U.S. patent application number 10/173509 was filed with the patent office on 2003-01-23 for method of identification and cloning differentially expressed messenger rnas.
This patent application is currently assigned to New York Blood Center Inc.. Invention is credited to Belyavsky, Alexander V., Ivanova, Natalia B..
Application Number | 20030017490 10/173509 |
Document ID | / |
Family ID | 20157730 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017490 |
Kind Code |
A1 |
Belyavsky, Alexander V. ; et
al. |
January 23, 2003 |
Method of identification and cloning differentially expressed
messenger RNAs
Abstract
A method of identification of differentially expressed messenger
RNA (mRNA) which consists of synthesizing from a set of sequences
of mRNA sets of fragments of complementary DNA (cDNA), which are
separated with the aid of gel electrophoresis and the pictures of
separation of the cDNA from different types of cells are compared
and fragments with differential signal intensity are identified.
For formation of the set of fragments the cDNA is cleaved with the
aid of restriction nucleases. A method of cloning of differentially
expressed mRNAs consists of synthesizing from sets of sequences of
mRNAs from different types of cells sets of fragments of
complementary DNA (cDNA) which are separated with the aid of gel
electrophoresis, the pictures of the separation of the cDNA from
different types of cells are compared, fragments of cDNA with
different signal intensities are separated from the gel, amplified
with the aid of a polymerase chain reaction and cloned to a plasmid
or phage vector. For the formation of the set of fragments one
carries out cleavage of the cDNA with the aid of restriction
endonucleases and uses only those fragments of cDNA that correspond
to the 3' or 5' end regions of the mRNAs.
Inventors: |
Belyavsky, Alexander V.;
(Moscow, RU) ; Ivanova, Natalia B.; (Moscow,
RU) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
New York Blood Center Inc.
|
Family ID: |
20157730 |
Appl. No.: |
10/173509 |
Filed: |
June 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10173509 |
Jun 18, 2002 |
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09664534 |
Sep 18, 2000 |
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09664534 |
Sep 18, 2000 |
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09247871 |
Feb 11, 1999 |
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09247871 |
Feb 11, 1999 |
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08964143 |
Nov 6, 1997 |
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6120996 |
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08964143 |
Nov 6, 1997 |
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08499899 |
Jul 11, 1995 |
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5814445 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12N 15/1096 20130101;
C12N 15/1072 20130101; C12Q 1/6809 20130101; C12Q 1/6855
20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 1994 |
RU |
94-024056 |
Claims
1. A method of identification of differentially expressed messenger
RNA (mRNA) which consists of synthesizing from sets of mRNA sets of
fragments of complementary DNA (cDNA), which are separated with the
aid of gel electrophoresis, the separation pictures of the cDNA
from different types of cells are compared and one identifies
fragments with differential intensity of signals, and which is
distinguished by the fact that, for the formation of a set of
fragments, cleavage of the cDNA is carried out with the aid of
restriction endonucleases.
2. A method as in claim 1, which is distinguished by the fact that
for creation of the set of fragments of cDNA one uses only those
restriction fragments that correspond to the 3' or 5' and regions
of the mRNA and the fragments of cDNA are labeled by introducing at
one end a radioactive label, a chemical grouping or specific
sequence of nucleotides.
3. A method as in claim 2, which is distinguished by the fact that
for creation of the set of fragments of cDNA one uses specific
immobilization of and fragments on a solid carrier.
4. A method as in claim 3, which is distinguished by the fact that
formation of the set of fragments of cDNA is done by means of
ligation, to and fragments of cDNA that are immobilized on a solid
carrier and that correspond to the 3' or 5' ends of the mRNA, of a
double-chain adaptor or single-chain oligonucleotide and
reamplification of the set of fragments by means of a polymerase
chain reaction.
5. A method as in claim 4, which is distinguished by the fact that
for equalization of the degree of representation of different
sequences in the set of fragments single or multiple removal of the
most represented sequences in the set of fragments of cDNA is done
by means of hybridization of nonimmobilized chains of fragments of
cDNA with complementary chains immobilized on a solid carrier.
6. A method as in claim 4 or 5, which is distinguished by the fact
that, with the goal of improving the sensitivity and resolution of
the method, the initial set of fragments of cDNA is divided into
nonintersecting subsets of fragments.
7. A method as in claim 6, which is distinguished by the fact that
the division of the initial set of fragments of cDNA into subsets
is done by means of immobilization of the set of fragments labeled
at one end, by the other end on a solid phase and successive
cleavage with a set of restriction endonucleases.
8. A method as in claim 6, which is distinguished by the fact that
the division of the initial set into subsets is done with the aid
of several separate amplification reactions with paired
combinations of primers corresponding to and sequences that are
common for all of the fragments of cDNA, but that contain an
additional base of the 3' and, immediately after the regions that
are common for all the fragments of cDNA.
9. A method as in any of claims 4-8, which is distinguished by the
fact that radioactive .sup.32P or .sup.33P is used as the
label.
10. A method as in any of claims 4-8, which is distinguished by the
fact that a chemical grouping is used as the label and the
separated fragments after transfer to a membrane are detected with
the aid of a reagent that specifically binds this group and is
conjugated with the enzyme, by means of registration of the
enzymatic activity of this enzyme.
11. A method as in any of claims 4-8, which is distinguished by the
fact that the separated fragments are transferred to a membrane and
after immobilization visualization of the fragments is done by
means of hybridization with a labeled oligonucleotide corresponding
to the adaptor sequence.
12. A method as in any of claims 4-8, which is distinguished by the
fact that the separated fragments are transferred from membrane and
after immobilization visualization of the subsets of separated
fragments is done by means of hybridization of the membrane with a
set of labeled oligonucleotides that correspond to the adaptor
sequence and that carry at the 3' and one or several additional
bases.
13. A method as in any of claims 4-12, which is distinguished by
the fact that the fragments are separated by two-dimensional gel
electrophoresis.
14. A method as in claim 13, which is distinguished by the fact
that fragments are separated by length in one direction of
two-dimensional electrophoresis and by melting profile using a
denaturing gradient in the other direction.
15. A method as in claim 13, which is distinguished by the fact
that single-chain fragments are separated by length under
denaturing conditions in one direction of two-dimensional
electrophoresis and according to secondary structure in
nondenaturing conditions in the second direction.
16. A method as in claim 13, which is distinguished by the fact
that fragments are separated by length in the first direction of
two-dimensional electrophoresis and in the switch to the second
direction the fragments of cDNA are specifically cleaved in the gel
by a set of restriction endonucleases, after which a second
separation by length is carried out.
17. A method of cloning of differentially expressed mRNAs which
consists of synthesizing from a set of sequences of mRNAs from
different types of cells sets of fragments of complementary DNA
(cDNA) which are separated with the aid of gel electrophoresis, the
pictures of separation of the cDNA from different types of cells
are compared, the fragments of cDNA with different signal
intensities are separated from the gel, amplified with the aid of a
polymerase chain reaction and cloned to a plasmid or phage vector,
and which is distinguished by the fact that, for the formation of
the set of fragments one carries out cleavage of the cDNA with the
aid of restriction endonucleases and uses only those fragments of
cDNA that correspond to the 3' or 5' end regions of the mRNAs.
Summary A method of identification of differentially express
messenger RNA (mRNA) which consists of synthesizing from a set of
sequences of mRNA sets of fragments of complementary DNA (cDNA),
which are separated with the aid of gel electrophoresis and the
pictures of separation of the cDNA from different types of cells
are compared and fragments with differential signal intensity are
identified. For formation of the set of fragments the cDNA is
cleaved with the aid of restriction nucleases. A method of cloning
of differentially expressed mRNAs consists of synthesizing from
sets of sequences of mRNAs from different types of cells sets of
fragments of complementary DNA (cDNA) which are separated with the
aid of gel electrophoresis, the pictures of the separation of the
cDNA from different types of cells are compared, fragments of cDNA
with different signal intensities are separated from the gel,
amplified with the aid of a polymerase chain reaction and cloned to
a plasmid or phage vector. For the formation of the set of
fragments one carries out cleavage of the cDNA with the aid of
restriction endonucleases and uses only those fragments of cDNA
that correspond to the 3' or 5' end regions of the mRNAs.
Description
ART
[0001] This invention relates to the field of molecular biology and
can be used in medicine and molecular biology for analysis of gene
expression and diagnosis and identification of mechanisms of
pathology at the genetic level.
PRIOR ART
[0002] Differential expression of genes, which is realized via the
synthesis of various sets of messenger RNAs (mRNAs), is the basis
of the variety of phenotypes and functions of the cells of living
organisms. In this connection one can understand the importance of
methods that might allow identification and investigation of
differentially expressed mRNAs, i.e., those RNAs, whose
concentration in the cellular mRNA pool differs in two or more
types of cells or changes in dependence on the functional state of
the cells.
[0003] A number of methods have been described for the detection
and cloning of differentially expressed mRNAs. In most cases they
come down to the use of the method of hybridization of nucleic
acids. In the differential screening method (see literature [1]) by
means of transfer to nitrocellulose or nylon filters replicas of
libraries of complementary DNA (cDNA) from the population of cells
A sown in dishes are hybridized alternately with labeled
preparations of cDNA from the population of cells A, B, C and so
forth. Since the hybridization signal obtained from individual
clones theoretically should be proportional to the representation
of the corresponding cloned sequence in the cDNA preparation, one
can, from the level of the signal obtained from different
preparations, identify clones corresponding to genes that are
expressed differentially in the populations of cells A, B and so
forth. An important shortcoming of this approach is its low
sensitivity, since only highly represented sequences (as a rule,
those comprising at least 0.1% mRNA) can be detected. For less
common sequences the hybridization signal does not exceed the
background. In addition, in this approach one can examine only a
small number (from several thousands to several tens of thousands)
of clones. Another shortcoming is that information about the
individuality of the clone (the description of its location in the
dish or replica) is lost at the end of work with these replicas and
cannot be used in subsequent experiments. Moreover, cDNA libraries
from different types of cells are not comparable in that identical
sequences occupy different places on filters in different
libraries. Thus, conversion from one type of cells to other types
is essentially impossible. Recently attempts have been made to
create ordered cDNA libraries containing individual clones
distributed in the cells of 96-well planchets. However, this does
not eliminate the principal shortcoming of low sensitivity. In
addition, sets of clones obtained from one type of cell are still
not comparable to sets of clones from another type of cell.
[0004] The method of subtracted hybridization is used in order to
overcome the low sensitivity of the differential screening method
[1]; in this method a cDNA preparation from cells A is hybridized
with a cDNA or mRNA preparation from cells B, after which the
resulting hybrids are removed by one or another method. The
resulting preparation is rich in sequences that are specific for
preparation A. This "subtracted" preparation can be used both for
the creation of libraries and for differential screening, since it
makes it possible both to increase the probability of finding
differentially expressed sequences and to increase the sensitivity
of detection of poorly represented sequences. However, the use of
this procedure makes the approach lengthy and laborious and, in
addition, the gain in sensitivity is still insufficient for
detection of rare sequences and is accompanied by the loss of the
possibility of making a direct comparison of the level of gene
expression in different cells.
[0005] Recently a modification of the differential screening method
was proposed, in which instead of cloned sequences large orderly
arranged sets of oligonucleotides are used for immobilization on a
solid substrate, for example, all possible tetradecanenucleotides
containing the common sequence AATAAA, which is encountered in most
mRNAs [2]. This method theoretically provides an exhaustive search
of a much larger set of mRNA sequences than in standard
differential screening. The shortcoming of this approach, like
other differential screening methods, is that under conditions of
hybridization with an excessively complex mixture of cDNA
molecules, the frequencies of representation of which may vary by
factors of hundreds and thousands, the probability of the
appearance of false signals owing to cross hybridization becomes
significant, which makes reliable detection and quantitative
analysis of rare mRNA sequences problematical. An additional
problem is the need for synthesis and immobilization on a small
area of from several dozens to hundreds of thousands of
oligonucleotides, which requires a large initial investment. In
addition, the approach in and of itself does not provide the
possibility of cloning any long segments of differentially
expressed mRNAs, which requires a separate operation.
[0006] Closest to the present invention is a method which is called
"PCR mated with reverse transcription, with arbitrary primers"
(arbitrary primed RT-PCR). In this method cDNA synthesized by mRNA
is used to create a set of cDNA fragments of discrete length with
the aid of a polymerase chain reaction (PCR) using primers of a
random sequence under nonrigorous conditions which contribute to
priming and amplification not of one, as is usual in standard PCR,
but rather of a whole group of cDNA sequences. The set of discrete
cDNA fragments is separated by electrophoresis in acrylamide gel
and compared with a set of fragments obtained under identical
conditions from a different type of cell. By using many arbitrary
primers, one can compare one or several discrete cDNA fragments to
most mRNA species. Two principal modifications of this approach
have been proposed. One of them uses one or two "arbitrary" primers
[3,4]. The second approach uses one arbitrary and one oligo (dT)
primer that contains two additional bases at the 3' end [5,6,7]. In
this case the amplified segments are adjacent to the 3' and
(poly(A) tail) of the mRNA. An important shortcoming of this
approach is the fact that no more than a few dozen sequences are
amplified in a single reaction. Thus, for an exhaustive search of
10-15 thousand RNA sequences expressed in individual types of cells
or tissues, as a minimum several hundred (probably several
thousand) reactions must be carried out. In addition, the sampling
of sequences amplified in one reaction is random and, therefore, in
the exhaustive search of a significant portion of sequences of
cellular mRNA there will be a very significant accumulation of
excess information. Thus, after a survey of 50% of all sequences,
in each subsequent reaction only {fraction (1/2)} of the amplified
sequences will be now. At a depth of review of 90% the fraction of
now sequences will fall to {fraction (1/10)}. Probably the most
significant shortcoming of the approach is its poor
reproducibility, which is connected with the fact that under weakly
selective priming conditions small variations in the starting
conditions or the quality of the RNA preparations, primers and
other parameters will cause significant quantitative changes in the
spectrum of amplified cDNA fragments. Thus, if the optimum
annealing temperature is 42.degree. C. a shift of the annealing
temperature up or down by 2.degree. resulted in the appearance of a
substantial background (i.e., additional bands) or to disappearance
of some bands [7]. Second, according to the data of the authors of
[7] when parallel experiments were carried out, about 95% of bands
proved to be reproducible [6]. Thus, the level of
nonreproducibility (5%) is comparable to or even exceeds the level
of differences between mRNA populations. According to our data,
when this approach is used, the number of reproducible differential
bands is usually smaller than the number of nonreproducible bands.
It is probable that the sensitivity of the approach to the quality
of preparations of RNA, primers, annealing time and other
parameters may substantially prevent a comparison of results with
known data.
[0007] Essence of Invention
[0008] The basis of the invention was the task of supporting the
possibility of a direct qualitative and quantitative comparison of
the spectra of mRNAs synthesized in different types of cells of the
same organism, the possibility of detection of differentially
expressed RNAs, including those with a low level of representation,
the possibility of correlating these RNAs with known ones, and also
the possibility of cloning fragments of such RNAs. The problem is
resolved by the fact that the set of messenger RNAs expressed in a
cell by means of synthesis of complementary DNA (cDNA) with
subsequent fragmentation of the cDNA by frequently cleaving
restriction endonucleases (having four- or five-letter recognition
sites) and use of the cDNA fragments corresponding to the 3' or 5'
ends of the mRNA, is represented in the form of a set of fragments
of cDNA of discrete length, no more than one fragment for each
species of mRNA, and at least one of the ends of the fragments
carries the marker or group necessary for detection. The generated
fragments of cDNA originate from the 3' end (adjacent to the
poly(A) tail) or 5' end regions of the mRNAs and support
representation, depending on the specific embodiment of the
invention, of from 90% to essentially 100% of mRNA sequences. For
an increase of the sensitivity of the approach and for unambiguous
identification of individual fragments the set of cDNA fragments
may be divided into several nonintersecting subsets of fragments.
The sets or subsets are divided by one- or two-dimensional gel
electrophoresis and detection of the marker and comparison of the
separation pictures is carried out. In this case the intensity of
the signal from each fragment of cDNA varies for mRNA preparations
from different cells in proportion to the representation of the
sequence corresponding to it in the mRNA pool. If necessary in the
detection stage, by means of transfer of the separated fragments to
a membrane and sequential hybridization with a set of
oligonucleotides, which partially overlap the common end sequence
of the fragments, additional analysis of the separation picture is
possible. The separated cDNA fragments, after additional operations
can be amplified and investigated by means of restriction and
hybridization analysis and also sequencing, and can be cloned to a
plasmid or phage vector.
[0009] Thus, in the proposed invention, in contrast to the approach
using arbitrary primers, at least one of the boundaries of the
fragments is specified by the position of the restriction site*; in
addition, in the proposed approach a set of cDNA fragments that is
as representative as possible is created at first and then divided
into nonintersecting subsets and than separated with the aid of gel
electrophoresis. The use of the reaction of cleavage of DNA by
restriction nucleases, which is characterized by high specificity,
completeness of cleavage and low sensitivity to variations of
temperature conditions, makes it possible to make the approach much
more reproducible and, therefore, will make possible a reliable
comparison with independently conducted experiments. These
characteristics, together with the high resolution of the method,
will make it possible to begin the creation of databases of gel
coordinates of expressed sequences. The proposed method makes it
possible to eliminate the excess of information that is
characteristic for the method with arbitrary primers. In addition
the presence of one common population of cDNA fragments in the
initial stages makes it possible to conduct such operations as
frequency equalizations by means of self-hybridization of the
population of cDNA fragments [8], which will make it possible to
reduce substantially the level of representation of the most
frequently encountered mRNA species and will help the isolation of
very rare mRNA sequences owing to a reduction of interference from
frequently encountered sequences.
LIST OF FIGURES
[0010] FIG. 1 shows one version of implementing the invention by
means of the formation of a set of 3' and labeled fragments of
cDNA, dividing it into subsets of fragments with the aid of
immobilization on a solid support and sequential treatment with a
series of restriction nucleases, and separation of the resulting
subsets by electrophoresis.
[0011] FIG. 2 shown a scheme of amplification and cloning of cDNA
fragments obtained and separated in gel with the aid of the method
shown in FIG. 1.
[0012] FIG. 3 shows a picture of separation in denaturing
polyacrylamide gel electrophoresis of .sup.32P-labeled fragments of
cDNA from preparations of RNA of mouse thymus (a) and mouse spleen
(b) obtained with the aid of the method presented in FIG. 1. The
first cleavage was done with the enzymes Sau3A (2-5) and BamHI (6),
the second with the enzymes EcoRV (2), PstI (3, 6), mspI (4) and
Hin PI (5). Microfragments of DNA are plotted on the left (1) and
their lengths in nucleotides are indicated.
[0013] FIG. 4 represents hybridization of cloned fragment 1 (a) and
fragment 2 (b) with preparations of poly(A).sup.+ RNA from mouse
spleen (1) and thymus (2) separated by electrophoresis and
transferred to a membrane. The picture of hybridization confirms
the specificity of expression that follows from FIG. 3. On the
right are plotted marker single-chain DNA fragments and their
lengths and nucleotides are indicated.
[0014] FIG. 5 shows an alternative method of dividing the set of 3'
and fragments of cDNA obtained by the method shown in FIG. 1 into
four subsets by means of amplification of the set of fragments by a
polymerase chain reaction with adaptor primers that contain a
supplemental base at the 3' end beyond the limits of the part that
is common for all fragments.
[0015] FIG. 6 shows a method of creating a set of 5' end fragments
of cDNA.
[0016] One variation of the announced method is presented in more
detail below.
[0017] Synthesis of the first chain of cDNA is accomplished by the
enzymatic route with the aid of reverse transcriptase and oligo(dT)
primer immobilized on microgranular solid support or of
oligo(dT)-containing primer carrying a biotin group at the 5' end.
For synthesis of the second chain of cDNA, preliminary suspension
of the oligo(dG) tail at the 3' end of the first chain of cDNA with
the aid of terminal transferase is carried out. Synthesis of the
second chain is carried out with modified DNA polymerase of phage
T7 sequenase (United States Biochemicals, USA) using an
oligo(dC)-containing primer as primer. Complete hydrolysis of the
cDNA with restriction endonucloase having a four-letter recognition
site (for example, Sau3A), rinsing of the released cleavage
products and ligation of the adaptor complementary to the cleavage
site are carried out. If a biotinylized primer is used for
synthesis of the first chain, the cDNA before or after cleavage
with restrictase is bonded to a streptavidin-containing solid
support (Streptavidin MagneSphere, Promega, USA). The ability of
streptavidin to bind the biotin group rapidly and stably is used in
this operation. To increase the above material, amplification of
the fragments of cDNA is carried out by means of PCR using the
primer that is in the adaptor and modified oligo(dT) primer
containing the biotin group at the 5' end. After immobilization of
the fragments in the streptavidin-containing solid support the free
ends are removed by treating with 100 mM NaOH, and then the primer
is annealed in the immobilized chains, the label is added to the 3'
end of the primer by the chain lengthening reaction with DNA
polymerase using radioactive .alpha.-.sup.32P or .alpha.-.sup.33P
DATP, after which unlabeled deoxynucleotide triphosphates are added
and complete chains of cDNA are completed. The sequence of primer
and segment cDNA annealed with it and also the conditions of the
labeling reaction are such that the region labeled is limited to
the adaptor sequence. After adding the marker, sequential
exhaustive cleavage with 8-10 restrictases is carried out, with
restrictases having 6-letter recognition sites being used first,
followed by 5-letter and finally 4-letter recognition sites. The
released labeled fragments of cDNA after each reaction are
collected separately. Separation of the fragments is done with one
of the systems for electrophoresis in polyacrylamide or similar gal
(Hydrolink, AT Biochem, USA). The system of two-dimensional
electrophoresis of DNA, in which separation of the double-chain DNA
along the length is carried out in the first reaction, and
separation with regard to composition is done in the second
direction (more precisely, over the melting profile) with a
denaturing gradient [9]. According to literature data, even after
transfer to a membrane, which significantly degrades resolution,
this kind of system makes it possible to separate at least 625
fragments of DNA on one gel [10]. To all appearances the upper
limit of resolution is 1 to 1.5 thousand DNA fragments. If the
population of cDNA fragments is divided into 8-12 nonintersecting
sets, one can separate up to 10-15 thousand fragments, i.e.,
essentially all mRNAs expressed in cells of a single type. The
signal from the separated fragments is detected by means of
autoradiography on x-ray film. The pictures obtained from
preparations of RNA from different types of cells are compared with
each other, the correspondence between individual fragments of cDNA
from different cells is established from the position in one- or
two-dimensional electrophoresis and fragments giving a differential
signal are identified.
[0018] In order to ascertain the sequence of known sequences to
which one or another spot on the two-dimensional electrophoreogram
may correspond, one first, based on cloned sequences of mRNAs in
the data bank, determines which sequences may give fragments of the
corresponding link and then, by using the algorithm for prediction
of mobilities of fragments of DNA in a denaturing gradient [11],
one can chose from those sequences the one whose melting profile
most accurately corresponds to the position occupied by the
fragment in the second direction.
[0019] In order to obtain the needed fragment in a quantity
sufficient for analysis and cloning, the fragment is eluted from
the gel, the oligo(dG) tail is suspended with the aid of terminal
transferase, and then amplified by means of PCR using adaptor
primer and oligo(dC) (FIG. 2). The identical nature of the fragment
of the known sequence can be verified by means of restriction
analysis or by determining the nucleotide sequence directly in the
amplified fragment. In addition, the amplified fragment may be
easily cloned to the plasmid or phage vector.
[0020] Our computer analysis of 659 cloned sequences of mouse mRNA
having a sequestered region belonging to the poly(A) tail showed
that when the restriction endonuclease Sau3A (recognition site:
GATC) was used for cleavage of the cDNA 93% of all mRNAs will be
cleaved by the enzyme and, therefore, will participate in the
analysis. The fraction of sequences of mRNAs participating in the
analysis can be increased to 99%-99.5%, if cleavage is done with a
second restrictase having a different recognition site of those
sequences of cDNA that were not cleaved by the first restrictase.
The computer analysis also shows that second cleavage of
immobilized fragments of cDNA with a set of restriction
endonucleases can reach the overwhelming majority of fragments (96%
if tan four-letter restrictases are used). Thus, from 90 to 96% of
all mRNA sequences of the cell will be represented in the form of
discrete fragments of cDNAs and will participate in the analysis
period.
[0021] The proposed method has high sensitivity. Using only 1 .mu.g
of cDNA fragments for labeling it is easy to obtain inclusion to
10.sup.8 count/min. This level of labeling makes it possible to
detect mRNA sequences that make up from 0.001% to 0.0001% of the
mRNAs of the cell.
[0022] Besides the variation described above, there are also other
variations of the invention. Synthesis of the second chain of cDNA
can be accomplished by other known methods by using as primers for
synthesis the chain or RNA in hybrid cDNA-RNA that has been cleaved
with the aid of RNAse H or by using self-priming of the first cDNA
chain after hydrolysis of the RNA chain [12]. The most important
modification is the use of the alternative method of dividing the
set of 3' end fragments of cDNA into nonintersecting subsets of
fragments by means of amplification with the aid of 12 different
pairs of primers, which include a) four versions of a modified
adaptor primer, which includes an adaptor end sequence that is
common for all fragments of cDNA and that each contains one
additional base at the 3' end; b) three versions of oligo(dT)
containing primer that contains one additional bass at the 3' end.
In this case one employs the property of Taq DNA polymerase to
extend only those primers that contain a completely paired 3' and
base [13]. In this version it is not necessary to carry out second
cleavage with the set of restrictases and, therefore, the share of
mRNA sequences that participate in the analysis can essentially
reach 100%.
[0023] One further important version of the approach is creation of
a set of fragments of cDNAs corresponding to the 5' end of the
mRNAs (FIG. 6). For this one uses specific labeling of the 5' end
of the mRNAs with an oligonucleotide primer (primer 1) in
accordance with the procedure of [14]. After this, one synthesizes
the first chain with the aid of a set of random haxamer primers,
synthesizes the second chain with the aid of primer 1 containing a
biotin group at the 5' end, immobilizes the cDNAs in streptavidin
microgranules, followed by hydrolysis of the cDNAs with
restrictase, ligation of the adaptor and other procedures, as
indicated above. Dividing the set of 5' end fragments of cDNAs into
subsets is accomplished either by immobilization of the labeled
fragments and sequences by treatment with restrictases or with the
aid of separate reactions of amplifications with and primers that
contain an additional base at the 3' end, as indicated above.
[0024] Besides two-dimensional electrophoresis using a denaturing
gradient in the second direction, it is also possible to use
systems of two-dimensional electrophoresis that use in the second
direction separation of single-chain DNA under nondenaturing
conditions owing its conformational polymorphism [15] or cleavage
of DNA after the first direction by frequently cleaving
restrictases [16] and separation by length in the second
direction.
[0025] Besides the use of radioactive .sup.32P or .sup.33P markers
with subsequent autoradiography for detection, a nonradioactive
variation of detection may also be accomplished; in this version
labeling of the fragments of cDNA is done with biotin groups or
other chemical groups that after transfer of the fragments to the
membrane and immobilization are detected with one of the commercial
systems of nonradioactive detection using chemiluminescence.
[0026] Detection of the separated unlabeled elements of cDNA can be
done by transfer of fragments to a membrane and hybridization with
a labeled adaptor primer. In this case, after hybridization with
the adaptor primers that contain additional basis at the 3' and,
selected visualization of the subsets of fragments separated in the
given gel is possible.
[0027] Information Confirming the Possibility of Implementation of
the Invention
[0028] To support the possibility of identification and cloning of
differentially expressed mRNAs, the experiments presented in the
following example were carried out.
EXAMPLE
[0029] Identification and cloning of mRNAs differentially expressed
in mouse thymus and spleen.
[0030] Preparation of total RNA are extracted from mouse thymus and
spleen in parallel using extraction with acid phenol [17].
Synthesis of the first cDNA chain was accomplished under the
following conditions: 37.degree. C., 60 min, reaction volume 20
.mu.L, 5 .mu.g total RNA, 200 U reverse transcriptase Superscript
(Gibco-BRL, USA), 10 pmol (T)-primer, biotinylized at the 5' end
(sequence 5'-biotin-GGGAGGCCC(T).sub.13), 30 U RNAse inhibitor from
human placenta, dATP, dGTP, dCTP, dTTP (1 mM each), 1.times. buffer
of reverse transcriptase in accordance with manufacturer's
recommendations make up the reaction mixture. Removal of the primer
is done with the aid of reprecipitation using
cetyltrimethylammonium bromide [18], after which additional
purification is carried out on a Wizard column (Promega, USA)
according to manufacturer's recommendations. The purified
preparation is precipitated with 3 volumes of ethanol using 2 .mu.g
glycogen (Boehringer-Mannheim, Germany) as carrier.
[0031] Suspension of the oligo(dT) tail is done under the following
conditions: 37.degree. C., 20 min, reaction volume 20 .mu.L, with
the reaction mixture containing hybrid mRNA-first chain of cDNA, 20
U terminal transferase (Gibco-BRL, USA), 0.02 mM dGTP, 1.times.
buffer of terminal transferase in accordance with manufacturer's
recommendations. Synthesis of the second chain of cDNA is done
under the following conditions: denaturation 98.degree. C., 1.5
min, annealing 60.degree. C., 2 min, elongation 72.degree. C., 20
min, reaction volume 25 .mu.L, reaction mixture contains: hybrid
mRNA-cDNA, 10 pmol (C)-primer (sequence 5'-AAGGAATT(C).sub.13),
dATP, dGTP, dCTP, dTTP (0.1 mM each), 1.5 U DNA polymerase Bio-Taq
(Biomaster, Russia), 1.times. buffer of Bio-Taq in accordance with
manufacturer's recommendations. Cleavage of cDNA by restriction
endonuclease is done under the following conditions: 37.degree. C.,
60 min, reaction volume 20 .mu.L, 4 U restriction endonuclease
Sau3A (New England Biolabs, USA), 1.times. buffer of enzyme
(according to manufacturer's recommendations). After hydrolysis the
reaction is stopped by the addition of EDTA to 20 mM and the 3' and
fragments of cDNA are immobilized on Streptavidin microbeads
(Promega, USA) in accordance with manufacturer's recommendation.
The adaptor is added:
[0032] 1. 5'-GATCGTTTTTTGAAGCTTGGAGCCCAC-3'
[0033] 2. 3'-CAAAAAACTTCGAACCTCGGGTG-5'
[0034] and ligated at 12.degree. C. overnight. Reamplification of
the cDNA fragments with the aid of PCR is done using Bio-(T.sub.13)
primer and primer 1 under the following conditions: denaturation
95.degree. C., 1.2 min, annealing 55.degree. C., 1.5 min,
elongation 72.degree. C., 3 min* reaction volume 100 .mu.L,
reaction mixtures contains: 30 pmol Bio-(T) primer, 30 pmol primer
1, 2.5 U DNA polymerase Bio-Taq; the mixture of deoxynucleotide
triphosphates, 0.1 mM each, 1.times. buffer of Bio-Taq. After 15
PCR cycles the fragments are immobilized on Streptavidin microbeads
(Promega, USA), then the free chain is removed by treatment with
100 mM NaOH for 10 min, rinsing with a buffer of composition: 40 mM
Tris-Cl, pH 7.0, 20 mM MgCl.sub.2, 50 [?] NaCl, primer 3
(5'-GTGGGCTCCAAGCTTC) is annealed, radioactive .alpha.-.sup.32P
dATP is added and the marker is added over 5 min using modified DNA
polymerase of phage T7 Sequenase (United States Biochemicals, USA),
after which the mixture of dNTP is added (to 0.2 mM each) and
complete chains are finished. Cleavage of the fragments is done
successively with restriction endonucleases EcoRV, PstI, MspI, Hin
PI (New England Biolabs, USA). After each endonuclease treatment
the fragments are collected, denatured and applied to sequestering
polyacrylamide gel (5% acrylamide, 0.25% methylene-bisacrylamide,
7M urea, 1.times. TBE buffer). Separation of the fragments is done
under standard conditions after which the gel is fixed in 10%
acetic acid for 30 min, dried and autoradiographed. The sequences
of mRNAs that are differentially expressed in thymus and spleen are
identified by means of a direct comparison of the sets of bands of
cDNA obtained from thymus and spleen and separated in adjacent
tracks. For cloning of the differentially expressed sequences the
corresponding bands of gel are cut apart and the fragments are
eluted by incubation in 150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 10 mM
EDTA overnight. Than precipitation of the fragments is carried out
with three volumes of 96% ethanol using glycogen as carrier. The
oligo(dG) tail is suspended at the 3' end of the fragment using
terminal transferase, as described above, and the fragment that has
been processed in this way is amplified by means of PCR using (C)
primer and primer 1.
[0035] The amplified fragment is purified with the aid of
electrophoresis in agarose gel, transferred to low melting agarose
and recovered in pure form with the aid of phenol extraction [19].
For cloning of the fragments they are treated with restrictase
Sau3A and EcoRI and ligated to plasma vector pUC18 cleaved by
restrictase BamHI and EcoRI, after which they transform competent
bacteria. The presence of recombinant clones is verified with the
aid of amplification of insertions using PCR. Verification of the
specificity of the cloned fragments is done by means of
hybridization of .sup.32P-labeled insertions from clones with blots
of amplified cDNA and poly (A).sup.+ RNA from the corresponding
organs.
[0036] The data in FIG. 3 show that this method leads to the
formation of a multitude of labeled discrete fragments of cDNAs
which can be separated in gel electrophoresis, and in comparing the
pictures of the separation of cDNAs from two different organs one
can identify differential bands. The fragments corresponding to the
differential bands can be amplified and cloned by the method
indicated in FIG. 2. The data given in FIG. 4 show that the cloned
fragments of cDNAs are indeed mRNAs differentially expressed in two
organs.
References
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* * * * *