may be used in the
preparation of full-length cDNA.
[0015] A process for making a protein with hyperactive reverse
transcriptase activity may include the steps of: transforming a
host cell with the hyperactive RT comprising a mutation in the
processivity domain that comprises one or more of the following
mutations corresponding to the amino acids in MMLV-RT: H638G,
Y586A, D653N, D524N, D524E and E562D and further comprising a F155Y
mutation in the nucleotide selection domain of MMLV-RT and
culturing the host cell under conditions such that the hyperactive
reverse transcriptase is produced by the host cell.
[0016] The present invention also includes an isolated and purified
nucleic acid encoding a hyperactive reverse transcriptase with a
mutation in the processivity domain and/or in the nucleotide
selection domain. For example, the nucleic acid sequence may be
modified to encode a hyperactive reverse transcriptase having a
mutation that corresponds to and includes, e.g., an H638G mutation
of the MMLV-RT, an F155Y mutation or an F155Y mutation and an H638G
mutation. The nucleic acid has SEQ ID No.: 1 and further include,
e.g., a nucleic acid segment encoding a leader sequence and/or
encode a protein segment other than the hyperactive reverse
transcriptase to form, e.g., a fusion protein. Another embodiment
of the present invention is a vector that includes a nucleic acid
having a nucleic acid encoding a hyperactive reverse transcriptase
that encodes a mutation in the processivity domain and/or in the
nucleotide selection domain.
[0017] Yet another embodiment of the present invention is a host
cell transformed with an expression vector having a nucleic acid
encoding an amino acid of SEQ ID NO.: 2, for a hyperactive reverse
transcriptase. The host cell may be a bacteria, fungi, plant, or
even a mammalian cell. One example of a host is E. coli or even P.
pastoris. The host cell may even be transformed to express a
hyperactive reverse transcriptase. The host cell, vector and
constructs disclosed herein may be used in a process for making an
isolated hyperactive reverse transcriptase that includes the steps
of transforming a host cell with an isolated nucleic acid that
encodes a hyperactive reverse transcriptase; and culturing the host
cell under conditions such that the hyperactive reverse
transcriptase is produced.
[0018] The hyperactive reverse transcriptase may include one or
more mutations replace at least one of the amino acids of the
processivity domain and the nucleotide selection domain, with an
alternative naturally occurring L-amino acid, the replacement being
selected from the group consisting of: (1) a substitution of any of
isoleucine, valine, and leucine for any other of these amino acids;
(2) a substitution of aspartic acid for glutamic acid or vice
versa; (3) a substitution of glutamine for asparagine or vice
versa; (4) a substitution of serine for threonine or vice versa;
(5) a substitution of glycine for alanine or vice versa; (6) a
substitution of alanine for valine or vice versa; (7) a
substitution of methionine for any of leucine, isoleucine, or
valine and vice versa; and (8) a substitution of lysine for
arginine or vice versa. Alternatively, the replacement may be
selected from the group consisting of: (1) a substitution of any of
isoleucine, valine, or leucine for any other of these amino acids;
(2) a substitution of aspartic acid for glutamic acid or vice
versa; (3) a substitution of glutamine for asparagine or vice
versa; and (4) a substitution of serine for threonine or vice versa
and wherein the hyperactive reverse transcriptase comprises a
hyperactive reverse transcriptase.
[0019] The present invention also includes a variety of kits that
use the present invention, which will generally include
instructions for the use of the hyperactive reverse transcriptase
and a variety of buffers, controls and the like. One example of a
kit may be used to synthesize nucleic acid synthesis, and includes
in a suitable container: a hyperactive reverse transcriptase; and a
reaction solution for the reverse transcriptase. The kit may also
include information insert may include information for using the
reverse transcriptase, a reaction solution comprises a 10.times.
concentrated reverse transcriptase reaction buffer, a primer, a
reverse transcriptase buffer, a PCR buffer, a single contained with
a mix of nucleotides or containers that each hold individual
nucleotides, a buffer for in vitro transcription, a template
purification column and/or one or more magnetic particles suitable
for nucleic acid purification. Alternatively, the kit for nucleic
acid synthesis may include in a suitable container a hyperactive
reverse transcriptase comprising one point mutation in the
processivity domain; and a reaction solution for the reverse
transcriptase. Another kit may include suitable containers having a
hyperactive reverse transcriptase comprising one point mutation in
the processivity domain and one point mutation in the nucleotide
selection domain; and a reaction solution for the reverse
transcriptase.
[0020] The present invention also includes a method for RNA
amplification that includes the steps of, reverse transcribing an
RNA template into a single-stranded cDNA with a hyperactive reverse
transcriptase in the presence of an oligonucleotide comprising a
transcriptional promoter and a primer, purifying the
single-stranded cDNA; and generating amplified RNA (aRNA) using an
RNA polymerase. Alternatively, a method for RNA amplification may
include the steps of: reverse transcribing an RNA template into a
single-stranded cDNA with a hyperactive reverse transcriptase in
the presence of an oligonucleotide comprising a transcriptional
promoter and a primer, converting the single-stranded cDNA into
double-stranded cDNA using a DNA polymerase, purifying the
double-stranded cDNA and generating amplified RNA (aRNA) using an
RNA polymerase. The method may also include purifying the aRNA and
aRNA made using the methods disclosed herein.
[0021] Yet another kit may be for RNA amplification and includes in
one or more suitable containers a hyperactive reverse transcriptase
that includes one or more point mutations in the processivity
domain and one or more point mutations in the nucleotide selection
domain; an oligonucleotide with a transcriptional promoter region
and/or oligo(dT) region; a DNA polymerase; and an RNA polymerase.
The kit may also include one or more of the following: an insert
may be provided that includes information for using the optimized
reverse transcriptase, a 10.times. concentrated reverse
transcriptase reaction buffer, a primer, a reverse transcriptase
buffer, a DNA Polymerase buffer, a mix of nucleotides, separate
containers for individual nucleotides, a buffer for in vitro
transcription, a nucleic acid purification column and/or a magnetic
particle or particles suitable for nucleic acid purification.
[0022] Another kit for RNA amplification may include one or more
suitable containers that include: a hyperactive reverse
transcriptase with one or more point mutations in the processivity
domain; an oligonucleotide with a transcriptional promoter region
and oligo(dT) region; a DNA polymerase; and an RNA polymerase. The
kits, methods and compositions disclosed herein may be used to make
an aRNA including a ssDNA or a DNA:RNA hybrid made from an RNA
template by a hyperactive reverse transcriptase. Also included may
be an RT-PCR kit with one or more suitable containers: a
hyperactive reverse transcriptase, two or more primers,
nucleotides, a thermostable DNA polymerase and an RT-PCR buffer.
The same container or a separate container may also be provided
that includes one or more reverse transcriptases in addition to the
hyperactive reverse transcriptase of the present invention as a
control or to provide additional reverse transcriptase
activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
figures in which corresponding numerals in the different figures
refer to corresponding parts and in which:
[0024] FIG. 1 shows the domain structure of MMLV RT, point
mutations relevant herein are marked;
[0025] FIG. 2 shows the gene sequence of F155Y; H638G MMLV RT (SEQ
ID NO: 1);
[0026] FIG. 3 shows the protein sequence of F155Y; H638G MMLV RT
(SEQ ID NO: 2);
[0027] FIG. 4 is a gel that shows a comparison of cDNA Product
Lengths by Various Mutant MMLV RT Enzymes using RNA Templates from
0.5 to 9.0 kb in Size;
[0028] FIG. 5 is a gel of the cDNA synthesis products using a 9 kb
RNA template with MMLV RT mutants in the presence of MgCl.sub.2 or
MnCl.sub.2;
[0029] FIG. 6A and FIG. 6B are graphs that demonstrate single round
RNA Amplification with 100 ng (FIG. 6A) or 1 .mu.g (FIG. 6B) Rat
Thymus Total RNA: MMLV RT Mutant Comparisons., the aRNA yields were
determined by UV absorbance at 260 nm, samples were performed in
duplicate;
[0030] FIG. 7 is a graph of a single round RNA Amplification with
90 ng HeLa S3 Total RNA: MMLV RT Mutant Comparisons (the aRNA
yields were determined by UV absorbance at 260 nm, performed in
duplicate); FH=F155Y; H638G MMLV RT; SSII=SuperScript II;
MMLV=wild-type MMLV RT; AMV=Avian Myeloblastosis Virus RT;
[0031] FIG. 8 is a graph that demonstrates the yield from a two
round RNA Amplification with 10-1000 pg HeLa S-3 Total RNA: MMLV RT
Mutant Comparisons (AMV=Avian Myeloblastosis Virus RT;
FYHG=F155Y;H638G MMLV RT; SSII=SuperScript II; The aRNA yields were
determined by UV absorbance at 260 nm, performed in duplicate);
[0032] FIG. 9 is a graph that demonstrates the yield from Two Round
RNA Amplification Comparing F155Y;H638G MMLV RT and AMV RT (input
total RNA was HeLa-S3, at 1 ng and 10 pg);
[0033] FIG. 10 is a graph that shows compares a hyperactive reverse
transcriptase with the Standard Affymetrix aRNA Protocol Comparison
to MessageAMP containing F155Y; H638G MMLV RT; and
[0034] FIG. 11 is a graph that shows a Scatter plot comparing
F155Y; H638G MMLV RT (x axis) vs. SSII (y axis) Signal Intensities
from a Human Focus Array.
DETAILED DESCRIPTION OF THE INVENTION
[0035] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts which can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
DEFINITIONS
[0036] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
limit the invention, except as outlined in the claims. As used
throughout the present specification the following abbreviations
are used: kb, kilobase (pairs); kD, kilodalton; PCR, polymerase
chain reaction; RT, reverse transcriptase; MMLV, Murine Moloney
leukemia virus; AMV, avian myoblastosis virus; RSV, Rous sarcoma
virus; HIV, human immunodeficiency virus; HFV, human foamy
virus.
[0037] The invention relates to the production of hyperactive RTs.
The present invention also includes RTs having DNA polymerase
activity and substantially reduced RNase H activity made using
recombinant DNA techniques wherein the RT is modified using point
mutations. More particularly, the present invention includes RTs
with one or more point mutations in the nucleotide selection
domain, RTs with one or more point mutations in the processivity
domain, and hyperactive RTs that include RTs with mutations in both
the nucleotide selection and the processivity domains. The RTs
disclosed herein may be expressed in host cells using, e.g.,
recombinant plasmids constructed as described herein that provide
reverse transcriptase for use in recombinant DNA technology to
synthesize cDNA from mRNA without the unwanted effects of RNase H
activity, which can excessively degrade the mRNA template during
first-strand synthesis.
[0038] As used herein, the terms "hyperactive Reverse
Transcriptase," "hyperactive RT" and the like are used to describe
a hyperactive RT purified to near homogeneity and having the
ability to enable greater than 20% more amplified RNA that can be
generated by the polymerase activity of a wild-type RT DNA
polymerase domain from an input of 100 ng total RNA in an RNA
amplification reaction that includes a 4 hr in vitro transcription
reaction. For example, an isolated hyperactive Reverse
Transcriptase that includes one or more point mutations in the
"processivity domain" and one or more point mutations in the
"nucleotide selection domain" is able to generate a yield of
greater than 5 micrograms of aRNA in a single amplification
reaction from 100 ng of total RNA, e.g., in a single round RNA
amplification of 100 ng HeLa S-3 Total RNA or Rat Thymus Total RNA
against wild-type enzyme MMLV-RT, AMV RT or any other RT as
determined by, e.g., UV absorbance at 260 nm or other equivalent
methods known to the skilled artisan. As will be apparent to those
of skill in the art, the "hyperactivity" of the enzymes of the
present invention may be as quantitatively distinct due to, e.g.,
assay conditions, temperatures, times, salts, source of RNA,
quality of RNA, activity read-out and the like.
[0039] The term "processivity domain" is used to describe the
region of the RT that is responsible for maintenance of the
template integrity in a standard RT reaction. As defined herein,
the processivity domain includes amino acids 497-671. One
indication of processivity is the average length of the cDNA that
can be synthesized from a long mRNA target. The present invention
is distinct from the domains identified by, e.g., Gerard, et al.,
U.S. Pat. No. 5,668,005 and patents related thereto, which
functionally identified the region spanning MMLV RT amino acids 503
through 611 as critical for RNase H activity. In contrast to the
region identified by Gerard, et al., the present invention
identifies locations and mutations outside of this previously
characterized region as also affect RNase H activity and,
importantly, enzyme processivity. Indeed, the mutant RT enzymes
described herein catalyze yields of amplified RNA that are superior
to other, commercially available enzymes mutated in the RNase H
domain, such as SuperScript II. As a result, the inventors describe
novel mutations that enable a large and unexpected improvement in
the yield of amplified RNA in Eberwine-like RNA amplification
protocols. The hyperactive RT with mutations in the processivity
domain of the RTs may also include one or more point mutations in
other domains. The present inventors have identified one series of
mutants that can affect the sensitivity of the RT for
distinguishing or having a preference for ribonucleotides and/or
deoxyribonucleotides during DNA synthesis, which are described
herein as the "nucleotide selection domain." As used herein the
phrase "nucleotide selection domain" or "NSD" includes but is not
limited to, mutations in the following amino acids in MMLV-RT:
D153, A154, F155, F156, C157, or L158 and the equivalent mutations
in other RTs. The equivalent mutation in the other RTs may be
localized based on the crystal structure of MMLV RT, which reveals
a secondary structure motif that encompasses a 3.sub.10 helix
around the nucleotide selection domain namely, amino acids 153 to
158 of the MMLV-RT.
[0040] Described herein are point mutations that alone or in
combination significantly enhance the yield of amplified nucleic
acids used useful for, e.g., the amplification of isolated RNA for
use in nucleic acid microarrays. Another method to detect the
activity of the hyperactive RTs of the present invention is the
length of the cDNAs, wherein the hyperactive RTs are able to copy
an mRNA to a product length greater than 9, 11, 15 or even 20
kilobases.
[0041] As used herein, the term "substantially reduced RNase H
activity" is used to describe an RT purified to near homogeneity
and having an RNase H activity of between about 0.01%, 1, 3, 4, 6,
9, 10, 15, 20, 25 and 50% of the RNase H activity of a wild-type RT
RNase H domain. Described herein are point mutations that alone or
in combination reduce the level of degradation of the RNA template
used in an RT reaction, that is, without significant degradation of
the mRNA template during first-strand synthesis, but that maintain
"processive" activity. The term "processivity" as used herein is
used to describe the ability of the RT to elongate its nucleic acid
product to produce a longer product. This processivity domain
includes, but is not limited to, one or more of the following
mutations corresponding to the amino acids in MMLV-RT: H638G,
Y586A, D653N, D524N, D524E and E562D. The double mutants of the
present invention also include mutations to the processivity domain
corresponding to the amino acids in MMLV-RT: H638G, Y586A, D653N,
D524N, D524E and E562D and one or more mutations to the nucleotide
selection domain that include one or more of the following
mutations in the following amino acids in MMLV-RT: D153, A154,
F155, F156, C157, or L158.
[0042] The term "degenerate variants" as used herein describes
having variations in the DNA or amino acid sequence that vary the
amino acids at the processivity domain and the nucleotide selection
domain such that the activities described herein are maintained.
The term "codon-optimized" sequence is used to describe a
hyperactive RT in which at least a portion of the sequence has been
modified by directed sequence modification, for example, changes to
the sequence in one or more underlying sequences that may or may
not affect the amino acid sequence but that are used to, e.g.,
improve the expression of the protein by using codons that are more
commonly used in a particular host organism. By the term
"recombinant," "isolated," "cloned" hyperactive RT or grammatical
equivalents herein is meant a polypeptide having a modified nucleic
or amino acid sequence of a mature RT (for example, from about 85
to 100% identical) as described herein, as well as amino acid
sequence variants that are enzymatically active RNA directed, DNA
polymerases with a catalytic profile that is distinct from that of
wild type RT, e.g., AMV RT, MMLV RT and the like as defined
hereinabove. In addition, sequences may be the combination of
sequences from different organisms for the same or closely related
sequences to, e.g., modify the functionality of the final protein
by directed modifications or even to permit specific recombinant
modification or manipulation by the user.
[0043] As defined herein, a "wild type" sequence, whether found in
a coding, non-coding or interface sequence is an allelic form of
sequence that performs the natural or normal function for that
sequence. Therefore, as used herein a wild type sequence includes
multiple allelic forms of a cognate sequence; for example, multiple
alleles of a wild type sequence may encode silent or conservative
changes to the protein sequence that a coding sequence encodes. A
"mutant" sequence is defined herein as one in which at least a
portion of the functionality of the sequence has been lost, for
example, changes to the sequence in a promoter or enhancer region
will affect at least partially the expression of a coding sequence
in an organism. A "mutation" in a sequence as used herein is any
change in a nucleic acid sequence that may arise such as from a
deletion, addition, substitution, or rearrangement. The mutation
may also affect one or more steps that the sequence is involved in.
For example, a change in a DNA sequence may lead to the synthesis
of an altered protein, one that is inactive, or to an inability to
produce the protein. A "mutation frequency" as used herein is the
frequency or rate with which a particular mutation appears in a
particular dataset. Mutation frequency may also be the frequency at
which any mutation appears in the whole dataset.
[0044] A sample is any mixture of macromolecules obtained from a
solution, a cell culture, a supernatant, an animal, an
environmental sample, a food sample or even a patient. This also
includes separated fractions of all of the preceding. Examples of
samples include, but are not limited to, blood, plasma, urine,
semen, saliva, lymph fluid, meningeal fluid, amniotic fluid,
glandular fluid, and cerebrospinal fluid. "Sample" also includes
solutions or mixtures containing homogenized solid material, such
as feces, cells, tissues, and biopsy samples. Samples herein
include one or more that are obtained at any point in time,
including diagnosis, prognosis, and periodic monitoring.
[0045] The terms "a sequence essentially as set forth in SEQ ID
NO.: (#)", "a sequence similar to", "nucleotide sequence" and
similar terms, with respect to nucleotides, refers to sequences
that substantially correspond to any portion of the sequence
identified herein as SEQ ID NO.: 1 or the point mutants and
combination of point mutants of RTs described herein and the
functional counterparts in related RTs. These terms refer to
synthetic as well as naturally-derived molecules and includes
sequences that possess biologically, immunologically,
experimentally, or otherwise functionally equivalent activity, for
instance with respect to hybridization by nucleic acid segments, or
the ability to encode all or portions of an RT having DNA
polymerase and/or substantially reduced RNase H activity.
Naturally, these terms are meant to include information in such a
sequence as specified by its linear order.
[0046] The term "homology" refers to the extent to which two
nucleic acids are complementary. There may be partial or complete
homology. A partially complementary sequence is one that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid and is referred to using the
functional term "substantially homologous." The degree or extent of
hybridization may be examined using a hybridization or other assay
(such as a competitive PCR assay) and is meant, as will be known to
those of skill in the art, to include specific interaction even at
low stringency.
[0047] The inhibition of hybridization of the completely
complementary sequence to the target sequence may also be examined
using a hybridization assay involving a solid support (e.g.,
Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. Low stringency conditions may
be used to identify the binding of two sequences to one another
while still being specific (i.e., selective). The absence of
non-specific binding may be tested by the use of a second target
that lacks even a partial degree of complementarity (e.g., less
than about 30% identity). In the absence of non-specific binding,
the probe will not hybridize to the second non-complementary target
and the original interaction will be found to be selective. Low
stringency conditions are generally conditions equivalent to
binding or hybridization at 42 degrees Centigrade in a solution
consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l
NaH.sub.2PO.sub.4--H.sub.2O and 1.85 g/l EDTA, pH 7.4), 0.1% SDS,
5.times.Denhardt's reagent (50.times.Denhardt's contains per 500
ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)
and 100 micrograms/ml denatured salmon sperm DNA); followed by
washing in a solution comprising 5.times.SSPE, 0.1% SDS at 42
degrees Centigrade when a probe of about 500 nucleotides in length
is employed. The art knows that numerous equivalent conditions may
be employed to achieve low stringency conditions. Factors that
affect the level of stringency include: the length and nature (DNA,
RNA, base composition) of the probe and nature of the target (DNA,
RNA, base composition, present in solution or immobilized, etc.)
and the concentration of the salts and other components (e.g.,
formamide, dextran sulfate, polyethylene glycol). Likewise, the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
inclusion of formamide, etc.).
[0048] The term "gene" is used to refer to a functional protein,
polypeptide or peptide-encoding unit. As will be understood by
those in the art, this functional term includes genomic sequences,
cDNA sequences, or fragments or combinations thereof, as well as
gene products, including those that may have been altered by the
hand of man. Purified genes, nucleic acids, protein and the like
are used to refer to these entities when identified and separated
from at least one contaminating nucleic acid or protein with which
it is ordinarily associated. As used herein the terms "protein",
"polypeptide" or "peptide" refer to compounds comprising amino
acids joined via peptide bonds and are used interchangeably.
[0049] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The vector may be further defined as one designed to
propagate specific sequences, or as an expression vector that
includes a promoter operatively linked to the specific sequence, or
one designed to cause such a promoter to be introduced. The vector
may exist in a state independent of the host cell chromosome, or
may be integrated into the host cell chromosome.
[0050] The term "host cell" refers to cells that have been
engineered to contain nucleic acid segments or altered segments,
whether archeal, prokaryotic, or eukaryotic. Thus, engineered, or
recombinant cells, are distinguishable from naturally occurring
cells that do not contain genes introduced recombinantly through
the hand of man.
[0051] The term "altered", or "alterations" or "modified" with
reference to nucleic acid or polypeptide sequences is meant to
include changes such as gross or point: insertions, deletions,
substitutions, fusions with related or unrelated sequences, such as
might occur by the hand of man, or those that may occur naturally
such as polymorphisms, alleles and other structural types.
Alterations encompass genomic DNA and RNA sequences that may differ
with respect to their hybridization properties using a given
hybridization probe. Alterations of polynucleotide sequences for a
hyperactive reverse transcriptase, or fragments thereof, include
those that increase, decrease, or have no effect on functionality.
Alterations of polypeptides refer to those that have been changed
by recombinant DNA engineering, chemical, or biochemical
modifications, such as amino acid derivatives or conjugates, or
post-translational modifications.
[0052] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it effects
the transcription of the sequence; or a ribosome binding site is
operably linked to e coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in same reading frame Enhancers do
not have to be contiguous. Linking is accomplished by ligation at
convenient restriction sites. If such sites do not exist, then
synthetic oligonucleotide adaptors or linkers are used in accord
with conventional practice.
[0053] As used herein, the expressions "cell" and "cell culture"
are used interchangeably end all such designations include progeny.
Thus, the words "transformants" and "transformed cells" include the
primary subject cell and cultures derived therefrom without regard
for the number of transfers. It is also understood that all progeny
may not be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included. Different designations are will be
clear from the contextually clear.
[0054] "Plasmids" are designated by, e.g., a lower case p preceded
and/or followed by capital letters and/or numbers. The starting
plasmids herein are commercially available, are publicly available
on an unrestricted basis, or can be constructed from such available
plasmids in accord with published procedures. In addition, other
equivalent plasmids are known in the art and will be apparent to
the ordinary artisan.
[0055] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method is selected based
on the host cell being transformed and may include, but is not
limited to, viral infection, electroporation, lipofection, and
particle bombardment. Such "transformed" cells include stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome.
[0056] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector." The term "vector" as used herein also includes
expression vectors in reference to a recombinant DNA molecule
containing a desired coding sequence and appropriate nucleic acid
sequences necessary for the expression of the operably-linked
coding sequence in a particular host organism. Nucleic acid
sequences necessary for expression in prokaryotes usually include a
promoter, an operator (optional), and a ribosome binding site,
often along with other sequences. The choice of a suitable vector
depends on a number of considerations known to one of ordinary
skill in the art, such as the size of the fragment, nature of the
host, number and position of restriction sites desired, and the
selection of marker and markers desired for selection. Expression
of the RT genes may also be placed under control of other
regulatory sequences homologous or heterologous to the host
organism in its untransformed state as will be known to the skilled
artisan. The selection of the host cell for transformation may
influence the decision of which vector and/or regulatory sequences
are provided along with the RT construct. Eukaryotic cells are
known to utilize promoters, enhancers, and termination and
polyadenylation signals.
[0057] As used herein, the term "amplify", when used in reference
to nucleic acids refers to the production of a large number of
copies of a nucleic acid sequence by any method known in the art.
Amplification is a special case of nucleic acid replication
involving template specificity. Template specificity is frequently
described in terms of "target" specificity. Target sequences are
"targets" in the sense that they are sought to be sorted out from
other nucleic acid. Amplification techniques have been designed
primarily for this sorting out.
[0058] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer may be
single stranded for maximum efficiency in amplification but may
alternatively be double stranded. If double stranded, the primer is
first treated to separate its strands before being used to prepare
extension products. The primer must be sufficiently long to prime
the synthesis of extension products in the presence of the inducing
agent. The exact lengths of the primers will depend on many
factors, including temperature, source of primer and the use of the
method.
[0059] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g. ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0060] As used herein, the term "target" when used in reference to
the polymerase chain reaction, refers to the region of nucleic acid
bounded by the primers used for polymerase chain reaction. Thus,
the "target" is sought to be sorted oat from other nucleic acid
sequences. A "segment" is defined as a region of nucleic acid
within the target sequence.
[0061] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195,
4,683,202, and 4,965,188, hereby incorporated by reference, which
describe a method for increasing the concentration of a segment of
a target sequence in a mixture of genomic DNA without cloning or
purification. This process for amplifying the target sequence
consists of introducing a large excess of two oligonucleotide
primers to the DNA mixture containing the desired target sequence,
followed by a precise sequence of thermal cycling in the presence
of a DNA polymerase. The two primers are complementary to their
respective strands of the double stranded target sequence. To
effect amplification, the mixture is denatured and the primers then
annealed to their complementary sequences within the target
molecule. Following annealing, the primers are extended with a
polymerase so as to form a new pair of complementary strands. The
steps of denaturation, primer annealing and polymerase extension
can be repeated many times (i.e., denaturation, annealing and
extension constitute one "cycle"; there can be numerous "cycles")
to obtain a high concentration of an amplified segment of the
desired target sequence. The length of the amplified segment of the
desired target sequence is determined by the relative positions of
the primers with respect to each other, and therefore, this length
is a controllable parameter. By virtue of the repeating aspect of
the process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified". With PCR, it is possible to amplify a single copy
of a specific target sequence in genomic DNA to a level detectable
by several different methodologies (e.g., hybridization with a
labeled probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of
.sup.32P-labeled deoxynucleotide triphosphates, such as dCTP or
dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide sequence can be amplified with the appropriate set
of primer molecules. In particular the amplified segments created
by the PCR process itself are, themselves, efficient templates for
subsequent PCR amplifications.
[0062] The reverse transcriptase gene (or the genetic information
contained therein) can be obtained from a number of different
sources, e.g., Moloney Murine leukemia virus (M-MLV); human T-cell
leukemia virus type I (HTLV-I); bovine leukemia virus (BLV); Rous
Sarcoma Virus (RSV); human immunodeficiency virus (HIV); yeast,
including Saccharomyces, Neurospora, Drosophila; primates; and
rodents. See, e.g., Weiss et al., U.S. Pat. No. 4,663,290 (1987);
Gerard, G. R., DNA 5:271-279 (1986); Kotewicz, M. L., et al., Gene
25:249-258 (1985); Tanese, N., et al., Proc. Natl. Acad. Sci. (USA)
82:4944-4948 (1985); Roth, M. J., et al., J. Biol. Chem.
260:9326-9335 (1985); Michel, F., et al., Nature 316:641-643
(1985); Akins, R. A., et al., Cell 47:505-516 (1986), EMBO J.
4:1267-1275 (1985); and Fawcett, D. F., Cell 47:1007-1015 (1986).
For instance, the gene may be obtained from public sources, e.g.,
ATCC, or may even be purified from eukaryotic cells infected with a
retrovirus, or from a plasmid that includes a portion the
retrovirus genome that includes the RT.
[0063] The mutation(s) for producing a hyperactive polymerase
domain as described herein may be obtained by point mutation(s) in
the processivity domain as described and disclosed herein.
Likewise, RT genes having DNA polymerase activity and substantially
reduced RNase H activity may be obtained by point mutation(s) of
the nucleotide selection domain. The plasmid thus obtained may then
be used to transform hosts which may then be screened for
hyperactive RT activity. RT RNase H activity may also be assayed by
template solubilization as compared to, e.g., a wild-type AMV, MMLV
or other RT.
[0064] The invention also includes fusion proteins that include the
hyperactive reverse transcriptase of the invention with, e.g., a
carrier protein or other anchor domain that permits isolation and
purification. It is also possible to prepare fusion proteins of the
hyperactive reverse transcriptase that are substituted at the amino
or carboxy termini with polypeptides which stabilize or change the
solubility of the reverse transcriptase. Amino-terminal and
carboxy-terminal gene fusion protein domains are well known in the
art.
[0065] The transformed hosts of the invention may be cultured
under, e.g., protein producing conditions according to any of the
methods that are known to those skilled in the art or protein
production, purification, isolation and characterization. Of
particular use may be host cells that have reduced endogenous RNase
activity, e.g., as taught in U.S. Pat. No. 5,998,195, relevant host
cells, constructs, vectors and methods incorporated herein by
reference. The hyperactive RT of the present invention may be
isolated according to conventional methods known to those skilled
in the art. For example, after protein expression the host cells
may be collected by centrifugation, washed with suitable buffers,
lysed, and the reverse transcriptase isolated by column
chromatography, for example, on DEAE-cellulose, phosphocellulose or
other standard isolation and identification techniques using, for
example, polyribocytidylic acid-agarose, or hydroxylapatite or by
electrophoresis or immunoprecipitation.
[0066] The hyperactive RT of the present invention may be used with
any assay and included in any kit that calls for an RT, e.g., it
may be used to prepare cDNA from RNA by hybridizing a primer, e.g.,
an oligo(dT) primer, or other complementary primers with the mRNA.
The hyperactive RT of the present invention is particularly useful
for the synthesis of full-length and/or extra-long length, complete
cDNA by adding the hyperactive RT and all four deoxynucleoside
triphosphates under conditions that permit elongation. Using the
hyperactive RT produced by the present invention allows for the
preparation of cDNA from mRNA with reduced degradation of the mRNA,
which results in cDNA synthesis of messages exceeding 9, 12 or even
15 kilobases.
[0067] The hyperactive RT of the present invention is suited for
incorporation into a kit for the preparation of cDNA from RNA, for
aRNA synthesis and for amplification of mRNA for microarray
analysis. Such a kit will generally include one or more containers,
such as vials, tubes, and the like, and the kit will containers
that have alone or in combination one or more of the separate
elements of the method used, e.g., to prepare cDNA from RNA or for
amplification of the RNA. For example, the kit may include one vial
that has the hyperactive RT in solution. Separate containers may
include suitable buffers, substrates for DNA synthesis such as the
deoxynucleotides, oligo(dT) primer, and even a control RNA for use
as a standard.
[0068] The reverse transcriptase may be present in the solution at
a concentration of 5, 10, 100, 200, 400 or more units/ml. The
reverse transcriptase may also be lyophilized in a plate well, and
its activity reconstituted upon hydration of the lyophilized
enzyme. Deoxynucleotides may be present either in lyophilized form,
as part of a buffer or in solution at a concentration of, e.g.,
about 0.5 mM to about 2 mM each. A suitable buffer, present at 2,
5, 10, 50 and/or 100 times the final concentration of use may be,
e.g., 250 mM Tris-HCl (pH 7.5 to 8.3), 375 mM KCl, 15 mM
MgCl.sub.2, and 50 mM dithiothreitol. An oligo (dT) may be present
at a concentration of 5 .mu.g/ml to 20 .mu.g/ml. Control RNA, such
as 2.3, 9.0 or greater kilobase control RNA, may be present at a
concentration of 10 .mu.g/ml to 20 .mu.g/ml.
[0069] Reverse transcriptase-polymerase chain reaction (RT-PCR)
and/or simultaneous DNA cleavage and reverse transcription may be
conducted using the hyperactive RT. The hyperactive RT of the
present invention may be used in conjunction with standard RT-PCR
techniques. RT-PCR is a common molecular biology procedure that
typically requires DNA-free RNA. DNase I digestion of contaminating
DNA is the method of choice for eradicating DNA in RNA preparations
destined for reverse transcription and PCR.
Example 1
[0070] The present inventors recognized that current RT mutants
fail to provide the best combination of amplification,
processivity, fidelity and ease of use. As such, the inventors
focused on the creation of plasmids expressing H638G MMLV RT, Y586A
MMLV RT, D653N MMLV RT, D524N MMLV RT, D524E MMLV RT, and E562D
MMLV RT. Efforts to develop an improved RT for RNA amplification
began with strategies for modulating RT-associated RNase H
activity. MMLV and AMV-related RTs with no RNase H activity are
known to synthesize longer cDNA products than their RNase H.sup.+
counterparts. However, a complete loss of RNase H activity may have
untoward effects in some applications (for an example, see
Biotechniques 2002 June; 32(6): 1224-5). In fact, RNase H treatment
of first strand cDNA is an obligate step in the aRNA synthesis
procedure. Thus, an appropriately balanced ratio of RNase H to
polymerase activity was potentially desirable.
[0071] As MMLV RT is modified easily by molecular techniques, this
enzyme was the target for improvement efforts. FIG. 1 shows the
relative domain structure of MMLV RT with the relevant point
mutations of the present invention marked in relation to the
domains. The present inventors identified a number of amino acid
residues within the DNA polymerase domain and carboxy from the
RNase H domain of MMLV RT as potential targets for site-directed
mutagenesis. This domain is composed of a portion of the
carboxy-terminus of the RT. For example, one group has identified
amino acid residues 503-611 as critical for RNase H activity by
gross deletion. Although the three dimensional structure of MMLV RT
RNase H domain has not been solved, the corresponding structure of
E. coli RNase H1 is known (Science. 1990 September 21;
249(4975):1398-405). However, E. coli RNase H1 shares only 30%
identity with the MMLV RNase H (Proc Natl Acad Sci USA. 1986
October; 83(20): 7648-52), which include essential metal binding
and active site residues. Several "support" residues not directly
involved in catalysis were also identical in the two enzymes.
Several mutants of E. coli RNase H1 have been identified that
exhibit reduced RNase H activity. The data provided herein support
the choice of 9-10 mutants that were found to enhance the ability
of the RT to maintain template interactions, significantly reduce
the RNase H activity of MMLV RT to a level in the 1-50% range as
compared to the wild-type MMLV RT, but not having the deleterious
effects of deletion mutants or mutants having no RNase H activity.
The results disclosed herein demonstrate that reduced activity (but
not eliminated or no RNase H activity), is desirable for the aRNA
synthesis application and the creation of a hyperactive RT.
[0072] The present inventors have developed a series of point
mutants, e.g., H638G MMLV RT, Y586A MMLV RT, D653N MMLV RT, D524N
MMLV RT, D524E MMLV RT, and E562D MMLV RT using pSE380 containing
the MMLV RT gene (pSE380-MMLV RT) and the mutagenic primers given
in Table 1. The nucleic acid sequence for one such mutant is shown
in FIG. 2, with the amino acid sequence described in FIG. 3.
Amplification of the mutant sequences was accomplished via PCR
using the Quick Change mutagenesis kit (Stratagene). The resulting
PCR product was transformed and plated onto solid media containing
ampicillin. Plasmid DNA from selected clones was prepared with the
QIAprep Spin Miniprep Kit. In the case of Y586A MMLV RT and H638G
MMLV RT, the presence of the correct mutation was diagnosed after
restriction digest with Sma I. Clones containing D653N, D524N MMLV
RT, D524E MMLV RT, and E562D MMLV RT were screened by sequencing.
In each case, sequencing across the MMLV gene confirmed the desired
mutations.
TABLE-US-00001 TABLE 1 Mutagenic Primers Used to Create H638G MMLV
RT, Y586A MMLV RT, and D653N MMLV RT. "F" and "R" refer to
"forward" and "reverse" primers, respectively. H638G-F (SEQ ID NO.:
3) CTTAGCATAATCCATTGTCCCGGGGGTCAAAAGGGACACAGCGC; H638G-R (SEQ ID
NO.: 4) GCGCTGTGTCCCTTTTGACCCCCGGGACAATGGATTATGCTAAG; Y586A-F (SEQ
ID NO.: 5) GAAGCTAAATGTTTATACTGATTCCCGGGCTGCTTTTGCTACTGCCC; Y586A-R
(SEQ ID NO.: 6) GGGCAGTAGCAAAAGCAGCCCGGGAATCAGTATAAACATTTAGCTTC;
D653N-F (SEQ ID NO.: 7) GGCAACCGGATGGCTAACCAAGCGGCCCGAAAG; D653N-R
(SEQ ID NO.: 8) CTTTCGGGCCGCTTGGTTAGCCATCCGGTTGCC; D524E-F (SEQ ID
NO.: 9) CACACCTGGTACACGGAAGGAAGCAGTCTCTTAC; D524E-R (SEQ ID NO.:
10) GTAAGAGACTGCTTCCTTCCGTGTACCAGGTGTG; D524N-F (SEQ ID NO.: 11)
CACACCTGGTACACGAATGGAAGCAGTCTCTTAC; D524N-R (SEQ ID NO.: 12)
GTAAGAGACTGCTTCCATTCGTGTACCAGGTGTG; E562D-F (SEQ ID NO.: 13)
CGCTCAGCGGGCTGATCTGATAGCACTCACCC; and E562D-R (SEQ ID NO.: 14)
GGGTGAGTGCTATCAGATCAGCCCGCTGAGCG.
Example 2
[0073] Creation of plasmids expressing F155Y MMLV RT, R301L MMLV
RT, and F309A MMLV RT. Clones F155Y MMLV RT, R301L MMLV RT, and
F309A MMLV RT were created using pSE380 containing the MMLV RT gene
(pSE380-MMLV RT) and the mutagenic primers given in Table 2.
Amplification of the mutant sequences was accomplished via PCR
using the Quick Change mutagenesis kit (Stratagene). The resulting
PCR product was transformed and plated onto solid media containing
ampicillin. Plasmid DNA from selected clones was prepared with the
QIAprep Spin Miniprep Kit. For each mutant, sequencing across the
MMLV gene confirmed the desired mutations.
TABLE-US-00002 TABLE 2 Mutagenic Primers Used to Create F155Y MMLV
RT, R301L MMLV RT, and F309A MMLV RT. "F" and "R" refer to
"forward" and "reverse" primers, respectively. F155Y-F (SEQ ID NO.:
15) GATTTAAAGGATGCCTATTTCTGCCTGAGACTC; F155Y-R (SEQ ID NO.: 16)
GAGTCTCAGGCAGAAATAGGCATCCTTTAAATC; R301L-F (SEQ ID NO.: 17)
GACCCCTCGACAACTACTGGAGTTCCTAGGGACGGC; R301L-R (SEQ ID NO.: 18)
GCCGTCCCTAGGAACTCCAGTAGTTGTCGAGGGGTC; F309A-F (SEQ ID NO.: 19)
TCCTAGGGACGGCAGGCGCCTGTCGCCTCTGGATCCCTG; and F309A-R (SEQ ID NO.:
20) CAGGGATCCAGAGGCGACAGGCGCCTGCCGTCCCTAGGA.
Example 3
[0074] Creation of Multiple Mutated MMLV RT Enzymes: Combined
Polymerase and RNase H Mutations. To create combined MMLV RT
mutants, plasmids containing the single mutations were used as
templates for a second-round and/or third-round mutagenesis
reaction. For example, to create the F155Y;H638G MMLV RT mutant,
the following changes to the RT gene were made, beginning with
wild-type MMLV RT (Accession number J02255):
1) Wild-type MMLV RT gene.fwdarw.Change F155 to Y155
2) F155Y MMLV RT.fwdarw.Change H638 to G638
Example 4
[0075] Expression and Purification of MMLV RT Mutants. Plasmids
carrying each mutated MMLV RT gene were transformed into XL-1 Blue
E. coli cells. Single colonies were picked, and cultured overnight
in LB media containing Ampicillin. The next day, 5 ml of the
culture was used to inoculate 0.5-4 L of LB-Amp. Cells were grown
to A600-0.4 at -29.degree. C. with 250 rpm shaking, and then
induced with IPTG. After 12-16 hr growth at -29.degree. C., cell
pellets were harvested for purification.
[0076] The MMLV mutants may be isolated and purified using a
multitude of techniques known by the skilled artisan depending,
e.g., in the level of purity desired and the expected uses of the
MMLV. Examples of methods of purification include, e.g., crude
filtration, column purification, epitope tagging, isolation by
specific or non-specific binding to resins, selective secretion and
the like. In one example, purification of the MMLV RT mutants was
accomplished by resuspending frozen cells from cultures in a
buffered, ionically controlled solution, e.g., a buffer containing
20 mM KPi pH 7.0, 500 mM NaCl and a protease inhibitor, e.g., 1 mM
PMSF. The contents of the resuspended cells are then extracted
using standard methods, e.g., French press, shearing or even
lysozyme digestion 4 C for 30 min, followed by sonication or other
forms of mechanical stress. The cell debris may then be cleared by
centrifugation or filtration. Examples of well-known techniques for
cellular content release such as cellular permeabilization are
summarized in, e.g., U.S. Pat. No. 6,630,333, relevant portions
incorporated herein by reference.
[0077] Following the release of the hyperactive reverse
transcriptase enzymes of the present invention, whether alone or as
fusion proteins, a variety of protein purification techniques may
be followed that are well-known to one of ordinary skill in the
art. Suitable techniques for purification include, but are not
limited, e.g., ammonium sulfate and/or ethanol precipitation, acid
extraction, preparative gel electrophoresis, immunoadsorption,
anion or cation exchange chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity
chromatography, immunoaffinity chromatography, size exclusion
chromatography, liquid chromatography (LC), high performance LC
(HPLC), fast performance LC (FPLC), hydroxylapatite chromatography,
lectin chromatography, binding to Glutathione-S-Transferase-resin
(GST-resin), Maltose-resin and immobilized metal affinity
chromatography (IMAC). Generally, the hyperactive reverse
transcriptase will be purified by a combination of liquid
chromatographic techniques including ion exchange, affinity and
size exclusion. When using a tagged fusion protein, the hyperactive
reverse transcriptase may be released by, e.g., protease digestion
of a linker, addition of a competitor (GST or Maltose), addition of
a chelating agent (IMAC) and the like, depending on the system
used.
[0078] Alternative chromatographic solid supports, mobile phases
and associated methods may be equivalently used and will be
well-known to one of ordinary skill of protein isolation and
purification. The invention thus provides for substantially
isolated and purified hyperactive reverse transcriptase.
Substantially pure a used herein refers to a preparation or sample
which is substantially free of contaminating components, proteins,
etc., which may adversely affect the activity or performance of the
hyperactive reverse transcriptase in the use of the enzyme such as
in amplification or synthesis. If the hyperactive reverse
transcriptase if produced as a fusion protein, the skilled artisan
may select any of a number of well-known fusion partners, e.g.,
GST, MBP, FLAG, myc, His or other tagging methodologies and/or
techniques. Depending on the fusion partner and/or tag, the
supernatant is loaded on the appropriate column under specific
ionic and buffering conditions and the hyperactive RT protein
allowed to bind, followed by isolation of the hyperactive RT of, or
from, the fusion peptide/protein carrier. For example, in some
cases the hyperactive RT remains on the column and the fusion
protein/peptide carrier is in the flow-through or vice versa, as
will be known to the skilled artisan. In some embodiment, the
hyperactive RT may still be used while on or about the resin, that
is, as a hyperactive RT resin.
Example 5
[0079] Measurement of RT-associated RNase H Activities.
Characterization of the RNase H activity of the MMLV RT mutants was
shown using an assay that reports cleavage of RNA from an RNA:DNA
hybrid. Briefly, a hybrid substrate was created by annealing a 1.5
kb RNA with an internal 20-base DNA oligonucleotide to a sequence
that is 500 bp from the end of the RNA. Scission of the RNA results
in two fragments, 1.0 kb and 0.5 kb, which are resolved and
quantified on an RNA LabChip. The assay (5.mu.) contains 2 .mu.M
DNA, 100 ng/.mu.l RNA, in 1.times.RT buffer. RNase H activity of
each mutant MMLV RT was compared by monitoring the fraction of
cleavage as a function of time.
[0080] Table 4 shows the RNase H activity of MMLV RT Mutants. RNase
H activity is expressed as the number of polymerase unit enzyme
equivalents that achieved 50% cleavage of the RNA:DNA hybrid
substrate. In most cases, cleavage was quantified at -20-35%
cleavage (when the assay was truly linear with respect to time and
input protein) and extrapolated to 50% to provide a convenient
mathematical reference point. ND=Not detected.
TABLE-US-00003 TABLE 4 # Pol Units to (U/.mu.g) Achieve % of
Specific RNase H Source 50% Cleavage wt-MMLV Activity* E. coli
RNase H (Ambion- 0.005 Cloned) AMV (Ambion) 2.0 35 MMLV-RT (Ambion)
0.7 100 200 Powerscript (Clontech) ND <<<<1%** 100
U/rxn Superscript II (Invitrogen) ND <<<<1%** 400 U/rxn
D524E MMLV RT 5 U/rxn ND ~1% 121 D524N MMLV RT 5 U/rxn ND <1%
147 E562D MMLV RT 5 U/rxn 23 ~3% 135 D653N MMLV RT 4.5 16 192 H638G
MMLV RT 5.2 13 236 F155Y; H638G-MMLV RT 7.0 10 240 F155Y MMLV RT
0.8 85 240 *Units/.mu.g protein. **No detectable activity.
Example 6
[0081] Analysis of cDNA Product Synthesis by MMLV RT Mutants. An
important property of RT enzymes is that they are able to synthesis
cDNA products from mRNA that faithfully maintains the original
information content of the transcript. In other words, these
enzymes should exhibit a high apparent processivity. Several of the
MMLV RT single mutants (D524E, D524N, E562D, H638G, D653N, R301L
and F309A) were compared with wild type MMLV-RT (Ambion),
wt-MMLV-RT-His and SuperScript II and III for their ability to make
long cDNA products using Ambion's Millenium Marker RNA templates
(ranging in size from 0.5 kb to 9.0 kb). The results are shown in
FIG. 4, which is a gel that shows a comparison of cDNA product
lengths by various mutant MMLV RT enzymes using RNA templates from
0.5 to 9.0 kb in size.
[0082] Briefly, 20 .mu.l reactions included 500 ng of Millenium
marker and 10 picomole of oligo dT (annealing at 70.degree. C. for
5 min and cool to 42.degree. C.), 250 .mu.M dGTP, dCTP, dTTP, 25
.mu.M dATP, 0.5 .mu.l .alpha.-.sup.32P dATP (3000 .mu.Ci/mM), 8
units of RIP and the indicated amount (10 or 100 U) of MMLV-RT
enzyme in Ambion's RetroScript buffer. The reaction was incubated
at 42.degree. C. for 1 hr and stopped by heating at 95 C for 5 min.
A total of 5 .mu.l of sample was mixed with an equal volume of
glyoxal loading dye, heated at 50.degree. C. for 30 min, and
resolved on 1% Agarose-glyoxal gel. The products were transferred
to a nylon membrane prior to exposure to film. Lanes 1 and 11:
Ambion MMLV-RT; lanes 2 and 12 MMLV-RT-His; lanes 3 and 13 D524E
MMLV RT; lanes 4 and 14 D524N MMLV RT; lane 5 and 15: E562D MMLV
RT; lanes 6 and 16: H638G MMLV RT; lanes 7 and 17: D653N MMLV RT;
lanes 8 and 18: R301L MMLV RT; lanes 9 and 19: F309A MMLV RT; lanes
10 and 20: SuperScript II. Lane 18* has only 20 U D653N MMLV RT
instead of 100 U owing the lower stock concentration of this
mutant.
[0083] Other MMLV RT mutants were characterized in similar assays.
For example, cDNA products by F155Y MMLV RT, H638G MMLV RT, D524E
MMLV RT, D524N MMLV RT, E562D MMLV RT, D653N MMLV RT and the double
mutant F155Y;H638G MMLV RT were assayed in a reaction that uses a
higher concentration of evenly balanced dNTP's and a single, 9.0 kb
RNA template. FIG. 5 is a gel that shows a comparison of cDNA
product lengths by various mutant MMLV RT enzymes using a 9.0 kb
RNA template. For these reactions, template-primer was incubated
for 4 min at 70.degree. C., and then added to the reaction
mastermix at 42.degree. C. Reactions were initiated by adding 50U
of enzyme and incubating for 30 min at 42.degree. C. Residual,
unhybridized RNA template was removed by treating all reactions
with 500 pg/.mu.l bovine RNase A treat in combination with a
1:10,000 dilution of SYBR Gold (to stain the cDNA products).
Samples were treated with RNase for 30 min at 37.degree. C. A total
of 1 .mu.L of 10.times.DNA loading dye was added, and one-half of
the reaction mixture loaded onto 0.7% agarose gel. The cDNA
products were resolved after electrophoresis for 40 min at 90V.
[0084] The top half of the gel in FIG. 5 shows the cDNA products
with these RT enzymes in a buffer containing 200 ng 9 kb RNA
template, 5 .mu.M oligo dT primer, 50 mM Tris pH 8.3, 75 mM KCl, 5
mM DTT, 0.5 mM of each dNTP, 10U RIP, and 50 U RT enzyme in a 20
.mu.l reaction volume. The bottom half of the gel shows cDNA
products from identical reactions, except that 3 mM MnCl.sub.2 was
used instead of 3 mM MgCl.sub.2. This change in the divalent ion
changes the product profile significantly, since Mn.sup.2+ is known
to dramatically enhance RNase H activity. As a result, Mn.sup.2+
causes the cDNA products to be shorter in proportion to the extent
of RNase H activity extant in each RT. As a result, those mutants
with the greatest amount of RNase H activity make the shortest cDNA
products, and those enzymes that have even 10-15% RNase H activity
are readily distinguished from enzymes with <1% RNase H
activity.
[0085] It is significant to note that although the F155Y;H638G MMLV
RT mutant fails to demonstrate an increase in this cDNA length
assay with this limited length template (9 kb), it was found to
outperform all other MMLV RT mutant enzymes in RNA
amplification.
Example 7
[0086] RNA Amplification Properties of MMLV RT Mutants. The RNA
amplification reagents used were from Ambion's MessageAmp kit
(Ambion, Inc., Austin, Tex., Cat#1750). The reactions were
performed according to the instruction manual with 100 units of RT
and indicated amount of template. Briefly, the MessageAmp (Ambion,
Inc., Austin, Tex.) procedure is based on antisense RNA (aRNA)
amplification and involves a series of enzymatic reactions
resulting in linear amplification of exceedingly small amounts of
RNA for use in array analysis. Unlike exponential RNA amplification
methods, such as NASBA and RT-PCR, aRNA amplification maintains
representation of the starting mRNA population.
[0087] The procedure begins with total or poly(A) RNA that is
reverse transcribed using a primer containing both oligo(dT) and a
T7 RNA polymerase promoter sequence. After first-strand synthesis,
the reaction is treated with RNase H to cleave the mRNA into small
fragments. These small RNA fragments serve as primers during a
second-strand synthesis reaction that produces a double-stranded
cDNA template for transcription. Contaminating rRNA, mRNA fragments
and primers are removed and the cDNA template is then used in a
large scale in vitro transcription reaction to produce linearly
amplified aRNA. The aRNA can be labeled with biotin rNTPS or amino
allyl-UTP during transcription. Alternatively, unlabeled aRNA can
be used as a template for a reverse transcription with
CyDye.TM.-labeled dNTPs to generate labeled cDNA. The
RETROscript.TM. Kit (Ambion, Inc.) may be used for this purpose.
For increased yields, the aRNA can also be used as template for
cDNA synthesis followed by a second round of amplification using
MessageAmp.
[0088] FIG. 6A and FIG. 6B are graphs of a single round RNA
amplification with: 100 ng (FIG. 6A) or 1 .mu.g (FIG. 6B) Rat
Thymus Total RNA, comparing different MMLV RT mutants versus
wild-type. The aRNA yields were determined by UV absorbance at 260
nm. Samples were performed in duplicate compares the aRNA yields
from each of the MMLV RT mutants. Significantly, when using 100 ng
total RNA, the double mutant F155Y;H638G MMLV RT produced about 3
to 5 times more aRNA than several other enzymes tested, such as
MMLV RT (Ambion), AMV RT (Ambion), and Superscript II (Invitrogen).
In a separate study using 100 ng of human HeLa-S3 cell total RNA,
the F155Y;H638G MMLV RT produced 1.5 to 2.2 times more aRNA
compared to other RTs, including AMV RT and SSII, after one round
of amplification. FIG. 7 is a graph of a single round RNA
amplification with 100 ng HeLa S-3 Total RNA, again comparing
different MMLV RT Mutants versus the wild-type enzyme, and AMV RT.
The aRNA yields were determined by UV absorbance at 260 nm and were
performed in duplicate. Although the F155Y;H638G MMLV RT produced
the most aRNA in this experiment, it is important to note that the
single mutant H638G MMLV RT produced almost as much aRNA as the
F155Y;H638G double mutant, and thus represents a noteworthy
improvement over the currently available reverse transcriptase
tools.
[0089] Comparable 2- to 4-fold enhancements in aRNA yield by
F155Y;H638G MMLV RT were observed in two round RNA amplification
reactions, starting with 1 ng, 100 pg, or 10 pg total HeLa-S3 RNA.
FIG. 8 is a graph that shows a two round RNA amplification with
10-1000 pg HeLa S-3 Total RNA, again comparing different MMLV RT
Mutants versus wild-type. AMV=Avian Myeloblastosis Virus RT;
FYHG=F155Y;H638G MMLV RT; SSII=SuperScript II. The aRNA yields were
determined by UV absorbance at 260 nm and were performed in
duplicate.
[0090] In another study, the aRNA yield by F155Y; H638G MMLV RT was
compared in MessageAMP with AMV RT using 100 ng and 1 .mu.g input
total RNA. As shown in Table 6, F155Y;H638G MMLV RT produced 12%
more aRNA from 1 .mu.g of total RNA, and 2.6-fold more aRNA from
100 ng total RNA (both at 200 U F155Y;H638G MMLV RT).
TABLE-US-00004 TABLE 5 Yields of aRNA by F155Y; H638G MMLV RT
compared to AMV RT at 100 ng and 1 .mu.g of HeLa-S3 Total RNA. RT,
Units 1 .mu.g 100 ng F155Y; H638G, 100 U 56.2 6.1 F155Y; H638G, 100
U 54.1 6.6 F155Y; H638G, 100 U 54.0 7.7 F155Y; H638G, 200 U 66.6
17.6 F155Y; H638G, 200 U 65.8 16.2 F155Y; H638G, 200 U 59.6 16.5
Wild-type AMV 56.8 6.2 Wild-type AMV 57.9 16.5
[0091] In another study, the aRNA yield by F155Y;H638G MMLV RT was
compared with AMV RT in a two round amplification using MessageAMP
using 10 pg input total HeLa-S3 RNA. FIG. 9 is graph that shows the
yield from a two round RNA amplification comparing F155Y;H638G MMLV
RT and AMV RT. The input total RNA was HeLa-S3, at 1 ng and 10 pg.
As shown in FIG. 9, F155Y;H638G MMLV RT produced 22-fold more aRNA
than the RT provided in the kit, and nearly 40-fold more aRNA than
wild-type AMV RT.
[0092] In yet another study, the aRNA yield by F155Y;H638G MMLV RT
was compared with the Affymetrix aRNA standard protocol which
recommends SuperScript II. In this case, the F155Y;H638G MMLV RT
mutant generated 7% more aRNA from 1 .mu.g of total RNA (HeLa-S3),
or 20% more aRNA from 100 ng of total RNA, in a single round of RNA
amplification. FIG. 10 is a graph that shows a comparison of the
present invention with the Standard Affymetrix aRNA Protocol
Comparison to MessageAMP containing F155Y;H638G MMLV RT. An input
1000 and 100 ng of HeLa-S3 total RNA was amplified using the
Affymetrix aRNA Protocol or Ambion's MessageAMP protocol using
F155Y;H638G MMLV RT. aRNA labeling was accomplished through 8 hr
biotin CTP/UTP IVT reactions. All reactions were performed in
duplicate. Average peak sizes of the aRNA exceed 1700 nucleotides
in each case. SSII=SuperScript II; DM=Double mutant, F155Y;H638G
MMLV RT.
Example 8
[0093] Performance of aRNA produced by F155Y;H638G MMLV RT and SSII
on Affymetrix GeneChips. RNA amplified in a single round by
F155Y;H638G MMLV RT (using the MessageAMP II protocol (see
www.ambion.com)) or the RT provided in the kit (using the
Affymetrix aRNA protocol (see www.Affymetrix.com)) was
biotin-labeled and hybridized to an Affymetrix Human Focus Array
for detection and analysis. The concordance between the two RT
enzymes was 93.69% using all 8794 elements on the array. FIG. 11 is
a scatter plot of F155Y;H638G MMLV RT (x axis) vs. SSII (y axis)
Signal Intensities from a Human Focus Array. The signal intensity
correlation is shown. Lines represent 2-, 3-, 10- and 30-fold
differences. Red dots are Present-Present calls, Black are
Absent-Absent calls, and Dark Blue are Present-Absent (or
Absent-Present). This is graph is used to visualize the concordance
between arrays. The region on the top, right half contains the P
calls (most important). DM=Double Mutant, F155Y;H638G MMLV RT.
[0094] Elimination of Absent and Marginal calls increases the
concordance substantially. The average signal was 1348 for
F155Y;H638G MMLV RT, and 1291 for the Affymetrix standard protocol.
Percent present calls were slightly higher for F155Y;H638G MMLV RT
than the standard protocol (Table 6), whereas the beta-actin ratio
was also slightly more favorable for F155Y;H638G MMLV RT than the
RT provided in the kit (Table 7).
TABLE-US-00005 TABLE 6 Percent Present Calls on the Human Focus
Array by RT Enzyme RT Enzyme % Present Calls F155Y; H638G MMLV RT
54.0% SSII 53.8%
TABLE-US-00006 TABLE 7 3'/5' Ratios for GAPDH and beta-Actin Genes
on the Human Focus Array by RT Enzyme. SSII F155Y; H638G MMLV RT
GAPDH 0.79 0.80 Beta-Actin 1.24 1.12
[0095] While this invention has been described in reference to
illustrative embodiments, this description is not intended to be
construed in a limiting sense. Various modifications and
combinations of the illustrative embodiments, as well as other
embodiments of the invention, will be apparent to persons skilled
in the art upon reference to the description. It is therefore
intended that the appended claims encompass any such modifications
or embodiments.
Sequence CWU 1
1
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atc ctg cta cag tac gtg 672Ala Asp Phe Arg Ile Gln His Pro Asp Leu
Ile Leu Leu Gln Tyr Val 210 215 220 gat gac tta ctg ctg gcc gcc act
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290 295 300 cta ggg acg gca ggc ttc tgt cgc ctc tgg atc cct ggg ttt
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cct tgg cgt cgg ccg gtg gcc tac ctg tcc aaa aag cta 1200Lys Leu Gly
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405 410 415 att gcc gta ctg aca aag gat gca ggc aag cta acc atg gga
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Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser 515 520
525 ctc tta caa gag gga cag cgt aag gcg gga gct gcg gtg acc acc gag
1632Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540 acc gag gta atc tgg gct aaa gcc ctg cca gcc ggg aca tcc
gct cag 1680Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser
Ala Gln 545 550 555 560 cgg gct gaa ctg ata gca ctc acc cag gcc cta
aag atg gca gaa ggt 1728Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu
Lys Met Ala Glu Gly 565 570 575 aag aag cta aat gtt tat act gat agc
cgt tat gct ttt gct act gcc 1776Lys Lys Leu Asn Val Tyr Thr Asp Ser
Arg Tyr Ala Phe Ala Thr Ala 580 585 590 cat atc cat gga gaa ata tac
aga agg cgt ggg ttg ctc aca tca gaa 1824His Ile His Gly Glu Ile Tyr
Arg Arg Arg Gly Leu Leu Thr Ser Glu 595 600 605 ggc aaa gag atc aaa
aat aaa gac gag atc ttg gcc cta cta aaa gcc 1872Gly Lys Glu Ile Lys
Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala 610 615 620 ctc ttt ctg
ccc aaa aga ctt agc ata atc cat tgt ccc ggg ggt caa 1920Leu Phe Leu
Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly Gly Gln 625 630 635 640
aag gga cac agc gcc gag gct aga ggc aac cgg atg gct gac caa gcg
1968Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655 gcc cga aag gca gcc atc aca gag act cca gac acc tct acc
ctc ctc 2016Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr
Leu Leu 660 665 670 cac cac cac cac cac cac taa 2037His His His His
His His 675 2678PRTMoloney murine sarcoma virus 2Met Thr Leu Asn
Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys 1 5 10 15 Glu Pro
Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln 20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro 35
40 45 Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
Gln 50 55 60 Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro
His Ile Gln 65 70 75 80 Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys
Gln Ser Pro Trp Asn 85 90 95 Thr Pro Leu Leu Pro Val Lys Lys Pro
Gly Thr Asn Asp Tyr Arg Pro 100 105 110 Val Gln Asp Leu Arg Glu Val
Asn Lys Arg Val Glu Asp Ile His Pro 115 120 125 Thr Val Pro Asn Pro
Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His 130 135 140 Gln Trp Tyr
Thr Val Leu Asp Leu Lys Asp Ala Tyr Phe Cys Leu Arg 145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro 165
170 175 Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
Gly 180 185 190 Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His
Arg Asp Leu 195 200 205 Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile
Leu Leu Gln Tyr Val 210 215 220 Asp Asp Leu Leu Leu Ala Ala Thr Ser
Glu Leu Asp Cys Gln Gln Gly 225 230 235 240 Thr Arg Ala Leu Leu Gln
Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser 245 250 255 Ala Lys Lys Ala
Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr 260 265 270 Leu Leu
Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr 275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe 290
295 300 Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
Glu 305 310 315 320 Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly
Thr Leu Phe Asn 325 330 335 Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln
Glu Ile Lys Gln Ala Leu 340 345 350 Leu Thr Ala Pro Ala Leu Gly Leu
Pro Asp Leu Thr Lys Pro Phe Glu 355 360 365 Leu Phe Val Asp Glu Lys
Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln 370 375 380 Lys Leu Gly Pro
Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu 385 390 395 400 Asp
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala 405 410
415 Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430 Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys
Gln Pro 435 440 445 Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His
Tyr Gln Ala Leu 450 455 460 Leu Leu Asp Thr Asp Arg Val Gln Phe Gly
Pro Val Val Ala Leu Asn 465 470 475 480 Pro Ala Thr Leu Leu Pro Leu
Pro Glu Glu Gly Leu Gln His Asn Cys 485 490 495 Leu Asp Ile Leu Ala
Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp 500 505 510 Gln Pro Leu
Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser 515 520 525 Leu
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu 530 535
540 Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560 Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met
Ala Glu Gly 565 570 575 Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr
Ala Phe Ala Thr Ala 580 585 590 His Ile His Gly Glu Ile Tyr Arg Arg
Arg Gly Leu Leu Thr Ser Glu 595 600 605 Gly Lys Glu Ile Lys Asn Lys
Asp Glu Ile Leu Ala Leu Leu Lys Ala 610 615 620 Leu Phe Leu Pro Lys
Arg Leu Ser Ile Ile His Cys Pro Gly Gly Gln 625 630 635 640 Lys Gly
His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala 645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660
665 670 His His His His His His 675 344DNAArtificialoligonucleotide
primer 3cttagcataa tccattgtcc cgggggtcaa aagggacaca gcgc
44444DNAArtificialoligonucleotide primer 4gcgctgtgtc ccttttgacc
cccgggacaa tggattatgc taag 44547DNAArtificialoligonucleotide primer
5gaagctaaat gtttatactg attcccgggc tgcttttgct actgccc
47647DNAartificialoligonucleotide sequence 6gggcagtagc aaaagcagcc
cgggaatcag tataaacatt tagcttc 47733DNAArtificialoligonucleotide
sequence 7ggcaaccgga tggctaacca agcggcccga aag
33833DNAArtificialoligonucleotide primer 8ctttcgggcc gcttggttag
ccatccggtt gcc 33934DNAArtificialoligonucleitide primer 9cacacctggt
acacggaagg aagcagtctc ttac 341034DNAArtificialoligonucleotide
primer 10gtaagagact gcttccttcc gtgtaccagg tgtg
341134DNAArtificialoligonucleotide primer 11cacacctggt acacgaatgg
aagcagtctc ttac 341234DNAArtificialoligonucleotide primer
12gtaagagact gcttccattc gtgtaccagg tgtg
341332DNAartificialoligonucleotide primer 13cgctcagcgg gctgatctga
tagcactcac cc 321432DNAartificialoligonucleotide primer
14gggtgagtgc tatcagatca gcccgctgag cg
321533DNAArtificialoligonucleotide primer 15gatttaaagg atgcctattt
ctgcctgaga ctc 331633DNAArtificialoligonucleotide primer
16gagtctcagg cagaaatagg catcctttaa atc
331736DNAartificialoligonucleotide primer 17gacccctcga caactactgg
agttcctagg gacggc 361836DNAartificialoligonucleotide primer
18gccgtcccta ggaactccag tagttgtcga ggggtc
361939DNAartificialoligonucleotide primer 19tcctagggac ggcaggcgcc
tgtcgcctct ggatccctg 392039DNAartificialoligonucleotide primer
20cagggatcca gaggcgacag gcgcctgccg tccctagga 39
* * * * *
References