U.S. patent application number 14/745012 was filed with the patent office on 2016-01-21 for cdna synthesis improvements.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Christian Gruber, Joel Jessee, Wu-Bo LI.
Application Number | 20160017391 14/745012 |
Document ID | / |
Family ID | 22402470 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160017391 |
Kind Code |
A1 |
LI; Wu-Bo ; et al. |
January 21, 2016 |
cDNA Synthesis Improvements
Abstract
The present invention generally relates to methods of making
cDNA molecules and cDNA libraries. The invention also relates to
cDNA molecules and cDNA libraries produced according to these
methods, as well as to vectors and host cells containing such cDNA
molecules and libraries. The invention also relates to kits for
making the cDNA molecules and libraries of the invention.
Inventors: |
LI; Wu-Bo; (N. Potomac,
MD) ; Jessee; Joel; (Mount Airy, MD) ; Gruber;
Christian; (Frederick, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
22402470 |
Appl. No.: |
14/745012 |
Filed: |
June 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14276944 |
May 13, 2014 |
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14745012 |
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13018419 |
Jan 31, 2011 |
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14276944 |
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12623375 |
Nov 21, 2009 |
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13018419 |
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11480575 |
Jul 5, 2006 |
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12623375 |
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09515513 |
Feb 29, 2000 |
7074556 |
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11480575 |
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60122395 |
Mar 2, 1999 |
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Current U.S.
Class: |
506/26 ; 435/188;
435/91.51 |
Current CPC
Class: |
C12P 19/34 20130101;
C07K 16/40 20130101; C12N 15/1096 20130101 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A method for synthesizing one or more cDNA molecules or
population of cDNA molecules comprising: mixing at least one mRNA
or poly A RNA template or population of such templates with at
least one polypeptide having reverse transcriptase activity; and
incubating said mixture under conditions sufficient to increase the
amount or percentage of full-length cDNA molecules synthesized.
2. The method of claim 1, wherein said conditions reduce or
substantially reduce internal priming.
3. The method of claim 1, wherein said polypeptide is a reverse
transcriptase selected from the group consisting of M-MLV RT, RSV
RT, AMV RT, RA V RT, MA V RT, and HIV RT, and derivatives,
fragments, mutations and variants thereof.
4. The method of claim 3, wherein said reverse transcriptase is
reduced or substantially reduced in RNase H activity.
5. The method of claim 2, wherein said conditions comprise
annealing or hybridizing one or more primers to said templates at
elevated temperatures.
6. The method of claim 5, wherein said elevated temperature ranges
from about 20.degree. C. to about 90.degree. C.
7. The method of claim 2, wherein said conditions comprise lowering
the amount of primer relative to the amount of said template.
8. The method of claim 7, wherein the ratio of said primer to said
template ranges from about 5:1 to about 1:20.
9. The method of claim 1, wherein said conditions comprise the use
of an inhibitor of the polypeptide having reverse transcriptase
activity.
10. The method of claim 9, wherein said inhibitor is an antibody or
antibody fragment.
11. The method of claim 10, wherein said antibody or antibody
fragment is polyclonal or monoclonal.
12. The method of claim 2, wherein said conditions comprise the use
of a primer having a high specificity.
13. The method of claim 2, wherein said conditions comprise
increasing the length of said primer.
14. The method of claim 13, wherein the length of said primer which
hybridizes to said template ranges from about 20 bases to about 60
bases.
15. The method of claim 1, wherein said method further comprises
incubating at least one of said cDNA molecules under conditions
sufficient to make at least one second nucleic acid molecule
complementary to all or a portion of said at least one cDNA
molecule, thereby producing one or more double stranded cDNA
molecules.
16. The method of claim 15, wherein said conditions for making said
second nucleic acid molecule increases the amount or percentage of
full-length double stranded cDNA molecules.
17.-28. (canceled)
29. A composition for making an increased amount or percentage of
full-length cDNA comprising at least one component selected from
the group consisting of one or more primers, one or more reverse
transcription inhibitors, one or more reverse transcription
enzymes, one or more nucleotides, one or more cap binding
molecules, one or more reverse transcription buffers.
30.-32. (canceled)
33. The composition of claim 29, wherein said one or more reverse
transcriptase inhibitors is an antibody or antibody fragment.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit, under 35 U.S.C.
.sctn.119(e), of U.S. Provisional Application No. 60/122,395, filed
on Mar. 2, 1999, which is fully incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the field of molecular and
cellular biology. The invention generally relates to methods of
synthesizing cDNA. More specifically, the present invention relates
to methods of increasing the average cDNA insert size and more
particularly, to increasing the percentage of full-length cDNA
present within cDNA libraries. Thus, the present invention provides
improved cDNA libraries useful in gene discovery.
[0003] In examining the structure and physiology of an organism,
tissue or cell, it is often desirable to determine its genetic
content. The genetic framework of an organism is encoded in the
double-stranded sequence of nucleotide bases in the
deoxyribonucleic acid (DNA) which is contained in the somatic and
germ cells of the organism. The genetic content of a particular
segment of DNA, or gene, is only manifested upon production of the
protein which the gene encodes. In order to produce a protein, a
complementary copy of one strand of the DNA double helix (the
"coding" strand) is produced by polymerase enzymes, resulting in a
specific sequence of ribonucleic acid (RNA). This particular type
of RNA, since it contains the genetic message from the DNA for
production of a protein, is called messenger RNA (mRNA).
[0004] Within a given cell, tissue or organism, there exist many
mRNA species, each encoding a separate and specific protein. This
fact provides a powerful tool to investigators interested in
studying genetic expression in a tissue or cell. mRNA molecules may
be isolated and further manipulated by various molecular biological
techniques, thereby allowing the elucidation of the full functional
genetic content of a cell, tissue or organism.
[0005] A common approach to the study of gene expression is the
production of complementary DNA (cDNA) clones. In this technique,
the mRNA molecules from an organism are isolated from an extract of
the cells or tissues of the organism. This isolation often employs
solid chromatography matrices, such as cellulose or agarose, to
which oligomers of thymidine (T) have been complexed. Since the 3'
termini on most eukaryotic mRNA molecules contain a string of
adenosine (A) bases, and since A binds to T, the mRNA molecules can
be rapidly purified from other molecules and substances in the
tissue or cell extract. From these purified mRNA molecules, cDNA
copies may be made using the enzyme reverse transcriptase (RT) or
DNA polymerases having RT activity, which results in the production
of single-stranded cDNA molecules. The single-stranded cDNAs may
then be converted into a complete double-stranded DNA copy (i.e., a
double-stranded cDNA) of the original mRNA (and thus of the
original double-stranded DNA sequence, encoding this mRNA,
contained in the genome of the organism) by the action of a DNA
polymerase. The protein-specific double-stranded cDNAs can then be
inserted into a vector, which is then introduced into a host
bacterial, yeast, animal or plant cell, a process referred to as
transformation or transfection. The host cells are then grown in
culture media, resulting in a population of host cells containing
(or in many cases, expressing) the gene of interest or portions of
the gene of interest.
[0006] This entire process, from isolation of mRNA to insertion of
the cDNA into a vector (e.g., plasmid, viral vector, cosmid, etc.)
to growth of host cell populations containing the isolated gene or
gene portions, is termed "cDNA cloning." If cDNAs are prepared from
a number of different mRNAs, the resulting set of cDNAs is called a
"cDNA library," an appropriate term since the set of cDNAs
represents a "population" of genes or portions of genes comprising
the functional genetic information present in the source cell,
tissue or organism. Genotypic analysis of these cDNA libraries can
yield much information on the structure and function of the
organisms from which they were derived.
[0007] The ability to increase the total amount of cDNA produced,
and more particularly to produce a cDNA libraries having an
increase in the average size of the cDNA molecules and/or to
produce cDNA libraries having an increase in the percentage of
full-length cDNA molecules would provide a significant advance in
cDNA library construction. Specifically, such advances would
greatly improve the probability of finding full-length genes of
interest.
[0008] Ideally, synthesis of a cDNA molecule initiates at or near
the 3' termini of the mRNA molecules. Priming of cDNA synthesis at
the 3' termini at the poly A tail using an oligo(dT) primer ensures
that the 3' message of the mRNAs will be represented in the cDNA
molecules produced. Priming which occurs within the mRNA molecules
(internal priming) results in synthesis of cDNA molecules which do
not contain the full-length message for the genes of interest. That
is, internal priming results in truncated cDNA molecules which
contain only a portion of the gene or genes of interest. Typically,
internal priming causes a loss of the 3' sequences from the message
population. Thus, internal priming lowers the total amount of cDNA
produced, decreases the average insert size of cDNA molecules for a
cDNA library and/or decreases the percentage of full-length cDNA
molecules in a given cDNA library. Sequencing analysis has
indicated that many eukaryotic mRNAs have internal poly adenylation
stretches which may serve as a priming site when an oligo(dT)
primer is used for first strand cDNA synthesis with reverse
transcriptase. Moreover, research has shown that some mRNAs can
have as many as 16 internal priming sites (Lovett, M., et al., The
construction of full-length cDNA libraries by conventional methods
and a novel double capture technique, University of Texas
Southwestern Medical Center, Dallas, Tex., presented at the
48.sup.th Annual Meeting held by The American Society of Human
Genetics, Oct. 27-31, 1998, Denver, Colo.). Thus, internal priming
of the primer to such internal poly A sequences may adversely
affect cDNA synthesis.
[0009] The present invention alleviates, prevents, reduces or
substantially reduces internal priming thereby providing
improvements in cDNA and cDNA library construction. Accordingly,
the present invention greatly facilitates gene discovery by
providing cDNA libraries containing a greater percentage of
full-length genes.
[0010] The present invention therefore relates to synthesizing a
cDNA molecule or molecules from an mRNA template or population of
mRNA templates under conditions sufficient to increase the total
amount of cDNA produced, increase the length of the cDNA molecules
produced, and/or increase the amount or percentage of full-length
cDNA molecules produced. In accordance with the invention, any
conditions which inhibit, prevent, reduce or substantially reduce
internal priming may be used. Such conditions preferably include
but are not limited to optimizing primer concentrations, optimizing
reaction temperatures and/or optimizing primer length or
specificity. Such result may also be accomplished in accordance
with the invention by optimizing the reverse transcription
reaction, preferably by inhibiting or preventing reverse
transcription until optimum or desired reaction conditions are
achieved.
[0011] Conventional methods for constructing cDNA libraries use a
molar ratio of oligo(dT) primer/mRNA template of 15:1 for first
strand cDNA synthesis. The use of such excess amounts of oligo(dT)
primer allows internal priming of one or more primers to one or
more of the mRNA templates in the reaction. According to a
preferred aspect of the present invention, the amount of oligo(dT)
primer is reduced for synthesis of first strand cDNA to inhibit,
prevent, reduce or substantially reduce internal priming. Preferred
molar ratios of primer to template range from about 12:1; 10:1;
9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1:1; 1:2; 1:3; 1:4; 1:5;
1:6; 1:7; 1:8; 1:9; 1:10 and 1:12. Preferably, molar ratios of
primer (e.g., oligo(dT)) to template (e.g., mRNA) range from about
5:1 to about 1:20, although lower molar ratios of primer to
template may be used in accordance with the invention.
Specifically, molar ratios of primer to template may be below about
1:10; 1:15; 1:20; 1:25; 1:50; 1:75; and 1:100. Preferably, ranges
of molar ratios are below about 5:1; 4:1; 3:1; 2:1; 1:1; 1:2; 1:3;
1:4; and 1:5. Most preferably, ratios of primer to template range
from about 10:1 to 1:10; 5:1 to 1:10; 4:1 to 1:10; 3:1 to 1:10;
2.5:1 to 1:10; 2:1 to 1:10; 1.5:1 to 1:10; and 1:1 to 1:10. The
optimum ratios of primer to template may vary depending on the
primer, mRNA, reverse transcription enzyme and reaction conditions
(annealing temperature, buffering salts, etc.). The desired primer
to template ratios can be readily determined by one skilled in the
art.
[0012] In conventional methods of cDNA library construction,
annealing or hybridizing primer to template is not carried out at a
temperature which prevents, inhibits, reduces or substantially
reduces internal priming. Typically, the mixture (e.g., mRNA and
oligo(dT) primer) is chilled on ice after denaturation or heating.
This process typically causes annealing or hybridization of the
primer to internal sites. According to a preferred aspect of the
present invention, the temperature during the annealing or
hybridization between the primer and the template is maintained so
that internal priming is inhibited, prevented, reduced or
substantially reduced. In accordance with the invention, such a
result is accomplished by carrying out primer annealing or
hybridization at higher temperatures. Such conditions may also
reduce the formation of mRNA secondary structures during cDNA
synthesis. Preferably, temperatures for annealing or hybridizing
primers to the templates range from about 10.degree. C. to about
90.degree. C.; more preferably about 10.degree. C. to about
80.degree. C.; still more preferably about 20.degree. C. to about
75.degree. C.; more preferably about 25.degree. C. to about
75.degree. C.; still more preferably about 30.degree. C. to about
65.degree. C.; still more preferably about 37.degree. C. to about
60.degree. C.; still more preferably about 40.degree. C. to about
60.degree. C.; still more preferably about 45.degree. C. to about
60.degree. C.; still more preferably about 45.degree. C. to about
55.degree. C.; and most preferably about 45.degree. C. to about
65.degree. C. The temperature used may vary depending on the type
and amount of primer and template and depending on the temperature
optimum of the reverse transcription enzyme. The optimum
temperature or temperature ranges can be readily determined by one
skilled in the art.
[0013] Conventional methods for cDNA synthesis typically requires
the use of oligo(dT) primers of a particular length (12-18 bases or
mer). Such primer length, however, lowers specificity of the primer
thereby allowing internal priming. Thus, the invention also relates
to increasing specificity of the primers to prevent, inhibit,
reduce or substantially reduce internal priming. In a preferred
aspect, primer specificity is increased by increasing the length of
the primer. Thus, for cDNA synthesis, longer oligo(dT) primers may
be used in accordance with the invention. Preferably, primer length
ranges from about 20 to about 100 bases, about 20 to about 75
bases, about 20 to about 60 bases, and about 20 to about 50 bases;
more preferably about 20 to about 45 bases; more preferably about
20 to about 40 bases; and most preferably about 25 to about 35
bases. In a preferred aspect, the length of the primers are greater
than 19 bases; more preferably greater than about 20 bases; more
preferably greater than about 25 bases; and still more preferably
greater than about 30 bases. Such primer lengths refer to the
length of the primers which anneal or hybridize to the template.
Optimum length and content (nucleotide sequence) of the primers may
vary depending on the type of template, the desired reaction
conditions, and the reverse transcription enzyme. In accordance
with the invention, additional sequences and/or modified
nucleotides may be included in the primers of the invention. For
example, additional sequences (which do not necessarily anneal or
hybridize to the template) may be included in the primers of the
invention to assist in cDNA synthesis including sequences
comprising one or more restriction endonuclease sites, one or more
derivative nucleotides (e.g., hapten containing nucleotides such as
biotinylated nucleotides), and the like. The type and length of the
primers used in accordance with the invention can be readily
determined by one or more skilled in the art.
[0014] Conventional cDNA synthesis methods do not control or vary
activity of the reverse transcription enzyme to optimize the
reverse transcription reaction. In accordance with the invention,
the activity of the reverse transcriptase is preferably controlled
to start synthesis at a desired time in the reaction. In a
preferred aspect, reverse transcriptase activity is inhibited or
prevented until optimum or desired reaction conditions are
achieved. Such a result is accomplished in accordance with the
invention by the use of inhibitors (such as antibodies or antibody
fragments) which inhibit reverse transcriptase activity. Such
reverse transcriptase inhibitors prevent or inhibit reverse
transcriptase activity at low temperatures such that internal
priming is prevented, inhibited, reduced or substantially reduced.
In accordance with the invention, such inhibitors preferably
prevent reverse transcriptase activity below 35.degree. C., below
40.degree. C., below 45.degree. C., below 50.degree. C., below
55.degree. C., below 60.degree. C., below 65.degree. C., below
70.degree. C., below 75.degree. C., below 80.degree. C., below
85.degree. C. and below 90.degree. C. Depending on the
thermostability of the enzyme having reverse transcriptase
activity, the inhibitor may be designed to inhibit activity of the
enzyme at a point at or near the temperature optimum for the enzyme
of interest Preferably, the inhibitor is inactivated at a
temperature below or near the temperature optimum of the enzyme
used, thereby allowing reverse transcription to take place. Thus,
the invention generally relates to the use of reverse transcriptase
inhibitors in cDNA synthesis. The type and amount of inhibitor may
vary depending on the type and amount of reverse transcription
enzyme and depending on the reaction conditions to be used. The
type of inhibitor and conditions used with such inhibitor can be
readily determined by one of ordinary skill in the art.
[0015] In accordance with the invention, any one or a combination
of the above improvements to cDNA synthesis may be used. Using any
one or a combination of these improvements provides for improved
first strand cDNA synthesis (e.g., more total cDNA, larger cDNA
and/or more full-length cDNA). In accordance with the invention,
the first strand cDNA molecules may be used as templates to make
one or more double stranded nucleic acid molecules (e.g., double
strand cDNA molecules) by incubating one or more of the first
strand cDNA molecules produced by the methods of the invention
under conditions sufficient to make one or more nucleic acid
molecules complementary to all or a portion of the first strand
cDNA molecules. Conditions for making double stranded nucleic acid
molecules preferably include incubation with one or more components
consisting of one or more DNA polymerases, one or more nucleotides,
one or more buffering salts, and one or more primers. In another
aspect of the invention, such conditions are modified to provide an
increase in the total amount of double stranded cDNA produced, an
increase in the length or size of the double stranded cDNA molecule
produced, and/or an increase in percentage full-length double
stranded cDNA molecule produced. Preferably, such conditions relate
to optimization of ribonuclease (RNase) digestion after first
strand cDNA synthesis. During first strand cDNA synthesis, if a
full-length cDNA molecule complementary to the mRNA template is not
made, a single stranded mRNA containing the cap structure will be
present at the 5' end of the mRNA of the mRNA/cDNA hybrid. If a
full-length cDNA is produced, a double stranded mRNA/cDNA hybrid is
produced with no single stranded mRNA present. Preferably, such
digestion conditions are optimized so that the single stranded mRNA
of the mRNA/cDNA double stranded molecules formed during first
strand cDNA synthesis is subject to RNase digestion. In this
manner, cap structure from mRNA/cDNA hybrids which are not
full-length are removed while full-length mRNA/cDNA hybrids will
retain the cap structure. Thus, cap capture can be used to select
for full-length molecules and select against molecules which are
not full-length. In a preferred aspect, the conditions are such
that the single stranded mRNA of the mRNA/cDNA hybrid is digested
or degraded while the mRNA of the double stranded mRNA/cDNA hybrid
is not degraded or not substantially degraded. Thus, such RNase
digestion is conducted under conditions such that second strand
synthesis is not substantially adversely affected. That is, second
strand synthesis in accordance with the invention produces larger
double stranded cDNA molecules compared to conventional techniques.
Conventional RNase I conditions typically range from 25 .mu.g to 40
u/.mu.g mRNA at 37.degree. C. and RNase A conditions typically are
1000 ng/.mu.g mRNA at 37.degree. C. Using conventional RNase
digestion, the average size of double stranded cDNA molecules
produced is about 200 bases. According to the present invention the
average size of double stranded cDNA molecules produced is
preferably greater than about 300 bases, greater than about 400
bases, greater than about 500 bases, greater than about 600 bases,
greater than about 700 bases, greater than about 800 bases, greater
than about 900 bases, greater than about 1 kilobase, greater than
about 1.5 kilobases, and greater than about 2 kilobases. In one
embodiment of the invention, the concentration of the ribonuclease,
the type of ribonuclease and reaction conditions are optimized to
improve double stranded cDNA synthesis in accordance with the
invention. Preferred ribonucleases for use in ribonuclease
digestions include ribonuclease A (RNase A) and/or ribonuclease I
(RNase I). Generally, lower temperatures (about 4.degree. C. to
about 50.degree. C.) and higher salt concentrations (about 5 mM to
about 5 M) will assist in inhibiting or controlling RNase digestion
in accordance with the invention. Salts used may include sodium
chloride, potassium, chloride, magnesium chloride, sodium acetate
etc. Additionally, lowering RNase amounts or concentrations may be
used to accomplish the desired result. Such concentrations for
RNase A may range from about 0.001 ng/.mu.g mRNA to about 500
ng/.mu.g of mRNA and for RNase I may range from about 0.001 u/.mu.g
mRNA to about 500 u/.mu.g mRNA. The incubation temperature, RNase
concentration and salt concentration may be readily determined by
one skilled in the art. In a preferred aspect, concentration of the
RNase A include ranges from 0.1 ng/.mu.g mRNA to 10 ng/.mu.g mRNA
in TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA) at 37.degree. C.
Alternatively, the concentration of the RNase A can include ranges
from 0.1 ng/.mu.g mRNA to 500 ng/.mu.g mRNA in 10 mM Tris, pH 7.5
buffer containing 250 mM NaCl at 25.degree. C. for 30 minutes.
Preferably, concentration of the RNase I used ranges from 0.1
unit/.mu.g mRNA to 1.0 unit/.mu.g mRNA in 10 mM Tris-HCl (pH 7.5),
5 mM EDTA (pH 8.0), 200 mM sodium acetate at 37.degree. C.
Alternatively, the concentration of the RNase I can be used at
ranges from 1.0 unit/.mu.g mRNA to 100 units/.mu.g mRNA in the same
buffer at 25.degree. C. for 30 minutes.
[0016] In another aspect, the invention relates to capture or
binding of the cap structure (e.g., m.sup.7GpppN) of the mRNA
before, during or after first strand cDNA synthesis. Thus, the
invention relates to selection of mRNA (before first strand
synthesis) or mRNA/cDNA hybrids (after or during first strand
synthesis) which have the cap structure in carrying out the methods
of the invention. Such selection or capture may be accomplished
with any cap binding molecule such as eIF4E, eIF4E peptides, eIF4E
peptide fragments (see WO 98/08865) and antibodies or antibody
fragments specific for cap structure. In a preferred aspect,
selection of the cap structure is accomplished after first strand
synthesis. More preferably, such cap capture occurs after
ribonuclease digestion in accordance with the methods of the
invention. For example, mRNA/cDNA hybrids subjected to ribonuclease
digestion are captured and then used for second strand cDNA
synthesis according to the invention.
[0017] Thus, the present invention is generally directed to methods
of synthesizing nucleic acid molecules. The present invention is
more specifically directed to methods of making one or more nucleic
acid molecules, especially cDNA molecules or cDNA libraries,
comprising mixing one or more nucleic acid templates (preferably
mRNA, poly A RNA or a population of mRNA molecules) with at least
one polypeptide having reverse transcriptase activity, and
incubating the mixture under conditions sufficient to make one or
more first nucleic acid molecules (e.g., first strand cDNA)
complementary to all or a portion of the one or more nucleic acid
templates. In accordance with the invention, such conditions
provide for an increased total amount of nucleic acid molecule
(cDNA) produced, compared to conventional procedures which do not
employ the improved modifications or conditions of the invention.
The invention also provides for an increase of length or average
size of the nucleic acid molecules (cDNA) produced and/or an
increase in the percentage or amount of full-length nucleic acid
molecules (cDNA) produced, compared to conventional procedures
which do not employ the improved modifications or conditions of the
invention. Determining the amount, length and full-length content
of the cDNA produced can be determined by conventional techniques
well known in the art and as described herein. The percentage or
average percentages of full-length cDNA in cDNA libraries produced
in accordance with the invention are preferably above about 15%,
more preferably above about 20%, more preferably above about 25%,
more preferably above about 30%, more preferably above about 40%,
more preferably above about 50%, more preferably above about 60%,
more preferably above about 70%, more preferably above about 80%
and most preferably above about 90%. Such full-length percentages
are preferably determined by random selection of a portion of the
clones of the cDNA library of interest (e.g., 100 to 1000 clones),
sequencing the clones and comparing the sequences to known sequence
data bases.
[0018] In preferred aspects of the invention, the improved results
of the invention are preferably accomplished by one or a
combination of modifications to the conditions for nucleic acid or
cDNA synthesis. Such conditions preferably include modifications
for improving first strand cDNA synthesis and/or improving second
strand cDNA synthesis.
[0019] In a preferred aspect, the invention specifically relates to
methods of making one or more double stranded cDNA molecules
comprising incubating one or more mRNA molecules (preferably a
population of mRNA molecules) with one or more primers of the
invention at temperatures and primer concentrations to prevent,
inhibit, reduce or substantially reduce internal priming prior to
or during first strand cDNA synthesis. Such reaction is preferably
conducted in the presence of one or more inhibitors of reverse
transcriptase activity in accordance with the invention.
Ribonuclease digestion is preferably conducted before second strand
cDNA synthesis and at ribonuclease concentrations sufficient to
increase the length, amount and/or size of double stranded cDNA
molecules produced during second strand synthesis. In accordance
with the invention, cap capture is preferably accomplished during
or after the ribonuclease digestion.
[0020] The invention is also directed to nucleic acid molecules and
cDNA molecules or populations of cDNA molecules (single or
double-stranded) produced according to the above-described methods
and to vectors (particularly expression vectors) comprising these
nucleic acid molecules and cDNA molecules. The invention also
relates to host cells containing such cDNA molecules and/or
vectors.
[0021] The invention is also directed to kits for use in the
methods of the invention. Such kits can be used for making single
or double-stranded nucleic acid molecules. The kits of the
invention comprise a carrier, such as a box or carton, having
therein one or more containers, such as vials, tubes, bottles and
the like. Such kits may comprise at least one component selected
from the group consisting of primers (preferably primers having
higher specificity and most preferably oligo(dT) primers having a
length equal to or greater than 20 bases), one or more polypeptides
having reverse transcriptase activity (reverse transcriptases and
DNA polymerases), one or more inhibitors of reverse transcription
(e.g., antibodies and antibody fragments directed against
polypeptides having RT activity), one or more cap binding molecules
(e.g., antibodies or antibody fragments directed against cap
structure), nucleic acid synthesis reaction buffers, one or more
nucleotides, one or more vectors, and instructions for carrying out
the methods of the invention.
[0022] The invention also relates to compositions for use in the
invention or made while carrying out the methods of the invention.
Such compositions may comprise at least one primer (e.g., oligo(dT)
or derivatives thereof) and at least one template in a sample or
reaction mixture in amounts or ratios in accordance with the
invention. Such composition may further comprise one or more
polypeptides having reverse transcriptase activity, one or more
reverse transcription inhibitors (e.g., anti-RT antibodies or
fragments thereof), one or more nucleotides, one or more cap
binding molecules (e.g., anti-cap antibodies for fragments
thereof), one or more buffering salts and the like. Such
compositions may also be maintained at a temperature to avoid
internal priming in accordance with the invention.
[0023] The compositions of the invention may also comprise amounts
of ribonuclease in accordance with the invention. Such compositions
may further comprise at least one component selected from one or
more mRNA/cDNA hybrids, one or more nucleotides, one or more
polypeptides having reverse transcriptase activity, one or more
buffering salts, one or more cap binding molecules (e.g., anti-cap
antibodies or fragments thereof) and the like.
[0024] The invention also relates to one or more antibodies
(monoclonal and polyclonal) and fragments thereof for use in the
methods, compositions and kits of the invention. Such antibodies,
include anti-cap and/or anti-RT antibodies and antibody
fragments.
[0025] Other preferred embodiments of the present invention will be
apparent to one of ordinary skill in the art in view of the
following drawings and description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is an autoradiograph of first strand cDNA synthesized
with SuperScript.TM. II (SS II) RT at 45.degree. C. with a 5/6 Kb
template with molar ratios of oligo(dT).sub.25-30/mRNA of 1:1,
2.5:1, 5:1, 10:1, and 50:1.
[0027] FIG. 2 is an autoradiograph of first strand cDNA synthesized
with ThermoScript.TM. II (TS II) RT at 45.degree. C., 50.degree. C.
and 55.degree. C. with a 5/6 Kb template with molar ratios of oligo
(dT).sub.25-30/mRNA of 1:1, 2.5:1, 5:1, 10:1, and 50:1.
[0028] FIG. 3 is an autoradiograph of first strand cDNA synthesized
with SS II RT using standard reaction temperatures and varying
reaction temperatures with a molar ratio of biotinylated-Not
I-oligo(dT).sub.25/mRNA of 0:1, 1:1 and 15:1.
[0029] FIG. 4 is an autoradiograph of first strand cDNA synthesized
with TS II RT using standard reaction conditions in which the
primer/template annealing is incubated on ice prior to cDNA
synthesis and using conditions according to the invention in which
annealing and the synthesis reaction temperatures are maintained
above 30.degree. C. (preferably above 37.degree. C.) with a molar
ratio of biotinylated-Not I-oligo(dT).sub.25/mRNA of 1:1 and 15:1.
Maintaining the annealing and reaction temperatures above
30.degree. C. (preferably above 37.degree. C.) in accordance with
the invention may also be referred to as "hot start."
[0030] FIG. 5 is an autoradiograph of second strand cDNA
synthesized using different amounts of RNase A.
[0031] FIG. 6 is an autoradiograph of second strand cDNA
synthesized using different amounts of RNase I.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0032] In order to provide a clearer and consistent understanding
of the specification and claims, including the scope to be given
such terms, the following definitions are provided.
[0033] Internal priming as used herein refers to hybridization or
annealing of one or more primers at one or more sites within one or
more mRNA molecules other than at the poly A tail located at the 3'
termini of the mRNA molecule.
[0034] Library as used herein refers to a set of nucleic acid
molecules (circular or linear) which is representative of all or a
portion or significant portion of the DNA content of an organism (a
"genomic library"), or a set of nucleic acid molecules
representative of all or a portion or significant portion of the
expressed genes (a "cDNA library") in a cell, tissue, organ or
organism. Such libraries may or may not be contained in one or more
vectors.
[0035] Vector as used herein refers to a plasmid, cosmid, phagemid
or phage DNA or other DNA molecule which is able to replicate
autonomously in a host cell, and which is characterized by one or a
small number of restriction endonuclease recognition sites at which
such DNA sequences may be cut in a determinable fashion without
loss of an essential biological function of the vector, and into
which DNA may be inserted in order to bring about its replication
and cloning. The vector may further contain one or more markers
suitable for use in the identification of cells transformed with
the vector. Markers, for example, include but are not limited to
tetracycline resistance or ampicillin resistance. Such vectors may
also contain one or more recombination sites, one or more
termination sites, one or more origins of replication, and the
like.
[0036] Primer as used herein refers to a single-stranded
oligonucleotide that is extended by covalent bonding of nucleotide
monomers during amplification or polymerization of a DNA molecule.
Preferred primers for use in the invention include oligo(dT)
primers or derivatives or variants thereof.
[0037] Oligonucleotide as used herein refers to a synthetic or
natural molecule comprising a covalently linked sequence of
nucleotides which are joined by a phosphodiester bond between the
3' position of the deoxyribose or ribose of one nucleotide and the
5' position of the deoxyribose or ribose of the adjacent
nucleotide.
[0038] Template as used herein refers to double-stranded or
single-stranded nucleic acid molecules which are to be amplified,
synthesized or sequenced. In the case of a double-stranded
molecules, denaturation of its stands to form a first and a second
strand is preferably performed before these molecules may be
amplified, synthesized or sequenced, or the double stranded
molecule may be used directly as a template. For single stranded
templates, a primer, complementary to a portion of the template is
hybridized or annealed under appropriate conditions and one or more
polymerases or reverse transcriptases may then synthesize a nucleic
acid molecule complementary to all or a portion of said template.
The newly synthesized molecules, according to the invention, may be
equal or shorter in length than the original template.
[0039] Incorporating as used herein means becoming a part of a DNA
and/or RNA molecule or primer.
[0040] Amplification as used herein refers to any in vitro method
for increasing the number of copies of a nucleotide sequence with
the use of a polymerase. Nucleic acid amplification results in the
incorporation of nucleotides into a DNA and/or RNA molecule or
primer thereby forming a new molecule complementary to a template.
The formed nucleic acid molecule and its template can be used as
templates to synthesize additional nucleic acid molecules. As used
herein, one amplification reaction may consist of many rounds of
replication. DNA amplification reactions include, for example,
polymerase chain reactions (PCR). One PCR reaction may consist of 5
to 100 "cycles" of denaturation and synthesis of a DNA
molecule.
[0041] Nucleotide as used herein refers to a base-sugar-phosphate
combination. Nucleotides are monomeric units of a nucleic acid
sequence (DNA and RNA). The term nucleotide includes ribonucleoside
triphosphate ATP, UTP, CTG, GTP and deoxyribonucleoside
triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or
derivatives thereof. Such derivatives include, for example,
[.alpha.S]dATP, 7-deaza-dGTP, 7-deaza-dATP, and biotinylated or
haptenylated nucleotides. The term nucleotide as used herein also
refers to dideoxyribonucleoside triphosphates (ddNTPs) and their
derivatives. Illustrated examples of dideoxyribonucleoside
triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP,
ddITP, and ddTTP. According to the present invention, a
"nucleotide" may be unlabeled or detectably labeled by well known
techniques. Detectable labels include, for example, radioactive
isotopes, fluorescent labels, chemiluminescent labels,
bioluminescent labels and enzyme labels.
[0042] Hybridization or annealing as used herein refers to base
pairing of two complementary single-stranded nucleic acid molecules
(RNA and/or DNA) to give a double-stranded molecule. As used
herein, two nucleic acid molecules may be hybridized or annealed,
although the base pairing is not completely complementary.
Accordingly, mismatched bases do not prevent hybridization or
annealing of two nucleic acid molecules provided that appropriate
conditions, well known in the art, are used. In the present
invention, the term hybridization or annealing preferably refers to
hybridization of one or more primers (e.g., oligo(dT) or
derivatives thereof) to one or more templates (e.g., mRNA).
[0043] Host cell as used herein refers to any prokaryotic or
eukaryotic cell that is the recipient of a replicable expression
vector or cloning vector. The terms "host" or "host cell" may be
used interchangeably herein. For examples of such hosts, see
Maniatis et al., "Molecular Cloning: A Laboratory Manual," Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).
Preferred prokaryotic hosts include, but are not limited to,
bacteria of the genus Escherichia (e.g., E. coli). Bacillus,
Staphylococcus, Agrobacter (e.g., A. tumefaciens), Streptomyces,
Pseudomonas, Salmonella, Serratia, Caryophanon, etc. The most
preferred prokaryotic host is E. coli. Bacterial hosts of
particular interest in the present invention include E. coli
strains K12, DH10B, DH5.alpha., Stb12 and HB 101, and others
available from Life Technologies, Inc. Preferred eukaryotic hosts
include, but are not limited to, fungi, fish cells, yeast cells,
plant cells and animal cells. Particularly preferred animal cells
are insect cells such as Drosophila cells, Spodoptera Sf9, Sf21
cells and Trichoplusa High-Five cells; nematode cells such as C.
elegans cells; and mammalian cells such as COS cells, CHO cells,
VERO cells, 293 cells, PERC6 cells, BHK cells and human cells.
[0044] Expression vector as used herein refers to a vector which is
capable of enhancing the expression of a gene or portion of a gene
which has been cloned into it, after transformation or transfection
into a host cell. The cloned gene is usually placed under the
control (i.e., operably linked to) certain control sequences such
as promoter sequences. Such promoters include but are not limited
to phage lambda P.sub.L promoter, and the E. coli lac, trp and tac
promoters. Other suitable promoters will be known to the skilled
artisan.
[0045] The nucleic acid templates suitable for reverse
transcription according to the invention include any nucleic acid
molecule or populations of nucleic acid molecules (preferably one
or more RNA molecules (e.g., one or more mRNA molecules or poly
A.sup.+ RNA molecules, and more preferably a population of mRNA
molecules) or one or more DNA molecules), particularly those
derived from a cell or tissue. In a preferred aspect, a population
of mRNA molecules (a number of different mRNA molecules) are used
to make a cDNA library according to the present invention.
[0046] To make the nucleic acid molecule or molecules complementary
to the one or more templates, a primer (e.g., an oligo(dT) primer)
and one or more nucleotides are used for nucleic acid synthesis
typically in the 3' to 5' direction. Nucleic acid molecules
suitable for reverse transcription according to this aspect of the
invention include any nucleic acid molecule, particularly those
derived from a prokaryotic or eukaryotic cell. Such cells may
include normal cells, diseased cells, transformed cells,
established cells, progenitor cells, precursor cells, fetal cells,
embryonic cells, bacterial cells, yeast cells, animal cells
(including human cells), avian cells, plant cells and the like, or
tissue isolated from a plant (e.g., corn, tomato, tobacco, potato,
soy bean, etc.) or an animal (e.g., human, cow, pig, mouse, sheep,
horse, monkey, canine, feline, rat, rabbit, bird, fish, insect,
etc.). Such nucleic acid molecules may also be isolated from
viruses.
[0047] The nucleic acid molecules that are used as templates to
prepare cDNA molecules according to the methods of the present
invention are preferably obtained from natural sources, such as a
variety of cells, tissues, organs or organisms. Cells that may be
used as sources of nucleic acid molecules may be prokaryotic
(bacterial cells, including but not limited to those of species of
the genera Escherichia, Bacillus, Serratia, Salmonella,
Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria,
Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas,
Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium,
Xanthomonas and Streptomyces) or eukaryotic (including fungi
(especially yeasts), plants, protozoans and other parasites, and
animals including insects (particularly Drosophila spp. cells),
nematodes (particularly Caenorhabditis elegans cells), and mammals
(particularly human cells)).
[0048] Mammalian somatic cells that may be used as sources of
nucleic acids include blood cells (reticulocytes and leukocytes),
endothelial cells, epithelial cells, neuronal cells (from the
central or peripheral nervous systems), muscle cells (including
myocytes and myoblasts from skeletal, smooth or cardiac muscle),
connective tissue cells (including fibroblasts, adipocytes,
chondrocytes, chondroblasts, osteocytes and osteoblasts) and other
stromal cells (e.g., macrophages, dendritic cells, Schwann cells).
Mammalian germ cells (spermatocytes and oocytes) may also be used
as sources of nucleic acids for use in the invention, as may the
progenitors, precursors and stem cells that give rise to the above
somatic and germ cells. Also suitable for use as nucleic acid
sources are mammalian tissues or organs such as those derived from
brain, kidney, liver, pancreas, blood, bone marrow, muscle,
nervous, skin, genitourinary, circulatory, lymphoid,
gastrointestinal and connective tissue sources, as well as those
derived from a mammalian (including human) embryo or fetus.
[0049] Any of the above cells, tissues and organs may be normal,
diseased, transformed, established, progenitors, precusors, fetal
or embryonic. Diseased cells may, for example, include those
involved in infectious diseases (caused by bacteria, fungi or
yeast, viruses (including AIDS, HIV, HTLV, herpes, hepatitis and
the like) or parasites), in genetic or biochemical pathologies
(e.g., cystic fibrosis, hemophilia, Alzheimer's disease, muscular
dystrophy or multiple sclerosis) or in cancerous processes.
Transformed or established animal cell lines may include, for
example, COS cells, CHO cells, VERO cells, BHK cells, HeLa cells,
HepG2 cells, K562 cells, 293 cells, L929 cells, F9 cells, and the
like. Other cells, cell lines, tissues, organs and organisms
suitable as sources of nucleic acids for use in the present
invention will be apparent to one of ordinary skill in the art.
[0050] Once the starting cells, tissues, organs or other samples
are obtained, nucleic acid molecules (such as mRNA) may be isolated
therefrom by methods that are well-known in the art (See, e.g.,
Maniatis, T., et al., Cell 15:687-701 (1978); Okayama, H., and
Berg, P., Mol. Cell. Biol. 2:161-170 (1982); Gubler, U., and
Hoffman, B. J., Gene 25:263-269 (1983); and Message Maker.TM. mRNA
Isolation System available from Life Technologies, Inc.). The
nucleic acid molecules thus isolated may then be used to prepare
cDNA molecules and cDNA libraries in accordance with the present
invention. The cDNA molecules and/or cDNA libraries produced in
accordance with the invention are preferably contained in one or
more vectors. Such vectors may be introduced into one or more host
cells by standard transformation or transfection techniques well
known in the art. Preferred host cells include prokaryotic host
cells such as cells of the genus Escherichia, particularly E.
coli.
[0051] Enzymes for use in the compositions, methods and kits of the
invention include any enzyme having reverse transcriptase activity.
Such enzymes include, but are not limited to, retroviral reverse
transcriptase, retrotransposon reverse transcriptase, hepatitis B
reverse transcriptase, cauliflower mosaic virus reverse
transcriptase, bacterial reverse transcriptase, Tth DNA polymerase,
Taq DNA polymerase (Saiki, R. K., et al., Science 239:487-491
(1988); U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne DNA polymerase
(WO 96/10640), Tma DNA polymerase (U.S. Pat. No. 5,374,553) and
mutants, fragments, variants or derivatives thereof (see, e.g.,
commonly owned, co-pending U.S. patent application Ser. Nos.
08/706,702 and 08/706,706, both filed Sep. 9, 1996, which are
incorporated by reference herein in their entireties). As will be
understood by one of ordinary skill in the art, modified reverse
transcriptases and DNA polymerase having RT activity may be
obtained by recombinant or genetic engineering techniques that are
well-known in the art. Mutant reverse transcriptases or polymerases
can, for example, be obtained by mutating the gene or genes
encoding the reverse transcriptase or polymerase of interest by
site-directed or random mutagenesis. Such mutations may include
point mutations, deletion mutations and insertional mutations.
Preferably, one or more point mutations (e.g., substitution of one
or more amino acids with one or more different amino acids) are
used to construct mutant reverse transcriptases or polymerases for
use in the invention. Fragments of reverse transcriptases or
polymerases may also be obtained by deletion mutation by
recombinant techniques that are well-known in the art, or by
enzymatic digestion of the reverse transcriptase(s) or
polymerase(s) of interest using any of a number of well-known
proteolytic enzymes.
[0052] Preferred enzymes for use in the invention include those
that are reduced or substantially reduced in RNase H activity. Such
enzymes that are reduced or substantially reduced in RNase H
activity may be obtained by mutating the RNase H domain within the
reverse transcriptase of interest, preferably by one or more point
mutations, one or more deletion mutations, and/or one or more
insertion mutations as described above. By an enzyme "substantially
reduced in RNase H activity" is meant that the enzyme has less than
about 30%, less than about 25%, less than about 20%, more
preferably less than about 15%, less than about 10%, less than
about 7.5%, or less than about 5%, and most preferably less than
about 5% or less than about 2%, of the RNase H activity of the
corresponding wildtype or RNase H.sup.+ enzyme such as wildtype
Moloney Murine Leukemia Virus (M-MLV), Avian Myeloblastosis Virus
(AMY) or Rous Sarcoma Virus (RSV) reverse transcriptases. The RNase
H activity of any enzyme may be determined by a variety of assays,
such as those described, for example, in U.S. Pat. No. 5,244,797,
in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988), Gerard,
G. F., et al., FOCUS 14(5):91 (1992), and in U.S. Pat. No.
5,668,005, the disclosures of all of which are fully incorporated
herein by reference.
[0053] Polypeptides having reverse transcriptase activity for use
in the invention may be obtained commercially, for example from
Life Technologies, Inc. (Rockville, Md.), Pharmacia (Piscataway,
N.J., Sigma (Saint Louis, Mo.) or Boehringer Mannheim Biochemicals
(Indianapolis, Ind.). Alternatively, polypeptides having reverse
transcriptase activity may be isolated from their natural viral or
bacterial sources according to standard procedures for isolating
and purifying natural proteins that are well-known to one of
ordinary skill in the art (see, e.g., Houts, G. E., et al., J.
Virol. 29:517 (1979)). In addition, the polypeptides having reverse
transcriptase activity may be prepared by recombinant DNA
techniques that are familiar to one of ordinary skill in the art
(see, e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265
(1988); Soltis, D. A., and Skalka, A. M., Proc. Natl. Acad. Sci.
USA 85:3372-3376 (1988)).
[0054] Preferred polypeptides having reverse transcriptase activity
for use in the invention include M-MLV reverse transcriptase, RSV
reverse transcriptase. AMV reverse transcriptase, Rous Associated
Virus (RAV) reverse transcriptase, Myeloblastosis Associated Virus
(MAV) reverse transcriptase and Human Immunodeficiency Virus (HIV)
reverse transcriptase, and others described in WO 98/47921 and
derivatives, variants, fragments or mutants thereof, and
combinations thereof. In a further preferred embodiment, activity,
and are most preferably selected from the group consisting of M-MLV
H.sup.- reverse transcriptase, RSV if reverse transcriptase, AMV
H.sup.- reverse transcriptase, RAV H.sup.- reverse transcriptase,
MAV H.sup.- reverse transcriptase and HIV H.sup.- reverse
transcriptase, and derivatives, variants, fragments or mutants
thereof, and combinations thereof. Reverse transcriptases of
particular interest include AMV RT and M-MLV RT, and more
preferably AMV RT and M-MLV RT having reduced or substantially
reduced RNase H activity (preferably AMV RT .alpha.H.sup.-/BH.sup.+
and M-MLV RT H.sup.-). The most preferred reverse transcriptases
for use in the invention include SuperScript.TM., SuperScript.TM.
II, ThermoScript.TM. and ThermoScript.TM. II available from Life
Technologies, Inc. See generally, WO 98/47921, U.S. Pat. Nos.
5,244,797 and 5,668,005, the entire contents of each of which are
herein incorporated by reference.
[0055] A variety of DNA polymerases are useful in accordance with
the present invention. Such polymerases include, but are not
limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus
aquaticus (Taq) DNA polymerase, Thermotoga neapolitana (Tne) DNA
polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus
litoralis (Tli or VENT.TM.) DNA polymerase, Pyrococcus furiosis
(Pfu) DNA polymerase, DEEPVENT.TM. DNA polymerase, Pyrococcus
woosii (Pwo) DNA polymerase, Bacillus sterothermophilus (Bst) DNA
polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus
acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac)
DNA polymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermus
ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME.TM.) DNA
polymerase, Methanobacterium thermoautotrophicum (Mth) DNA
polymerase, Mycobacterium spp. DNA polymerase (Mtb, Mlep), and
mutants, variants and derivatives thereof.
[0056] DNA polymerases used in accordance with the invention may be
any enzyme that can synthesize a DNA molecule from a nucleic acid
template, typically in the 5' to 3' direction. Such polymerases may
be mesophilic or thermophilic. Mesophilic polymerases include T4
DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Klenow
fragment DNA polymerase, DNA polymerase III, DNA polymerase I and
the like. Thermostable DNA polymerases include Taq, Tne, Tma, Pfu,
VENT.TM., DEEPVENT.TM., Tth and mutants, variants and derivatives
thereof (U.S. Pat. No. 5,436,149; U.S. Pat. No. 5,512,462; WO
92/06188; WO 92/06200; WO 96/10640; Barnes, W. M., Gene 112:29-35
(1992); Lawyer, F. C., et al., PCR Meth. Appl. 2:275-287 (1993);
Flaman, J.-M., et al., Nucl. Acids Res. 22(15):3259-3260
(1994)).
[0057] DNA polymerases for use in the invention may be obtained
commercially, for example from Life Technologies, Inc. (Rockville,
Md.), Perkin-Elmer (Branchburg, N.J.), New England BioLabs
(Beverly, Mass.) or Boehringer Mannheim Biochemicals (Indianapolis,
Ind.).
[0058] The present invention is also directed to nucleic acid
molecules produced by the methods of the invention, which may be
cDNA molecules, especially full-length cDNA molecules, to vectors
(particularly expression vectors) comprising these nucleic acid
molecules and cDNA molecules and to host cells comprising these
nucleic acid molecules, cDNA molecules, and/or vectors.
[0059] Recombinant vectors may be produced according to this aspect
of the invention by inserting, using methods that are well-known in
the art, one or more of the cDNA molecules or nucleic acid
molecules prepared according to the present methods into one or
more vectors. The vector used in this aspect of the invention may
be, for example, a phage or a plasmid vector, and is preferably a
plasmid. Preferred are vectors comprising cis-acting control
regions to the nucleic acid encoding the polypeptide of interest.
Appropriate trans-acting factors may be supplied by the host,
supplied by a complementing vector or supplied by the vector itself
upon introduction into the host.
[0060] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids or bacteriophages, and vectors
derived from combinations thereof, such as cosmids and phagemids,
and will preferably include at least one selectable marker such as
a tetracycline or ampicillin resistance gene for culturing in a
bacterial host cell. Prior to insertion into such an expression
vector, the cDNA or nucleic acid molecules of the invention should
be operatively linked to an appropriate promoter.
[0061] Among vectors preferred for use in the present invention
include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A, available from Stratagene; pcDNA3 available from
Invitrogen; pGEX, pTrxfus, pTrc99a, pET-5, pET-9, pKK223-3,
pKK233-3, pDR540, pRIT5 available from Pharmacia; and pSPORT1,
pSPORT2, pSV.SPORT1, pCMVSPORT6 and pCMVSPORT available from Life
Technologies, Inc. Other suitable vectors will be readily apparent
to the skilled artisan.
[0062] The invention may be used in conjunction with any methods of
cDNA synthesis that are well-known in the art (see, e.g., Gubler,
U., and Hoffman, B. J., Gene 25:263-269 (1983); Krug, M. S., and
Berger, S. L., Meth. Enzymol. 152:316-325 (1987); Sambrook, J., et
al., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold
Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp.
8.60-8.63 (1989); PCT US98/19948; and WO 98/51699) to produce cDNA
molecules or libraries. Other methods of cDNA synthesis which may
advantageously use the present invention will be readily apparent
to one of ordinary skill in the art.
[0063] Having obtained cDNA molecules or libraries according to the
present methods, these cDNAs may be isolated for further analysis
or manipulation. Detailed methodologies for purification of cDNAs
are taught in the GENETRAPPER.TM. manual (Life Technologies), which
is incorporated herein by reference in its entirety, although
alternative standard techniques that are known in the art (see,
e.g., Sambrook, J., et al., Molecular Cloning: A Laboratory Manual,
2.sup.nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press, pp. 8.60-8.63 (1989)) may also be used. The cDNA
molecules or libraries produced by the invention may also be
further manipulated by standard molecular biology techniques such
as two hybrid analysis, cDNA normalization, sequencing and
amplification. More particularly, the methods of the invention and
the cDNA molecules or libraries produced by such methods may be
used in combination with RT-PCR and 5' RACE technology (Life
Technologies, Inc.) and differential display.
[0064] A variety of inhibitors and binding molecules are suitable
for use in the present methods. Included among these inhibitors or
binding molecules are antibodies that bind to the above-described
polypeptides having reverse transcriptase activity (such as anti-RT
antibodies including anti-AMV RT antibodies, anti-M-MLV RT
antibodies or anti-RSV RT antibodies) or to cap structure (e.g.,
anti-cap antibodies), and fragments thereof (such as Fab or
F(ab').sub.2 fragments). Such antibodies may be polyclonal or
monoclonal, and may be prepared in a variety of species according
to methods that are well-known in the art. See, for instance,
Sutcliffe, J. G., et al., Science 219:660-666 (1983); Wilson et
al., Cell 37: 767 (1984); and Bittle, F. J., et al., J. Gen. Virol.
66:2347-2354 (1985). Antibodies specific for any of the
above-described reverse transcriptases or cap structures can be
raised against the intact polymerase polypeptide or cap structures
or one or more fragments thereof. These polypeptides or cap
structures or fragments thereof may be presented together with a
carrier protein (e.g., albumin) to an animal system (such as rabbit
or mouse) or, if they are long enough (at least about 25 amino
acids), without a carrier.
[0065] As used herein, the tee n "antibody" (Ab) may be used
interchangeably with the terms "polyclonal antibody" or "monoclonal
antibody" (mAb), except in specific contexts as described below.
These terms, as used herein, are meant to include intact molecules
as well as antibody fragments (such as, for example, Fab and
F(ab'), fragments) which are capable of specifically binding to a
polypeptide having reverse transcriptase activity (such as a DNA
polymerase or a reverse transcriptase) or cap structures or
portions thereof.
[0066] The antibodies used in the methods of the present invention
may be polyclonal or monoclonal, and may be prepared by any of a
variety of methods (see, e.g., U.S. Pat. No. 5,587,287). For
example, polyclonal antibodies may be made by immunizing an animal
with one or more polypeptides having reverse transcriptase activity
or cap structures or portions thereof according to standard
techniques (see, e.g., Harlow, E., and Lane, D., Antibodies: A
Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press (1988); Kaufman, R B., et al., In: Handbook of
Molecular and Cellular Methods in Biology and Medicine, Boca Raton,
Fla.: CRC Press, pp. 468-469 (1995)). Alternatively, monoclonal
antibodies (or fragments thereof) to be used in the present methods
may be prepared using hybridoma technology that is well-known in
the art (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur.
J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292
(1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell
Hybridomas, New York: Elsevier, pp. 563-681 (1981); Kaufman, P. B.,
et al., In: Handbook of Molecular and Cellular Methods in Biology
and Medicine, Boca Raton, Fla.: CRC Press, pp. 444-467 (1995)).
[0067] It will be appreciated that Fab, F(ab').sub.2 and other
fragments of the above-described antibodies may be used in the
methods described herein. Such fragments are typically produced by
proteolytic cleavage, using enzymes such as papain (to produce Fab
fragments) or pepsin (to produce F(ab').sub.2 fragments). Antibody
fragments may also be produced through the application of
recombinant DNA technology or through synthetic chemistry.
[0068] The invention also provides kits for use in accordance with
the invention. Such kits comprise a carrier means, such as a box or
carton, having in close confinement therein one or more container
means, such as vials, tubes, bottles and the like, wherein the kit
may comprise (in the same or separate more reverse transcription
inhibitors, one or more cap binding molecules, one or more DNA
polymerases, suitable buffers, one or more nucleotides and/or one
or more primers (e.g., oligo(dT) for reverse transcription). The
kits encompassed by this aspect of the present invention may
further comprise additional reagents and compounds necessary for
carrying out standard nucleic acid reverse transcription
protocols.
[0069] It will be readily apparent to one of ordinary skill in the
relevant art that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLES
Example 1
Comparison of First Strand cDNA Synthesis with Varying Ratios of
Oligo (dT) Primer/mRNA
[0070] This example compares first strand cDNA synthesis of the
MAP4 gene with various ratios of oligo dT primer/starting mRNA. All
components are available from Life Technologies, Inc., Rockville,
Md., unless specified otherwise.
[0071] The master mix for Superscript II reverse transcriptase (SS
II RT) was prepared as specified in Table 1 below.
TABLE-US-00001 TABLE 1 Component .mu.l .mu.l 5X SSII RT buffer 4 28
0.1M DTT 2 14 10 mM dNTP 1 7 .alpha.-.sup.32P dCTP 0.5 3.5 Water
1.5 10.5 Total volume 9 63
[0072] The master mix for ThermoScript.TM. II RT (TS RT) (AMV RT
.alpha.H.sup.-.beta.H.sup.+) (see WO 98/47921) was prepared as
specified in Table 2 below.
TABLE-US-00002 TABLE 2 Component .mu.l .mu.l 10X TS II buffer* 2 38
0.1M DTT 2 38 10 mM dNTP 2 38 .alpha.-.sup.32P dCTP 0.5 9.5 Rnase
OUT (40 u/.mu.l) 1 19 Water 1.5 28.5 Total volume 9 171 *10X TS II
buffer comprises 50 mM Tris-HCl (pH 8.4), 750 mM KCl, and 75 mM
MgCl.sub.2.
[0073] The master annealing mix was prepared by adding a 5 Kb MAP4
mRNA, oligo(dT).sub.25-30 and water to 5 tubes in the amounts
specified in Table 3 below.
TABLE-US-00003 TABLE 3 Ratio of oligo (dT)/mRNA 1:1 2.5:1 5:1 10:1
50:1 Component Volume (.mu.l) MAP4 mRNA (1 .mu.g/.mu.l) 5 5 5 5 5
Oligo(dT).sub.25-30 (10 ng/.mu.l) 2.6 6.5 13.1 -- --
Oligo(dT).sub.25-30 (100 ng/.mu.l) -- -- -- 2.6 13.1 Water 42.4
38.5 31.9 42.4 31.9 Total volume 50 50 50 50 50
[0074] The mixture was heated at 70.degree. C. for 10 minutes and
then chilled on ice for 5 minutes.
[0075] Synthesis of first strand cDNA was done by adding 9 .mu.l of
the appropriate reverse transcriptase master mix, 10 .mu.l of the
master annealing mix and 1 .mu.l of either SS II RT (200 units/up
or TS II RT (15 units/.mu.l) for a total volume of 20 .mu.l as
summarized in Table 4 below.
TABLE-US-00004 TABLE 4 Reverse Tube Transcriptase Temperature Ratio
of oligo (dT).sub.25-30/mRNA 1 SS II 45.degree. C. 1 2 2.5 3 5 4 10
5 50 1 TS II RT 45.degree. C. 1 2 2.5 3 5 4 10 5 50 6 50.degree. C.
1 7 2.5 8 5 9 10 10 50 11 55.degree. C. 1 12 2.5 13 5 14 10 15
50
[0076] The reactions were incubated for 1 hour at 45.degree. C. for
SS II RT and at 45, 50 or 55.degree. C. for TS II RT. The tubes
were placed on ice to complete the reaction. 18 .mu.l first stand
cDNA of the reaction tube was precipitated and re-suspended in 10
.mu.l of water. 5 .mu.l of the first strand cDNA was mixed with 5
.mu.l of standard loading buffer (60 mM NaOH, 4 mM EDTA, 0.1%
bromophenol blue), and loaded onto 1.4% alkaline agarose gel for
analysis. These results are shown in FIGS. 1 and 2.
[0077] FIG. 1 is an autoradiograph of first strand cDNA synthesized
with SS II RT at 45.degree. C. Lane M is the 1 kb DNA ladder. Lanes
1-5 represents reaction conditions with a molar ratio of
oligo(dT).sub.25-30/mRNA of 1:1, 2.5:1, 5:1, 10:1 and 50:1,
respectively. FIG. 2 is an autoradiograph of first strand cDNA
synthesized with TS II RT. Lane M is the 1 kb DNA ladder. Lanes 1-5
represent reaction conditions at 45.degree. C. with a molar ratio
of oligo(dT).sub.25-30/mRNA of 1:1, 2.5:1, 5:1, 10:1 and 50:1,
respectively. Lanes 6-10 represent reaction conditions at
50.degree. C. with a molar ratio of oligo (dT).sub.25-30/mRNA of
1:1, 2.5:1, 5:1, 10:1 and 50:1, respectively. Lanes 11-15 represent
reaction conditions at 55.degree. C. with a molar ratio of oligo
(dT).sub.25-30/mRNA of 1:1, 2.5:1, 5:1, 10:1 and 50:1,
respectively. The results show that by reducing the molar ratio of
oligo(dT) primer/mRNA (preferably to 1:1) internal priming with
reverse transcriptase was almost entirely eliminated.
Example 2
Comparison of First Strand cDNA Synthesis Under Standard and Hot
Start Conditions
[0078] This experiment was designed to compare first strand cDNA
synthesis of the MAP4 gene with standard reaction and hot start
conditions.
[0079] The annealing mix was prepared by mixing 1 .mu.g of MAP4
mRNA and biotinylated Not I oligo(dT).sub.25 primer ((Biotin).sub.4
GACTAGTTCTAGAT CGCGAGCGG CCGCCCTTTTT TTTTTTTTTTTT TTTTTTTT; see WO
98/51699) in the desired molar ratio of oligo (dT)/mRNA of 0:1, 1:1
or 15:1 in thin-walled PCR tubes and bringing the volume up to 10
.mu.l with water. If several tubes are identical, they may be made
in one batch and aliquotted accordingly. The annealing mix was kept
on ice.
[0080] The master mix for Superscript II reverse transcriptase (SS
II RT) was prepared as specified in Table 5 below.
TABLE-US-00005 TABLE 5 Component .mu.l .mu.l 5X SSII RT buffer 4 28
0.1M DTT 2 14 10 mM dNTP 1 7 .alpha.-.sup.32P dCTP 0.5 3.5 SSII RT
(200 u/.mu.l) 1 7 Water 1.5 10.5 Total volume 10 70
[0081] The SS II RT master mix was then divided into two equal
aliquots, one for processing with standard reaction temperatures
(batch 1) and one for processing with hot start reaction
temperatures (batch 2). To allow for condensation, an additional
10% volume of water was added to batch 2. All mixes were kept on
ice.
[0082] Synthesis of first strand cDNA was begun by briefly spinning
tubes containing annealing mix to collect droplets, placing the
tubes in a thermocycler and then heating them to 70.degree. C. for
10 minutes. After this 10 minute cycle at 70.degree. C., the tubes
of annealing mix for batch 1 were immediately removed to ice. The
tubes of annealing mix for batch 2 were allowed to cool to
45.degree. C. in the thermocycler while the batch 2 master mix was
placed in the thermocycler and incubated at 45.degree. C. for 5
minutes. After the 5 minute incubation, 11 .mu.l of the master mix
for batch 2 was added to each batch 2 annealing tube and mixed with
a pipette 2 times. Care was taken not to spin the tubes to avoid
lowering the temperature.
[0083] 10 .mu.l of the master mix for batch 1 was added to each
batch 1 annealing tube. The batch 1 tubes were lightly vortexed and
briefly centrifuged to collect condensation droplets. The batch 1
tubes were then returned to the thermocylcer and the tubes from
both batch 1 and 2 were incubated at 45.degree. C. for one
hour.
[0084] 5 .mu.l of the first strand cDNA from each tube was mixed
with 5 .mu.l of standard loading buffer (60 mM NaOH, 4 mM EDTA,
0.1% bromophenol blue) and loaded onto 1.4% alkaline agarose gel
for analysis. The results are shown in FIG. 3.
[0085] FIG. 3 is an autoradiograph of first strand cDNA synthesized
with SS II RT. Lanes 1, 3 and 5 represents batch 1 reaction
conditions with a molar ratio of biotinylated oligo(dT)/mRNA of
0:1, 1:1 and 15:1, respectively. Lanes 2, 4 and 6 represents batch
2 reaction conditions with a molar ratio of biotinylated
oligo(dT)/mRNA of 0:1, 1:1 and 15:1, respectively.
[0086] First strand cDNA was also synthesized with TS II RT using
15 units of TS II RT per .mu.g mRNA using a biotinylated
oligo(dT)/mRNA ratio of 1:1 and 15:1. The same protocol described
above was followed, except that the temperature was varied to
50.degree. C. The results are shown in FIG. 4. FIG. 4 is an
autoradiograph of first strand cDNA synthesized with TS II RT. Lane
M is the 1 kb DNA ladder. Lanes 1 and 3 represent reactions
conditions using standard reaction temperatures at a 1:1 ratio and
15:1 ratio, respectively. Lanes 2 and 4 represent hot start
reactions conditions at a 1:1 ratio and 15:1 ratio, respectively,
as described above.
[0087] The results indicated that by dropping the reaction
temperature to the reverse transcriptase reaction temperature after
denaturation of the primer and mRNA mixture, the reaction was
started directly and internal priming was avoided entirely.
Example 3
Synthesis of Double Strand cDNA by Controlling the Reaction
Temperature and the Concentration of Salt and RNase
[0088] This example describes the synthesis of double stranded cDNA
by controlling the reaction temperature and the concentration of
salt and different ribonuclease (RNases) during the treatment of
the cDNA/mRNA hybrids after first strand cDNA synthesis.
[0089] First strand cDNA was synthesized as described above in
Example 2 and digested with either RNase I or RNase A as further
described below.
[0090] RNase I digestion of first strand cDNA was done by
re-suspending the first strand cDNA in 180 .mu.l of water and 20
.mu.l of 10.times. RNase I buffer (100 mM Tris-HCl (pH 7.5), 50 mM
EDTA, 2 M sodium acetate). 2.5 units of RNase I (1 unit/.mu.g mRNA)
were added and the mixture was mixed well. The RNase I digestion
mixture was incubated at 25.degree. C. for 30 minutes and extracted
with phenol/chloroform once. The supernatant was precipitated with
1 .mu.l of glycogen, 100 .mu.l of ammonium acetate and 800 .mu.l of
ethanol.
[0091] RNase A digestion of first strand cDNA was done by
re-suspending the first strand cDNA in 200 .mu.l of digestion
buffer (10 mM Tris-HCl (pH 7.5), 250 mM NaCl). 12.5 ng of RNase A
(5 ng/.mu.g mRNA) were added and the mixture was mixed well. The
RNase A digestion mixture was incubated at 25.degree. C. for 30
minutes and extracted with phenol/chloroform once. The supernatant
was precipitated with 1 .mu.l of glycogen, 100 .mu.l of ammonium
acetate and 800 .mu.l of ethanol.
Example 4
Enrichment of the Full-Length cDNA Clones with Cap-Binding
Proteins
[0092] This example describes enrichment of full-length cDNA clones
with the cap-binding protein eIF4E.
[0093] cDNA was prepared by precipitating the RNase I treated first
strand cDNA described in Example 3 above and washing with 70%
ethanol. The resulting pellet was dried at room temperature for 5
minutes, and re-suspended in 210 .mu.l of 10 mM KPO.sub.4, 100 mM
KCl, 2 mM EDTA, 6 mM DTT and 5% glycerol. The cDNA was stored on
ice.
[0094] eIF4E glutathione sepharose 4B beads were prepared by first
mixing glutathione sepharose 4B beads (Pharmacia, Sweden) well. To
prepare eIF4E beads, a recombinant host cell expressing GST tagged
eIF4E protein (the eIF4E gene was cloned into a GST fusion vector
to create a N-terminal GST-eIF4E fusion gene) was grown and the
fusion protein was purified by standard techniques. Thus, the
invention also relates to recombinant host cells expressing eIF4E
protein (particularly as fusion proteins), to vectors comprising
the genes expressing such proteins or fusion proteins and to the
recombinant proteins or fusion proteins produced. In the present
invention any tag can be used (e.g., His Tag, GST tag, HA tag, Trx
tag, etc.). Such tags may be positioned at the carboxy and/or
N-terminal region of the eIF4E gene.
[0095] The GST-eIF4E fusion protein was complexed with sepharose 4B
beads by glutathione coupling using gluthionine sepharose 4B beads
(Pharmacia Biotech) following the manufacturers protocols. 200
.mu.l of the beads were transferred to a 1.5 ml microcentrifuge
tube, centrifuged for 1 second, and 75 .mu.l of supernatant was
removed. The beads were washed twice with 1 ml of reaction buffer
(10 mM KPO.sub.4, 100 mM KCl, 2 mM EDTA, 6 mM DTT and 5% glycerol),
and re-suspended in 258 .mu.l of reaction buffer, followed by the
addition of 42 .mu.l (18 pmoles/.mu.l) of eIF4E protein (600
pmoles/100 .mu.l beads). The mixture was mixed on a head to head
roller at 4.degree. C. for 30 minutes. The mixture was then
centrifuged for 1 second, and the supernatant was removed. The
beads were washed twice with 1 ml of reaction buffer and once with
1 ml of 25 .mu.g/ml yeast tRNA in reaction buffer, 20 .mu.l of
reaction buffer and 5 .mu.g of yeast tRNA were then added to the
beads. 200 .mu.l of RNase I treated cDNA was added to the beads,
and the content was mixed on a roller at room temperature for 1
hour. After 1 hour, the mixture was centrifuged for 1 second, and
the supernatant was removed. The beads were washed twice with 1 ml
of reaction buffer and once with 1 ml of 500 .mu.M GDP in reaction
buffer. The cDNA was eluted twice with 250 .mu.l of 500 .mu.M GDP
in reaction buffer. The eluted solutions were pooled and
centrifuged for 1 minute to remove the beads. The eluted cDNA was
extracted twice with an equal volume of phenol/chloroform. The cDNA
was divided into two tubes and precipitated with 1 .mu.l of
glycogen, 0.5 volume of 7.5 M ammonium acetate and 2.5 volume of
ethanol.
Example 5
Evaluation of the cDNA Library
[0096] To evaluate the quality of the cDNA libraries constructed
with the above-described full-length methods, the MAP4 gene (5-6
kb) and other genes was selected as the target genes. MAP4 and
other cDNA clones were isolated from libraries constructed by
standard methods well-known in the art (see SuperScript.TM. Plasmid
Manual, Life Technologies, Inc.) and the above-described
full-length methods with 3' and 5' GeneTrapper cDNA Positive
Selection System (Life Technologies, Inc., Rockville, Md.). The
positive clones were size analyzed by PCR. Tables 6 and 7 below
summarizes the results of the enrichment of full-length cDNA clones
in human fibroblast cDNA libraries constructed with methods
well-known in the art (control) and the full-length methods
described above (full-length method).
TABLE-US-00006 TABLE 6 % full-length with % full-length with 5'
GeneTrapper 3' GeneTrapper Gene control* full-length method
control* full-length method MAP4 12.8 90.3 6.25 37.5 (5-6 kb)
[0097] The control library was constructed with SS II RT using
known methods.
TABLE-US-00007 TABLE 7 Full-length of % Full-length by 5' Gene name
gene (Kb) GeneTrapper MAP4 (Microtubule-associated 5/6 90.3 protein
4) .beta.-Adaptin* 3.8/5.7 90.0 TR (Transferrin receptor) 5.0 45.0
PTK (Protein tyrosine Kinase) 3.0 84.4 RPA (DNA Replication protein
A) 1.4 98.0 *There are two members of the genes, 3.8 kb and 5.7 kb
in the family.
[0098] These results show that the full-length methods described
above yielded >90% full-length cDNA clones with the 5'
GeneTrapper system, compared to <13% using standard methods.
Furthermore, the above-described full-length methods yielded
>37% full-length clones with the 3' GeneTrapper system, as
compared to <7% using standard methods.
Example 6
First Strand cDNA Synthesis, RNase I Digestion and eIF-4E
Capture
[0099] All conditions and parameters described above in Examples 2,
3 (RNase I) and 4 were followed, except for the following: 4
reactions of 10 .mu.g of human fibroblast cytoplasmic mRNA were
used per reaction (see WO 98/45311); the biotinylated
primer-adapter
(Biotin).sub.4-GACTAGTTCTAGATCGCGAGCGGCCGCCC(T).sub.25 was used at
a 1:1 primer/mRNA molar ratio; TS II RT was used at 50.degree. C.;
and SS II RT was used at 45.degree. C. Table 8 below summarizes the
first strand cDNA and eIF-4E capture results.
Example 7
Second Strand cDNA Synthesis
[0100] Second strand cDNA was synthesized by first dissolving each
of the four reaction pellets obtained in Example 6 above in 104
.mu.l of DEPC-treated water and then adding the following reagents
to each reaction:
[0101] 4 .mu.l of 5.times. First Strand Buffer*
[0102] 30 .mu.l of 5.times. Second Strand Buffer*
[0103] 2 .mu.l of 0.1 M DTT
[0104] 4 .mu.l of 10 mM dNTPs
[0105] 1 .mu.l of E. coli DNA ligase (10 units/.mu.l)
[0106] 1 .mu.l of E. coli RNAse H (2 units/.mu.l)
[0107] 4 .mu.l of E. coli DNA polymerase (10 units/.mu.l)
see SuperScript Plasmid System manual (Life Technologies, Inc.,
Rockville, Md.)
[0108] These reactions mixtures were then incubated for 2 hours at
16.degree. C. 2 of T4 DNA polymerase (5 units/.mu.l) was added and
incubation at 16.degree. C. was continued for 5 more minutes.
Example 8
Streptavidin Bead Preparation
[0109] During the last 30 minutes of the 2 hour second strand
reaction described in Example 7 above, streptavidin paramagnetic
beads were prepared as follows.
[0110] Streptavidin paramagnetic beads (Seradyn) were gently mixed
by pipetting until the beads were completely re-suspended. 150
.mu.l of the mixed beads were transferred to the bottom of a
microcentrifuge tube for each reaction. The tubes were inserted
into a Magna-Sep Magnetic Particle
[0111] Separator (Life Technologies, Inc., Rockville, Md.) (the
magnet) and let sit for 2 minutes. While the tubes were in the
magnet, the supernatant was removed by pipetting and 100 .mu.l of
TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) was immediately
added to the beads.
[0112] The tubes were then removed from the magnet and the beads
were gently re-suspended by finger tapping or vortexing at the
lowest setting. The tubes were re-inserted into the magnet. After 2
minutes, the supernatant was removed, the beads were re-suspended
in 160 .mu.l of binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA,
1 M NaCl) and the tubes were placed into a microcentrifuge tube
rack.
Example 9
Capture of the Double-Stranded cDNA Library
[0113] After incubating the second strand reaction with T4 DNA
polymerase as described in Example 7 above, the reaction mixtures
were placed on ice and 10 .mu.l of 0.5 M EDTA was added. Then the
cDNA library was captured according to the following procedure (see
generally WO 98/51699).
[0114] The paramagnetic beads prepared according to Example 8 were
transferred to the second strand reaction mixture tubes and gently
mixed by pipetting and the suspension was incubated for 60 minutes
at room temperature. The tubes were then inserted into the magnet.
After 2 minutes, the supernatant was removed and discarded.
[0115] 100 .mu.l of wash buffer (10 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 500 mM NaCl) was added to the beads, the beads were
re-suspended by finger tapping or gently vortexing at the lowest
setting and the tubes were re-inserted into the magnet for 2
minutes. The supernatant was removed and discarded. This washing
step was repeated one more time and then 100 .mu.l of wash buffer
was added to the beads. The tubes were then again inserted into the
magnet for 5 minutes.
Example 10
Not I Digestion
[0116] After the 5 minute incubation described in the last step of
Example 9, the supernatant was removed and discarded from the
paramagnetic beads and 41 .mu.l of autoclaved, distilled water, 5
.mu.l of REact 3 buffer, 4 .mu.l of Not I was added and the beads
were mixed well by pipetting. The reaction was then incubated for 2
hours at 37.degree. C. The tubes were then inserted into the magnet
for 2 minutes and the supernatant containing the cDNA library was
transferred to fresh tubes.
[0117] 50 .mu.l of phenol:chloroform:isoamyl alcohol (25:24:1) was
added to the supernatant, the solution was vortexed thoroughly, and
then centrifuged at room temperature for 5 minutes at
14,000.times.g. 45 .mu.l of the upper, aqueous layer was carefully
removed and transferred to fresh microcentrifuge tubes. 23 .mu.l of
7.5 M ammonium acetate, 1 .mu.l of glycogen (20 .mu.g) and 172
.mu.l of ethanol (-20.degree. C.) was added. The solution was mixed
well and stored on dry ice (or -70.degree. C. freezer) for 15
min.
[0118] The ethanol solution was then centrifuged at 4.degree. C.
for 30 minutes at 14,000.times.g. The supernatant was carefully
removed from the small pellets. 100 .mu.l of 70% ethanol was added
and the tubes were centrifuged at room temperature for 2 minutes at
14,000.times.g. The ethanol was removed and the pellets were dried
in a speed-vac for 2 minutes or until dry. The pellets were then
dissolved in 20 .mu.l of TE buffer (10 mM Tris-HCl (pH 7.5), 0.1 mM
EDTA). The final yield of cDNA was determined by the Cerenkov
counts (see Table 8 below).
TABLE-US-00008 TABLE 8 standard (S) or Reverse varied (V) %
Incorporation Amount of cDNA after Transcriptase temperature (ng of
cDNA) eIF-4E capture TS II RT S 27% (2,720 ng) 512 ng TS II RT V
(hot start) 26% (2,640 ng) 473 ng SS II RT S 46% (4,560 ng) 306 ng
SS II RT V (hot start) 47% (4,730 ng) 363 ng
Example 11
Ligation of cDNA to the Vector and Introduction into E. coli
[0119] From 10 to 30 ng of the un-fractionated or size fractionated
(.gtoreq.1.5 kb by low melting gel electrophoresis) cDNA was
ligated into a vector pCMVSPORT 6 (Life Technologies, Inc.). This
ligation was introduced into E. coli by electroporation as
described in the SuperScript Plasmid System manual (Life
Technologies, Inc., Rockville, Md.), except that the cloning vector
was pre-digested with Not I and Eco RV.
[0120] Sequence analysis of randomly selected clones from the cDNA
library constructed (304 clones) were analyzed by 5' and 3'
sequencing to determine the total percentage of full-length random
clones in the cDNA library. Sequences were compared for homology
with GeneBank sequences. The results are summarized in Table 9
below. Based on the results, approximately 68% of the random clones
were full-length (including known full-length clones and unknown
full-length clones). Thus, approximately 17% unknown full-length
clones were obtained from the human fibroblast cytoplasmic mRNA
library.
TABLE-US-00009 TABLE 9 Number of Clones Percentage Total Sequences
304 73.3% Sequences with Homology 223 51% Full-Length Clones 114
17% Potentially Full-Length 39 17% Partial Clones 70 31%
Example 12
RNAse Assay
[0121] First strand cDNA was treated with RNase A at 1000 ng/.mu.g
mRNA in TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) and RNase I
25 to 40 u/.mu.g mRNA in TEN (10 mM Tris-HCl (pH 7.5), 5 mM EDTA
(pH 8.0), 200 mM Sodium Acetate) at 37.degree. C. essentially as
described in Example 3. However, this treatment with large amounts
of RNase at elevated temperatures resulted in libraries containing
very small average cDNA insert size (about 200 bp). Therefore, a
second strand cDNA assay was developed to determine the optimal
amount of RNase needed.
[0122] First strand cDNA (radioactively labeled and
non-radioactively labeled) was synthesized using HeLa mRNA at 500
ng of RNA/reaction. The first strand cDNA was precipitated with
ethanol and dissolved in DEPC-treated water. The cold first strand
cDNA was added to RNase buffer with different amounts of RNase.
After incubation for 30 minutes at 25.degree. C., the treated cDNA
was extracted with phenol:chloroform and precipitated with ethanol.
The treated cDNA was dissolved in DEPC-treated water, a second
strand cDNA reaction was performed with .sup.32P-dCTP plus and
minus RNase H. The reaction was extracted with phenol:chloroform
and precipitated with ethanol. Equal amounts of cpm was
electrophoresed into a 1.4% alkaline-agarose gel. The results are
shown in FIGS. 5 and 6.
[0123] FIG. 5 is an autoradiograph of second strand cDNA
synthesized using different amounts of RNase A. Lane M is the 1 kb
DNA ladder. Lane 1 represents untreated first strand cDNA. Lane 2
represents untreated second strand cDNA. Lanes 3, 5, 7 and 9
represent second strand cDNA synthesized without RNase H and with
RNase A concentrations of 0, 125 ng, 2.5 ng and 5 ng, respectively.
Lanes 4, 6, 8 and 10 represent second strand cDNA synthesized with
RNase H and with RNase A at concentrations of 0, 1.25 ng, 2.5 ng
and 5 rig, respectively.
[0124] FIG. 6 is an autoradiograph of second strand cDNA
synthesized using different amounts of RNase I. Lane M is the 1 kb
DNA ladder. Lane 1 represents untreated first strand cDNA, Lane 2
represents untreated second strand cDNA. Lanes 3, 5, 7 and 9
represent second strand cDNA synthesized without RNase H and with
RNase I concentrations of 0, 0.5 u, 1.25 u and 2.5 u, respectively.
Lanes 4, 6, 8 and 10 represent second strand cDNA synthesized with
RNase H and with RNase I at concentrations of 0, 0.5 u, 1.25 u and
2.5 u, respectively.
[0125] These gel analysis demonstrated that a concentration of 1.25
ng of RNAse A (see FIG. 5) or 0.5 units of RNAse I (see FIG. 6) may
be optimal to use with 500 ng of starting mRNA.
Example 13
Preparation of Antibodies Against Cap Structure
[0126] The antibody to cap was generated using m7guanosnine-KLH as
the antigen. 1200 hybridomas were plated and only 120 colonies were
generated. Of these only 6 colonies were positive for cap. After
further analysis, 3 were determined to have the affinity required.
The first screen ELISA consists of binding m7guanosine-BSA to an
ELISA plate, block with BSA, bind hybridoma supernatants, react
with secondary antibody and determine positives via a colorimetric
reaction with BCIPINPT. The secondary screen included incubating
appropriate dilutions of the hybridoma supernatants with either 0.1
mM m7GTP, 0.1 mM cap analog M.sup.7G.sup.5'ppp.sup.5'G, 0.5 mM
m7guanosine or 0.5 mM GTP. The pretreated supernatant was then used
in the standard ELISA procedure. The GTP did not compete with the
m7guanosine-BSA whereas the m7 versions all competed
efficiently.
[0127] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0128] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
1154DNAArtificialSynthetic oligonucleotide primer 1gactagttct
agatcgcgag cggccgccct tttttttttt tttttttttt tttt 54
* * * * *