Note: Descriptions are shown in the official language in which they were submitted.
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A method for amplifying low abundance nucleic acid
sequences and means for performing said method
s FIELD OF THE INVENTION
The present invention relates to methods as well as to nucleic
acid primers and kits containing the same for performing efficiently an
amplification of nucleic acid sequences from a sample, particularly of
io nucleic acid sequences that are initially poorly represented in said
sample.
BACKGROUND OF THE INVENTION
is DNA sequence information resulting from genome and
expressed sequence tag (EST) sequencing projects is expected to
provide the basis for further understanding of the control and mode of
action of individual, and groups of gene products.
In this respect, analysis and comparison of when, where and to
2o what degree genes are expressed, commonly known as expression,
profiling, is playing an essential role in the functional characterization of
newly identified genes.
Many tissue cellular systems, such as the immune and nervous
systems, are composed of highly heterogeneous cell populations. A key
2s factor lies in understanding their physiology, and the role of specific
gene
products expressed with the ability to examine gene usage in the context
of this cellular diversity.
In the past, low throughput and laborious methods such as
Northern Blotting and nuclease protection assays were employed to
3o study gene expression.
More recently, various methods have been developed for
assessing simultaneously the expression of large numbers of genes.
All these techniques, however, require relatively large amounts
of RNA and currently lack the sensitivity to analyze specimens derived
3s from small populations of cells or indeed from an individual cell.
CONFIRMATION COPY
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This is compounded by the fact that it is very difficult in the case of many
cell types to obtain enough specific cellular material for RNA
experimentation. Consequently many areas of investigation are
frustrated by lack of starting material.
s Thus in situ hybridization provides detailed information on the
cellular expression pattern of a gene in intact tissue. However, this
technique is laborious to perform, and does not allow the analysis of
more than a very small number of transcripts in a single preparation,
when performed in whole-mounts or tissue sections.
io The polymerase chain reaction (PCR) has been used
successfully to investigate gene expression in cytoplasmic samples,
particularly with the nested-primer approach which provides good
sensitivity, but restricts the analyses to a small number of closely related
genes from specific gene families.
is Some techniques allow detection of the expression of unrelated
genes in a single cell, such as T7 RNA polymerase amplification of
mRNA and PCR after prior homopolymeric tailing of the first strand
cDNA. However, neither of these approaches have been demonstrated
to allow the analysis of more than a small number of genes and are not
2o widely used.
The former is technically difficult, whilst the latter may be biased
against long transcripts and often requires subsequent cloning of the
amplified products.
Alternatively, a method for expression profiling in single cells
2s using 3' end amplification PCR has been developed by Dixon et al.
(1998, Nucleic acids research, vol. 26 (n°19): 4426-4431). This method
comprises a first step wherein mRNA species present in a cell are
reversed transcribed using a first heeled primer, thereby providing a
population of first strand cDNA species and a second step wherein
3o partial 3' end second cDNA strand populations are synthesized using a
second heeled primer population.
Using this technique of amplification, the authors have
succeeded in detecting, from a mRNA population contained in the
cytoplasm of a single cell, the presence of poorly expressed transcripts
3s in cholinergic interneurons such as the neurokinin type 1 receptor.
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However, one of the drawbacks of this technique is that it does
not allow the detection of more than 40 low abundance genes from a
single cell. This technique generates large amounts of high molecular
weight cDNA in gene specific PCR reactions. This not only reduces the
s sensitivity of the PCR assay but means that much of the amplified
product may not be assayed for gene sequence.
SUMMARY OF THE INVENTION.
io The inventors have developed sensitive methods for amplifying
mRNA species present in a sample that allows the detection and cloning
of one or several mRNA species of interest, particularly mRNA species
which are initially present at a low copy number in a sample to be
assayed.
is For instance, when applying the new method of the invention to
mRNA samples obtained from cholinergic neurones, the inventors have
succeeded in detecting the expression of low abundance A1 receptor
mRNA at levels 50 fold lower than those possible using previous
methods. In addition, when applying the method of the invention to 2.5
2o ng of total RNA (equivalent to that contained in approximately 250 cells),
specific gene sequences could be detected using one millionth of the
final product.
The present invention also relates to methods for increasing the
number of nucleotide sequences corresponding to the mRNA species
2s present initially at a low copy number in a sample to be assayed.
In addition, this technology allows high throughput analysis
systems, e.g. arrays or gene chips to be used to analyse gene
expression on extremely small samples, including analysing the
expression of genes in a single cell.
3o The invention also pertains to various technical means that are
necessary to perform these methods, and particularly to oligonucleotide
primers that are required to perform the methods of the invention.-
Additionally, other objects of the invention consist of kits that
are specially designed to perform the disclosed methods, particularly kits
3s containing the oligonucleotide primers mentioned above.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Detection of gene specific sequences after
amplification of cDNA derived from 100 pg of total RNA using the first
s embodiment of the method of the present invention.
Figure 2: Detection of gene specific sequences after
amplification of cDNA derived from total RNA using the second (l)and
third (II) embodiments of the method of the present invention.
to
Figure 3: Diagram to illustrate product priming/product repair
after amplification of small amounts of cDNA using the first and second
embodiment of the method of the present invention.
Figure 4: Detection of gene specific sequences after high
stringency amplification of cDNA derived from 1000pg of total RNA using
the third embodiment of the method of the present invention.
2o Figure 5: Detection of gene specific sequences after in vitro
transcription of RNA from amplified cDNA derived from liver total RNA
using the third embodiment of the method of the present invention.
Figure 6: Size distribution of the RNA produced after incubating
the amplification products obtained according to the third embodiment of
the present invention in the presence of T7 polymerase (complementary
RNA, left) or T3 polymerase (sense RNA right).
Figure 7A: Visualisation of the amplification products obtained
3o according to the third embodiment to step d) after gene specific
amplification with primers specific for tubulin, RL3, Synaptotagmin 1 and
A2A receptor.
Figure 7B: Visualisation of the amplification products obtained
according to the third embodiment of the method that have been
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transcribed in vitro into the corresponding sense RNA using T3 RNA
polymerase, and then reverse transcribed prior to gene specific PCR.
Throughout the specification, the following terms are defined as
s follows:
Low amounts of mRNA is intended to designate the amount of
mRNA present in a maximum of 1000 cells, 1 to 100 cells being
preferred, considering that in general, there are between 1 and 100
io copies of any given mRNA present in a given cell.
Increase the number of nucleotide sequences corresponding to
the mRNA species present in a sample is intended to designate an
increase in nucleotide sequence to obtain a number of copies which is
sufficient to allow at least one of the following methods:
is (i) detection of the sequence of interest with specific
oligonucleotide probes;
(ii) amplification of the sequence of interest with specific
oligonucleotide primers;
(iii) cloning of the DNA molecules obtained in a replication
2o and/or expression vector, or
(iv) In vitro RNA transcription, either for hybridization
assays of for further reverse transcription optionally using unlabelled or
labeled substrates followed by gene specific PCR or hybridization.
2s Sample: is intended to designate material which contains the
mRNA which is to be analyzed. For example a cellular extract obtained
from 1 to 1000 cells.
High molecular weight DNA is intended to designate any nucleic
3o acid species which is outside the expected range of molecular weight
observed for natural mRNA species. Preferably any nucleic acid
sequence with a size above 5kb.
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DETAILED DESCRIPTION OF THE INVENTION
Three different ways of amplifying low amounts of mRNA
present in a sample have been found, each of these methods being
s described in detail hereafter. Thus, these, ways of amplifying allow in
several instances to amplify all mRMA species which are present in the
sample of interest. Also included is a detailed description of reagents
and oligonucleotide primers required for amplifying low amounts of
mRNA
io
FIRST EMBODIMENT OF THE INVENTION.
The first embodiment of the amplification method takes
advantage of the generation high molecular weight DNA molecules are
is formed following amplification of the cDNA species obtained through
reverse transcription of the initial mRNA species present in the sample.
The inventors have found that these high molecular weight DNA
molecules or bridged products may result from the formation of partially
duplexed DNA molecules during the annealing step. These partially
2o duplexed DNA molecules would contain partially complementary
sequences that hybridize with one another in the low stringency
hybridization conditions used, thus forming bridges between two
structurally related or unrelated amplified cDNA molecules contained in
the amplification mixture. Repetitive amplification cycles result in large
2s nucleic acid molecules. Furthermore, the first embodiment makes use of
the high molecular weight DNA produced by this process to analyze
amplified species of interest.
The first embodiment makes use of these findings by providing
a process which by favoring an increase in the production of these high
3o molecular weight DNA molecules in particular, allows to amplify mRNA
species present in a low quantity in a sample to be analyzed.
More precisely, there is disclosed a method to increase the
number of nucleotide sequences corresponding to the mRNA species
present in a low quantity in a sample, comprising:
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a) reverse transcribing said mRNA species using a first heeled
primer population to provide first strand cDNA sequences;
b) synthesizing second cDNA strands from said first strand
s cDNA sequences using a second heeled primer population;
c) amplifying said first and second cDNA strands resulting from
step b) over a number of amplification cycles with the aid of a
thermoresistant DNA polymerase(s) with:
io (i) a first primer comprising at least a portion of the heel
sequence of the first heeled primer; and
(ii) a second primer comprising at least a portion of the heel
sequence of the second heeled primer,
is wherein said method is characterized in that it comprises the steps of:
d') increasing the proportion of high molecular weight DNA
molecules,
e') using or analyzing specific nucleic acid sequences present in
the high molecular weight DNA molecules,
PRIMERS
The term " heeled " primer will be readily understood in the art
to be a primer comprising a hybridizing region and a non-hybridizing
2s region, wherein the non-hybridizing region represents the " heel " of the
primer.
The first heeled primer is actually a population of individual
primer species. The first heeled primer population consists of a
3o population of nucleic acid sequences each comprising, from 5' end to 3'
end:
(i) a heel sequence of 15 to 22 nucleotides in length which does
not hybridize with the mRNA molecules initially present in the sample;
(ii) an oligo dT sequence of 15 to 25 nucleotides in length;
3s (iii) a nucleotide which should not be thymidine (A, C or G); and
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(iv) a variable sequence of 2-4 nucleotides in length.
The components described in iii and iv being capable of
hybridizing to a mRNA molecule at the 5' end of the poly-A tail thereof,
wherein substantially every possible variable sequence combination is
s found in said first heeled primer population .
In a specific embodiment of the first heeled primer, the variable
sequence of 2 to 4 nucleotides is selected among the following variable
nucleotides sequence:
5'-(A or G or C)-N1_3-, wherein N is a nucleotide selected from
to A, T, C or G.
In a preferably advantageous alternative, the first heeled primer
may also comprise an RNA polymerase binding site, such as the T7, T3
or SP6 promoter, located between the oligo dT sequence and the heel.
is The second heeled primer is also a population of individual primer
species. When the first strand cDNA population is contacted with the
second heeled primer population under appropriate hybridizing
conditions, each cDNA species will hybridize with at least one second
heeled primer, (partly because of the selection of nucleotide sequences
2o amongst the second heeled primers), second cDNA strand synthesis
then proceeds in a 5' to 3' direction from the hybridized second primer.
The second heeled primer population may comprise primers
differing by up to five nucleotide bases (differing in the hybridizing
region), .
2s the second heeled primer population preferably comprising a number of
primers in the range 1000 to 100,000 primers, more preferably in the
range 1024 to 65536 primers. In order to achieve this, the primers of the
second heeled primer population preferably each comprise a first
variable sequence of nucleotides in the range of 4 to 7 nucleotides 3' to
3o the heel and a second variable sequence of at least 5 nucleotides
contiguous 3' therewith. As will be appreciated, where there are 5
random nucleotides (which is preferred) there will be 45 (i.e. 1024)
possible pentamer sequences.
The second variable sequence of this primer may be selected
3s by sequence analysis of known sequences so as to promote the ability of
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the second heeled primer as a whole to hybridize to the transcribed
cDNA species. Sequence analysis can be carried out through databases
of DNA or RNA sequences. In particular, known sequences of the
organism of interest are preferably consulted. The second variable
s sequence of nucleotides preferably comprises a number of nucleotides
in the range 2 to 10 nucleotides. In a particularly preferred embodiment,
the second variable sequence of nucleotides may comprise a number of
nucleotides equivalent to the number of nucleotides in the first variable
sequence of this primer.
io The second variable nucleotide sequence of the second heeled
primers may be constant throughout the population of these primers and
it is selected so as to stabilize the primers and to ensure optimal
efficiency of hybridization to the target first strand cDNA species.
In a preferred embodiment, the second heeled primer from the
is population of second primers preferably hybridizes on average once in
every 1 kb portion of first strand cDNA species. This has been found to
produce optimal amplification of mRNA in a sample.
Particularly preferred second variable sequences of nucleotides
in the second primers are:
20 5'-CGAGA-3', 5'-CGACA-3', 5'-CGTAC-3' and 5'-ATGCG-3'
The non hybridizing heel regions of the first and second heeled
primers are preferably selected so that they lack the ability to hybridize to
mRNA or first strand cDNA. The heel regions, like the hybridizing or
2s variable sequence regions of the second primers, are selected by
analysis of known nucleotide sequences. In particularly preferred
embodiments, the heel regions preferably comprise sequences absent
from the mRNA species in the sample. However, the heel regions may
simply comprise sequences absent from the genome of the organism
3o from which the sample is taken. The heel regions preferably comprise a
number of nucleotides in the range 15 to 22, more preferably 18 to 20
nucleotides.
Preferably, the heel sequences are chosen among nucleic acid
sequences having a GC content of about 50%, or for example from
3s about 43% to about 55% of the heel sequence.
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A particularly preferred heel sequence of the second heeled
primer population is the following nucleic acid sequence of SEQ ID N°1:
5'-CTGCATCTATCTAATGCTCC-3'
PROCESS
The particular temperatures, enzymes and reagents (other than
the first heeled primer) used in the process of reverse transcription in
io step a) may be those already known in the art.
Preferably, step a) is performed at 37°C in the presence of a
reverse transcriptase.
The frequency with which an individual second heeled primer
is population species hybridizes along a given length of nucleic acid may
be adjusted by employing suitable hybridizing conditions. Preferably, the
hybridization conditions are of limited stringency so enabling efficient
hybridization of the first variable sequence to target cDNA. The degree of
stringency and the number of contiguous random bases in the second
2o heeled primers may be varied according to routine trial and error in order
to achieve the desired frequency of hybridization of second heeled
primer species along a given length of nucleic acid material.
Most preferably, the conditions for the hybridization between the
second heeled primer and the first cDNA strands obtained at step a) are
2s of low stringency.
In step b), synthesis of the second cDNA strands is performed
in the presence of DNA polymerase, preferably a Taq polymerase, in a
suitable elongation buffer solution.
3o Preferably, the amount of second heeled primers added to the
buffer solution vary from 0.01 ng to 10 ng in the elongation reaction
buffer solution.
Particularly, the annealing buffer may comprise a concentration
of magnesium, generally up to 6 mM magnesium, preferably between 1.5
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mM and 6 mM magnesium and most preferably about 4.5 mM
magnesium.
In the case wherein the concentration of magnesium in the
elongation buffer has been adjusted to 4.5 mM, the temperature of
s annealing between the second heeled primer and the first cDNA strands
is of about 50°C and the elongation temperature in the presence of the
suitable DNA polymerase is of about 72°C.
The cDNA molecules that are generated at the end of step b)
are highly representative of the spectrum of mRNA molecules in a
io sample, as mRNA species of low abundance are reverse-transcribed to
the same level of efficiency as more abundant mRNA species.
Step c):
The amplification reaction of step c) is performed with a pair of
is oligonucleotide primers that respectively comprise at least a portion of
the heel sequence of the first and second heeled primers that are
defined above.
The first primer of step c) is preferably the heel of the first
heeled primer. The second primer of step c) is preferably the heel of the
2o second heeled primer.
The second primer of step c) may be the same as the second
heeled primer and this can be advantageous in reducing the number of
reagents needed to perform the first embodiment
A further alternative is to use the second heeled primer as the
2s sole primer.
Preferably, the amplification reaction of step c) is performed
using low stringency hybridization conditions. For example, amplification
reactions are performed in the presence of a concentration of
3o magnesium generally up to 5 mM, preferably between 4 mM and 5 mM
magnesium and most preferably of about 4.5 mM magnesium. With the
latter magnesium concentration, each amplification cycle comprises a
denaturing step at 92°C, an annealing step at 60°C and an
elongation
step at 72°C.
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Advantageously, the amplification reaction of step c) comprises
from 30 to 50 amplification cycles, and most preferably comprises about
40 amplification cycles. However, other cycle numbers could be
envisaged. One parameter of the optimum number of cycles required is
s determined by the polymerase used.
In a first series of further process steps, the cDNA may be
submitted to in vitro transcription either immediately after step c) if the
appropriate concentration of cDNA is present in the sample or after
io further amplification, such as through step d'), steps d) and e) which
is/are discussed in more detail below. In this context, it is essential that
at least one of the primers used in step a), b) and/or c) comprises a RNA
polymerase binding site such as the T7 RNA polymerase promoter. The
RNA generated can then be subjected to further process steps, for
is instance by being labeled during reverse transcription and hybridized to
DNA arrays. Alternatively, cDNA generated in the presence or absence
of a labeled substrate can be used in gene-specific PCR experiments.
Step d'):
2o In the preferred embodiment of step d'), step d') is a
combination of the following steps d) and e).
Step d):
At the end of step c), the reaction mixture containing the
Zs population of amplified DNA molecules which include the "bridged
products" can be diluted to obtain a diluted cDNA solution containing a
cDNA concentration which is between 2 and 100 x inferior to the cDNA
concentration of the product of step c). Preferably the diluted cDNA
concentration ranges between about 2 and 100 times less, and most
3o preferably between 40 and 80 times less, than the initial cDNA
concentration found in the reaction mixture obtained at the end of step
c). This dilution step is essential for performing the further steps of the
method as it results in the almost complete elimination of the primers
initially added to the amplification mixture. The elimination of most of the
3s primers, which are not part of the original gene sequence to be detected,
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reduces the element of randomness which would be introduced in the
further amplification steps. This element of randomness arises from the
mis-hybridization occuring under the lower stringency conditions
employed.
s
Step e):
Preferably step a consists of adding a thermoresistant DNA polymerase
to the diluted cDNA solution of step d) and performing a further set of
amplification reaction cycles without adding further primers.
io Following dilution of DNA the amplification of step e) is
performed without adding any primers to the diluted cDNA solution
obtained at step d). Because no exogenous primers are added, the
annealing step results in the hybridization between different amplified
DNA molecules initially present in the diluted cDNA solution, which are
is then elongated before the resulting duplex elongated cDNA molecules
are denatured at the end of each amplification cycle.
Without wishing to be bound by any particular theory, it appears
that the "self priming" amplification of step e) also results in an increase
of the number of bridged DNA molecules having a high molecular weight
2o and therefore in an increase in the number of bridged but appropriately
amplified genes from the sample.
Preferably, the amplification reaction of step e) is performed for
a number of amplification cycles ranging from 20 to 40 amplification
cycles, more preferably from 25 to 35 amplification cycles and is most
2s preferably of about 30 amplification cycles. However, other cycle
numbers could be envisaged. One parameter of the optimum number of
cycles required is determined by the polymerase used.
Preferably, the amplification reaction of step e) is performed at
hybridization conditions of low stringency, but with a greater stringency
3o than the hybridization conditions used in the amplification reaction of
step c). Typically, the magnesium concentration generally used is up to
4,5mM, preferably between 1.5 mM and 4.5 mM magnesium and most
preferably about 3.5 mM. In these amplification conditions, each
amplification cycle comprises the following steps of:
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(i) obtaining single stranded DNA molecules by incubating the
sample at a temperature comprised between 85°C and 95°C;
(ii) annealing the single stranded DNA molecules obtained at
step (i) at a temperature comprised between 55°C and 75°C;
s (iii) elongating the annealed DNA molecules using a
thermoresistant DNA polymerase at a temperature comprised between
65°C and 75°C;
(iv) reiterating steps (i) through (iii) for the desired number of
cycles.
to In a most preferred embodiment, the amplification reaction of
step e) comprises a denaturation step at 92°C, an annealing step at a
temperature comprised between 55°C and 72°C, for example
55°C,
60°C, 65°C or 72°C, and an elongation step at 72°C
in the presence of a
suitable DNA polymerase.
Step e'):
A preferred embodiment of step e') comprise a combination of
the following steps f) and g).
2o Step f):
The amplification mixture which contains a population of
amplified heterogeneous cDNA molecules is then submitted to a further
step (step f) wherein the high molecular weight cDNA species, preferably
2s those having a length of at least 4.5 kb, are separated.
Step g):
In a preferred embodiment step g) consists of confirming the presence of
at least one nucleic acid sequence species contained in the high
3o molecular weight cDNA separated at step f)
The high molecular weight cDNA species previously separated
at step f) can readily be used, typically for detecting the presence of at
least one nucleic acid sequence of interest.
Alternatively, the amplification method may comprise an
3s additional amplification step following step f), which consists of
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submitting at least a part of the high molecular weight DNA molecules
separated at step f) to a further amplification reaction using a pair of
primers, wherein a first primer comprises a portion of the first heel
sequence and the second primer comprises a portion of the second heel
s sequence.
Step g) of the amplification method comprises anyone of the
following methods:
(i) detection of the sequences of interest with specific
io oligonucleotide probes;
(ii) amplification of the sequences of interest with specific
oligonucleotide primers;
(iii) cloning of the DNA molecules obtained in a replication
and/or expression vector; or
Is (iv) in vitro RNA transcription, either for hybridization assays or
for further reverse transcription optionally using unlabelled or labeled
substrate followed by gene specific PCR or hybridization.
In part (iv) of step g), the resulting cDNA may also be submitted
2o to in vitro transcription. In this context, it is essential that one of the
primers comprises a RNA polymerase binding site such as the T7 RNA
polymerase promoter. The RNA generated can then be subjected to
further process steps, for instance either by being labeled and attached
to DNA arrays for hybridization experiments or by being reverse
2s transcribed, optionally using a fluorescent, radioactive or otherwise
labeled substrate, to generate labeled cDNA strands. The resulting
labeled cDNA can then be hybridized to a DNA array or used in gene-
specific PCR experiments.
3o It is to be noted that the labeling of any of the reactants used in
any one of the 3 embodiments of the invention, although optional, can be
very useful in that it allows the skilled person to directly hybridize to a
DNA array the products of the process of the present invention.
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In conducting the series of experiments which lead to the first
embodiment of the invention described above, the inventors came to the
conclusion that even though the "bridged sequences" refered to
previously contain useful and exploitable information on the genes
s present in the sample to be analyzed, it would be useful to reduce
bridge formation in order to obtain individual gene sequences in better
yields and which could then be analyzed more specifically. Thus a key
element of embodiments II and III described below resides in preventing
or at least reducing the formation of "bridge sequences" to the largest
io extent possible. Therefore, the methods of embodiments II and III are
characterized in that they comprise a process step which allows either to
prevent or to reduce the formation of "bridged sequences" following
reverse transcription and amplification of the nucleic acid sequences
present in the sample to be analyzed.
is
SECOND EMBODIMENT OF THE INVENTION
In the second embodiment of the amplification method, the
generation of a large number of high molecular weight DNA molecules is
2o prevented or reduced by inserting a nucleic acid sequence encoding a
cleavage site, in particular a restriction endonuclease site, at least in the
heel sequence of the second heeled primer
Consequently, another object of this invention consists of a
method to increase the number of nucleic acid sequences corresponding
2s to the mRNA species present in a low quantity in a sample, wherein said
method comprises the steps of:
a) reverse transcribing said mRNA species using a first heeled
primer population to provide first strand cDNA sequences;
b) synthesizing second cDNA strands from said first strand
3o cDNA sequences using a second heeled primer population, wherein
each of the primers of said first, and/or second heeled primer population
optionally contains a rare cleavage site in particular a rare restriction site
located at the 3' end of its heel sequence;
c) amplifying the first and second cDNA strands resulting from
3s step b) over a number of amplification cycles with:
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(i) a first primer comprising at least a portion of the heel
sequence of the first heeled primer; and
(ii) a second primer comprising at least a portion of the heel
sequence of the second heeled primer;
s d') cutting any large DNA molecules and preventing bridge
formation in subsequent steps by suppressing the heel portions of at
least one said first or second heeled primer
e') increasing the amount of long double strand products with
sequences more 5' from the original mRNA.
io
Preferably, the synthesis of the first and second cDNA strands
in steps a) and b) are performed under the same conditions defined for
steps a) and b) of the first embodiment.
is Step c):
Typically, the first amplification reaction of step c) of this second
method is performed under low stringency hybridization conditions.
The low stringency hybridization conditions used at step c)
increase the chances to elongate any sequence present initially in the
2o sample containing the first and second cDNA strands population.
Preferably, the amplification reaction of step c) includes the
following steps of:
(i) obtaining single stranded DNA molecules at a temperature
comprised between 85°C and 95°C;
2s (ii) annealing the primers to the single stranded DNA molecules
at a temperature comprised between 45°C and 65°C;
(iii) elongating the annealed DNA molecules at a temperature
comprised between 65°C and 75°C, preferably between 70°C
and 75°C
in the presence of a concentration of 4.5mM magnesium;
30 (iv) reiterating steps (i) to (iii) for the desired number of cycles.
In a most preferred embodiment, the amplification reaction of
step c) includes a denaturation step at 92°C, an annealing step at
60°C
and an elongation step at 72°C.
In another preferred embodiment, the amplification reaction
3s step of the first and second cDNA strands comprises between 30 and 50
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amplification cycles, more preferably between 35 and 45 amplification
cycles, most preferably about 40 amplification cycles. However, other
cycle numbers could be envisaged. One parameter of the optimum
number of cycles required is determined by the polymerase used.
s
Step d'):
In a prefered embodiment of step d') consists of incubating the product
obtained at step c with at least one restriction enzyme that specifically
recognise the cleavage site in particular a rare restriction sites included
io in the primers.
Incubating the cDNA strands obtained at the end of the first
amplification reaction step c) as shown in embodiment I with the
corresponding cleavage agent such as a restriction endonuclease results
in the cleavage of the high molecular weight cDNAs produced at this
is step. This prevents an increase in the number of the high molecular
weight cDNA species that would have been generated during the second
amplification reaction step e) (of embodiment 1 ). This step also serves
to remove DNA sequences which can compete with the gene specific
primers used later on in gene specific PCR. Furthermore, cutting and
2o removing the resulting products increases the efficiency of in vitro RNA
transcription from amplified DNA.
According to a specific variant of the second embodiment, the
DNA molecules amplified in step c) are incubated in step d) with two
restriction enzymes recognizing the rare cleavage site in particular a
2s rare restriction sites of the first and the second heeled primers.
As a preferred variant, the rare cleavage site in particular a rare
restriction site sequence present at the 3' end of the heel of at least the
second heeled primer is a sequence recognized by the Mlul restriction
enzyme.
3o Restriction cleavage in step d) is performed using the
conventional restriction cleavage techniques well known to those skilled
in the art such as described for example in Sambrook, J., Fritsch, E.F. ,
and T. Maniatis, (1989, Molecular cloning: A laboratory Manual . 2nd ed.;
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
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Step e'):
In a preferred embodiment step e') is a combination of the following
steps e) and f).
s Step e):
The cleavage step can be followed by step e) wherein the
product of step d) is diluted by an order of magnitude of 2 to 100 times in
order to almost completely eliminate the primers used for the first
amplification of step d). This favours the phenomenon of self priming
io as shown in figure 3) in the further set of amplification reaction cycles
of
step f).
As shown in Figure 3, in the absence of added primers, and
after strand separation at 92°C, short strands (e.g. strand B) will be
able
to serve as primers on complementary longer strands (e.g. strand A),
Is resulting in an increase in the amount of double stranded gene specific
sequence 5' to the reverse transcription primer site. Note that removal of
the second strand primer heel, facilitates this process since the heel
primer sequence is not complementary to the gene sequence of strand
A. Thick bars on the right side of the diagram represent the reverse
2o transcription primer heel, while thick bars on the left represent the
second strand cDNA primer heel.
More preferably, the product of step d) is diluted 10 to 80 times
and is most preferably diluted about 40 times.
2s Step f):
In a preferred embodiment step f) consists of adding a
thermoresistant DNA polymerase to the diluted sample of step e) and
performing a further set of amplification cycles without adding further
nucleic acid primer.
3o Subsequently to the dilutions of step e), a further set of
amplification cycles without adding further nucleic acid primers can
advantageously be performed in a step f).
In a preferred embodiment, each amplification cycle of step f)
comprises the following steps of:
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(i) obtaining single stranded DNA molecules by incubating the
sample at a temperature comprised between 85°C and 95°C;
(ii) annealing the single stranded DNA molecules obtained at
step (i) at a temperature comprised between 55°C and 75°C;
s (iii) elongating the annealed DNA molecules using a thermo-
resistant DNA polymerase at a temperature comprised between 65°C
and 75°C;
(iv) reiterating steps (i) through (iii) for the desired number of
cycles.
io
In a preferred variant of this embodiment, the denaturation step
is performed at 92°C, the annealing step is performed at 55°C,
60°C,
65°C or 72°C and the elongation step is performed at
72°C.
In another preferred variant, the amplification cycles carried out
is in step f) compris between 10 and 40 cycles, more preferably between
25 and 35 cycles and most preferably about 30 cycles. However, other
cycle numbers could be envisaged. One parameter of the optimum
number of cycles required is determined by the polymerase used.
The set of amplification cycles carried out in step f) is preferably
Zo performed under low stringency hybridization conditions, the presence of
about 3.5 mM magnesium.
In a specific variant of the second embodiment, the method
comprises a further step wherein the DNA molecules obtained at step f)
having a length of less than 50 base pairs are discarded from the
2s reaction mixture.
Step g):
Furthermore, step g) can comprises one or several of the
so following methods:
(i) detection of sequences of interest with specific
oligonucleotide probes;
(ii) amplification of sequences of interest with specific
oligonucleotide primers;
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(iii) cloning of the DNA molecules obtained in a replication
and/or expression vector; or
(iv) in vitro RNA transcription, either for hybridization assays or
for further reverse transcription using unlabeled or labeled substrate
s followed by gene specific PCR or hybridization.
It is important to note that the amplified cDNA obtained from the
reverse transcription and amplification of the nucleic acid sequences of
the sample may be submitted to in vifro transcription either immediately
io after step d) if the appropriate concentration of cDNA is present in the
sample or after further amplification such as through steps e), f) and g).
In this context, it is essential that at least one of the primers comprises a
RNA polymerase binding site such as the T7 RNA polymerase promoter.
The RNA generated can then be subjected to further process steps, for
is instance either by being labeled and hybridized to DNA arrays or by
being reverse transcribed, optionally using a fluorescent, radioactive or
otherwise labeled substrate, to generate labeled cDNA strands. The
resulting labeled cDNA can then be hybridized to a DNA array or used in
gene-specific PCR experiments.
It is to be noted that the labeling of any of the reactants used in
the above method, although optional, can be very useful in that it allows
the skilled person to directly hybridize on a DNA array the products of the
process of the present invention.
PRIMERS
It is to be noted that although the presence of a cleavage site is
an important feature of the second embodiment, this cleavage site can
be located either on the first heeled primer, on the second heeled primer,
on both primers or on primers used in step c). However, it is necessary
that at least one primer comprise a cleavage site.
A) First heeled primers
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For performing this second embodiment of the method, the first
heeled primer population consists of a population of nucleic acids
comprising, from 5' end to 3' end:
(i) a heel sequence of 15 to 22 nucleotides in length which is
s not complementary to the mRNA molecules present in the sample or with
the first strand cDNA molecules synthesized at step a) and wherein the
heel sequence optionally includes the nucleotide sequence of a rare
cleavage site in particular a rare restriction site located at its 3' end;
(ii) An optional but preferably present RNA polymerase
io promoter sequence,
(iii) an oligo dT sequence of 15 to 25 nucleotides in length;
(iv) a variable sequence of 2-4 nucleotides in length. This
sequence is able to hybridize to a mRNA molecule at the 5' end of the
poly-A tail thereof, wherein substantially every possible variable
is sequence combination is found in said first heeled primer population.
Typically, the variable sequence of 2-4 nucleotides in length of
the first heeled primer is selected among the following variable
nucleotide sequence: 5'-(A or G or C)-N~_3-3', wherein N is a nucleotide
selected from A, T, C or G.
2o In a specific embodiment of this second amplification method,
the first heeled primer may therefore also include the sequence of a rare
cleavagecleavage site in particular a rare restriction site. cleavage site
in particular a rare restriction sitecleavage site in particular a rare
restriction sitecleavage site in particular a rare restriction site
2s The sequence of a rare cleavage site in particular a rare
restriction site is usually located within or close to the 3' end of its heel
sequence. In the context of the present invention, 'close to the 3' end' is
intended to designate that the cleavage site in particular a rare restriction
site is to be positioned so as to leave as few bases as possible from the
3o heel after restriction enzyme cutting so as to avoid subsequent aberrant
hybridization between the remaining and generated sequences.
Preferably, the cleavage site in particular a rare restriction site
is selected from the so-called 'rare cutter' the group which comprises, for
example, Not1 Bsshll, Narl, Mlul, Nrul and Nael.
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Preferably, the cleavage site in particular a rare restriction site
of the first heeled primer is identical to the cleavage site in particular a
rare restriction site of the second heeled primer.
Alternatively, the cleavage site in particular a rare restriction site
s of said first heeled primer may be different from the cleavage site in
particular a rare restriction site of the second heeled primer.
B) Second heeled primer
io The second heeled primer population consists of a population
of nucleic acid sequences each comprising, from 5' end to 3' end:
(i) a heel sequence of 15 to 22 nucleotides in length
which is not complementary to the mRNA molecules present in
the sample or with the first strand cDNA molecules synthesized
is at step a) wherein the heel optionally sequence includes the
nucleotide sequence of a rare cleavage site, in particular a
rarely used site, located at its 3' end;
(ii) An optional but preferably present RNA polymerase
promoter sequence,
20 (iii) a first variable sequence of 4 to 7 nucleotides in length
selected such that substantially every possible sequence combination of
4 to 7 nucleotides is found in said second heeled primer population; and
(iv) a second variable nucleotide sequence that was calculated
to hybridize on average once in every 1 kb portion of said first strand
2s cDNA molecules under low stringency hybridization conditions.
Preferably, the cleavage site is located within the heel
sequence. More preferably, the cleavage site in particular a rare
restriction site is located at the 3' end of the heel sequence of the second
heeled primer population, and is a rarely occurring cleavage site in
3o particular a rare restriction site in the genome from which the initial
mRNAs are expressed.
Most preferably, the cleavage site in particular a rare restriction
site is selected among the cleavage site in particular a rare restriction
sites that are found less than once every 20 kb in the genome of the
3s organism from which the cDNA amplification is sought.
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In mammals, and more particularly in the rat, such rare
cleavage site in particular a rare restriction site is selected from the so-
called 'rare cutter' group of cleavage site in particular a rare restriction
s sites which comprises, for example, Not1, Bsshll, Narl, Mlul, Nrul and
Nael.
Preferably, the heel sequence of the second heeled primer
consists of the nucleotide sequence of SEQ ID N°2,
CTGCATCTATCTAGTACGCGT.
to In a preferred embodiment of the second heeled sequence,
said second variable sequence is chosen from the group of sequences
consisting of 5'-CGAGA-3', 5'-CGACA-3', 5'-CGTAC-3' and 5'-ATGCG-
3', such that each of said second variable sequence is found in said
second heeled primer population.
KITS
The invention further relates to a kits for the amplification of the
mRNA species present in a sample, wherein said kit compris:
(i) a first heeled primer population; and
(ii) a second heeled primer population, as defined above for
either embodiments I or II.
The invention also pertains to a kit for the amplification of the
mRNA species presenting a sample wherein said kit further comprises:
(iii) a first primer consisting of the heel sequence of the first
2s heeled primer;
(iv) a second primer consisting of the heel sequence of the
second heeled primer.
In a specific embodiment, the mRNA amplification kit may
further comprise one or more restriction enzymes that recognize the rare
3o cleavage site in particular a rare restriction site sequence that may be
present in the heel sequence of the heeled primers.
In another preferred embodiment, said mRNA amplification kit
may further comprise a restriction enzyme that recognize the rare
cleavage site in particular a rare restriction site sequence that may be
3s present in the heel sequence of the first heeled primer.
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In yet another preferred embodiment, the kit may also include a
suitable RNA polymerase.
THIRD EMBODIMENT OF THE INVENTION
According to this third amplification method of the invention,
higher stringency hybridization conditions are used to prevent the
generation of bridged nucleic acids In the second embodiment, bridges
io were cleaved by using a primers containing a rare cleavage site in its
heel sequence. This allowed cleavage by a cleaving agent, preferably a
restriction endonuclease, of the long cDNA molecules formed during the
first set of amplification cycles.
The conditions for performing the third embodiment have been
is chosen to further reduce bridge formation. Such conditions include for
example, (apart from the optional presence of a restriction site on the
primer) increasing the stringency of hybridization with respect to the
stringency used in embodiment 1 or 2, for example by optimizing buffer
conditions which will in turn decrease mis-hybridizations and/or
2o increasing the GC content of the primers which allows elevated
annealing temperatures, which also reduces mis-hybridization and
increases the distance between hybridized paired oligonucleotides.
These higher stringency hybridization conditions may be met
according to two alternatives of this third embodiment which are
2s described hereunder.
The method of embodiment III is a method to increase the
number of nucleotide sequences corresponding to an mRNA species
present in a sample in a low quantity comprising the steps of:
3o a) reverse transcribing the mRNA species using first heeled
primer population to provide first strand cDNA species;
b) synthesizing second cDNA strands using a second heeled
primer population;
c) amplifying said second cDNA strands resulting from step b)
3s over a number of amplification cycles in order to generate second cDNA
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strands comprising heels at both ends and increasing the number of
second cDNA strands corresponding to long mRNA species present
initially in the sample to be assayed;
d) amplifying the DNA molecules resulting from step c) under
s hybridization conditions which are of a higher stringency than those of
step c) and which enable reduction of the synthesis of high molecular
weight cDNA molecules; and
e) recovering the population of DNA molecules obtained at step
d).
to
PROCESS
Step a)
Preferably, step a) is the same as for the first and the second
embodiments, except that the first heeled primer population comprises a
is heel sequence that must have a GC content ranging from 60% to 80%
and which is most preferably of about 75%, in order to permit an
increase in the stringency of the hybridization conditions used in the first
set of amplification cycles of step c), thereby reducing the formation of
nucleic acid bridges inside the amplified cDNA molecule.
Step b)
Step b) of synthesis of the second cDNA strands is also
performed at hybridization conditions of a higher stringency than the
hybridization conditions used in step b) as described for the first and the
2s second embodimentsof the invention.
Step b) is preferably performed at high stringency conditions.
A preferred example of high stringency conditions is as follows:
synthesizing second cDNA strands using a second heeled primer
population preferably at a concentration ranging between 0.02 to 200 ng
3o per reaction in the following conditions;
(i) adding the primers to the cDNA product obtained at step a);
(ii) obtaining single stranded DNA molecules at a temperature
comprised between 85°C and 95°C preferablyfor a period of time
which
ranges from 2 to 5 min,;
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(iii) adding a thermoresistant DNA polymerase and optionally a
thermoresistant proof reading enzyme to the mixture obtained at step (ii);
(iv) Optionally maintaining the temperature of the mixture at
approximately 94°C during a period of time up to 5 min
s (v) annealing the healed primers to said single stranded DNA at
a temperature comprised between 40°C and 72 °C;
(vi) elongating the annealed DNA molecules at a temperature
comprised between 60°C and 75 °C;
Hgh stringency hybridization conditions are notably obtained
io according to the specific structural features of the second heeled primer
used.
Preferably, step b) is performed in the presence of a
magnesium concentration generally up to 5 mM magnesium, preferably
between 3 and 5mM magnesium, most preferably of 3.5 mM.
is The thermoresistant DNA polymerase is preferably added at
step (b) in an amount that rangesfrom 3U to 5U, most preferably 4.5U
DNA polymerase in a volume of 1 NI. Optionally step (b) is performed in
the presence of both a thermoresistant DNA polymerase and a proof
reading enzyme.
2o This enzyme being added at the same time as the DNA
polymerase and in an amount which preferably ranges from 0.1 U to
0.5U, most preferably 0.25 U and is admixed with the DNA polymerase
in a volume of 1 NI.
With regard to step (b) (iv), it is performed for a period of time
2s preferably from 1 min to 3 min, most preferably during 2 min.
With regard to step (b) (v), it is preferably performed at a
temperature of 50°C for a period of time generally up to10 min,
preferably between 4 min and 10 min, more preferably 6 min and 8 min
and most preferably 7.5 min.
3o With regard to, step (b) (vi), it is preferably performed at a
temperature of 72°C for a period of time comprised between 1 min and 5
min, preferably between 2 min and 4 min and most preferably during 2.5
min.
High amounts of second heeled primer population used in steps
3s b) and c) increases the probability of annealing of at least one primer to
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every sequence contained in the first cDNA strands previously
synthesized at step a).
Step c)
s Although the inventors do not wish to be bound to any particular
theory, it appears that through the successive cycles of the amplification
reaction of step c), the sequences that contain at their 5'-end the heel
sequence of the second heeled primer will anneal to the first strand
cDNA in order to generate second cDNA strands comprising heels at
io both ends. These repetitive cycles of step c) increase the chances of
detecting every first strand present in the reaction mixture of step b).
As a consequence, complementary sequences to the 5'end of
the gene sequences present in the sample are generated.
is In a first preferred embodiment of step c), no denaturation is
performed during the successive cycles. This situation permits an
increased efficiency in long sequence elongation by allowing the
polymerase to work through several cycles without removing the primers
or short DNA sequences hybridized to the first strand. Furthermore, the
2o inventors believe that the polymerase may actually displace small
sequences hybridized to the first strand during the elongation to favor the
extension of longer sequences already hybridized to this first strand.
In a second preferred embodiment of step c), denaturation is
performed under mild temperature conditions, preferably in the range of
2s 80 to 85°C. In these conditions, small mismatched sequences,
generally
of less than 50 by in length and preferably at least the second heel
primers, are removed from their hybridization site on the first strand and
are thus available for further priming in subsequent reactions. The further
increases the yield in the amplification of the second strand cDNA.
3o In a third preferred embodiment of step c), denaturation is
performed under usual temperature conditions, preferably in the range of
85 to 95°C.
The first and second cDNA strands previously synthesized are
3s preferably amplified over a number of amplification cycles with the
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second heeled primer at a concentration ranging between 0.02 to 200 ng
per reaction, preferably 0.02 to 100 ng, more preferably between 1 and
50 ng and most preferably between 1 and 10 ng.
The preferred amount of second heeled primer population used
s in step c) increases the probability of annealing of at least one primer to
every sequence contained in the first and second cDNA strands
previously synthesized at steps a) and b).
Preferably, a population of approximately 4" primers is used
during the amplification reaction of step c). This increases the chances
io of each gene sequence annealing to at least one primer.
The availability of each primer is increased by multiplying the number of
cycles in the amplification reaction of step c).Preferably, step (c) is
performed in the presence of 4.5 mM magnesium between 30 and 50
amplification cycles, more preferably between 35 and 45 amplification
is cycles and most preferably about 40 amplification cycles.
Advantageously, the amplification reaction of step c) is
performed in the presence of both a thermoresistant DNA polymerase
and a thermoresistant proof reading enzyme.
The presence of a thermoresistant proof reading enzyme in the
2o amplification buffer allows a significant increase in the quality of the
sequences that are synthesized during the elongation step of each
amplification reaction cycle.
Most preferably, step c) comprises a step wherein the heeled
primers are elongated in the presence of the DNA polymerase and
zs optionally the proof reading enzyme at a temperature ranging between
40 and 72°C.
Optionally, an annealing step may be performed between the
denaturation step and the elongation step, at 40°C, a temperature
wherein the DNA polymerase is almost prevented to synthesize DNA.
3o Preferably, step c) comprises an elongation step wherein the
annealed DNA molecules are elongated at a temperature comprised
between 65 and 75°C in the presence of a thermoresistant DNA
polymerase.
In a preferred alternative variant, step c) can comprise
3s the step of amplifying second cDNA strands resulting from step b) over a
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number of amplification cycles with said second heeled primer preferably
at a concentration ranging between 0.02 to 200 ng per reaction in the
following conditions;
(i) optionally obtaining single stranded DNA molecules at a
s temperature comprised between 80°C and 95°C, in the presence
of a
thermoresistant DNA polymerase,
(ii) annealing the second strand primers to the first strand
(single stranded) DNA molecules at a temperature comprised between
40°C and 72°C; preferably between 40°C and 60°C,
io (iii) elongating the annealed DNA molecules at a temperature
comprised between 65°C and 75°C optionally in the presence of a
thermoresistant DNA polymerase;
(iv) repeating steps (ii) and (iii) (with (i) as an option) for the
desired number of cycles.
is Preferably, steps (c) (ii) to (iv) are repeated for 10 to 60 cycles,
preferably from 20 to 50 cycles and most preferably about 20 or about 40
cycles.
In a preferred variant of step (c), a population of second heeled
primers is added at step (b).
ao Preferably, step c) is performed in the presence of a
magnesium concentration up to 5 mM and~most preferably of 3.5 mM.
In a most preferred variant, step (c) is performed in the
presence of both a thermoresistant DNA polymerase and a proof reading
enzyme.
2s Advantageously, the proof reading enzyme is added at the
same time as the DNA polymerase and in an amount which ranges from
0.1 U to 0.5U, most preferably 0.25 U and is admixed with the DNA
polymerase in the volume of 1 NI.
Step d)
The third embodiment further comprises a second set of
amplification cycles which are performed at step d) (referred to above)
under more stringent hybridization conditions. This serves to amplify all
3s the cDNAs bearing heel sequences with minimum bridge formation and,
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due to the high stringency conditions used, at high efficiency, thus
increasing the yield of sequences initially present in the sample (when
compared to embodiments 1 and 2 above).
Preferably, each amplification reaction cycle of step d)
s comprises the following steps of:
(i) obtaining single stranded DNA molecules by incubating the
sample at a temperature comprised between 85°C and 95°C;
(ii) elongating the annealed DNA molecules using a
thermoresistant DNA polymerase at a temperature comprised between
io 65°C and 75°C;
(iii) reiterating steps (i) and (ii) for the desired number of
reaction cycles.
Preferably, the amplification reaction of step d) is performed in
the presence of 2.5mM magnesium, between 30 and 50 amplification
is cycles, more preferably between 35 and 45 amplification cycles and
most preferably about 40 amplification cycles. However, other
magnesium concentrations could be used, depending on the choice of
polymerase.
The denaturation temperature is preferably 95°C and the
2o elongation temperature is preferably 72°C.
Most preferably, the annealing and elongation step is performed
during a period of time which is sufficient for maximizing the annealing of
the primers to the single stranded cDNA molecules.
Typically, such annealing and elongation step ranges from 2.5
2s to 3.5 minutes and is most preferably about 3 minutes.
In another preferred embodiment, step d) can be
performed as follows:
amplifying said first and second cDNA strands resulting from step c) over
a number of amplification cycles with primers selected from the group
3o consisting of (1) a primer comprising a portion of the heel sequence of
the first heeled primer which portion is of a nucleotide length sufficient to
hybridize with its complementary sequence under the hybridization
conditions specified, (2) a primer comprising a portion of the heel
sequence of the second heeled primer which portion is of a nucleotide
3s length sufficient to hybridize with its complementary sequence under the
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hybridization conditions specified, and (3) a mixture of the primers (1)
and (2), wherein the total concentration of primers preferably ranges
between 0.02 and 500 ng per reaction in the following conditions:
(i) adding the primers to the cDNA product obtained at step c);
s (ii) obtaining single stranded DNA molecules at a temperature
comprised between 80°C and 95°C;
(iii) adding a thermoresistant DNA polymerise;
(iv) maintaining the temperature at a range from 80°C to 95°C
for a period of time preferably comprised between 5 sec to 15 min;
io (v) annealing the primers to the said single stranded DNA and
elongating the annealed DNA molecules at a temperature comprised
between 65°C and 75 °C;
(vi) carrying out steps (iv) and (v) for a desired number of
cycles.
is
With regard to step d), in a preferred embodiment, step d) (iv) is
performed at a temperature of 94°C. According to these conditions, the
reaction mixture contains essentially the single stranded cDNA products
obtained at step c), the amplification primers as well as the
zo thermoresistant DNA polymerise which is not active at this high
temperature.
Preferably, at the first occurrence of step d) (iv) in the
amplification method, the temperature is maintained in the range from
80°C to 95°C, most preferably 94°C, for a period of time
up to 3 minutes,
2s most preferably 2 minutes. For the further occurrences of step d) (iv)"
then the temperature ranges from 80°C to 95°C, most preferably
94°C,
and is maintained up to 60 sec, most preferably 20 sec.
At step d) (v) the primers are annealed to the single stranded
DNA molecules at a temperature wherein the thermoresistant DNA
3o polymerise is able to elongate the primers using the cDNA molecules as
templates.
Preferably, step d) (v) is performed at a temperature comprised
between 68°C and 74°C, most preferably 72°C.
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In a preferred embodiment, step d) (v) is performed during a
period of time comprised between 1 min and 10 min, most preferably 5
min.
In a preferred embodiment, the last occurrence of step d) (v)" is
s performed during a period of time comprised between 10 and 60 min,
preferably between 25 and 40 min, most preferably during 35 min.
Step d), (vi) preferably comprises between 10 and 50
amplification cycles.
to Preferably, the amplification reaction of step d) is performed in
the presence of both a thermoresistant DNA polymerase and a
thermoresistant proof reading enzyme, also step d) is preferably
performed in the presence of a concentration of magnesium comprised
between 2 and 5 mM.
In a preferred expression of the second alternative of the third
embodiment, the respective concentration of primers at step (d) ranges
from 0.02 to 500 ng.
Step d) which includes steps (i) to (vi) as described above, is
preferably performed when the initial sample contains a large amount of
mRNA, such as for example an amount of mRNA corresponding to the
whole mRNA that can be found after extraction from about 100 cells (e.g.
100 mammalian cells).
In this situation step d) is preferably performed in the presence
of a concentration of magnesium up to 3 mM, most preferably 2 mM
If the initial sample contained a small amount of mRNA, such as
the amount of mRNA that may be found after extraction from 1 to 10
3o cells, step d) above will preferably comprise further steps of amplifying
the products obtained at step d) (vi) of the second alternative.
In this situation, step d) further comprises the steps of
amplifying the DNA molecules obtained at step d) (vi) over a number of
amplification cycles with primers selected from the group consisting of
3s (1 ) a primer comprising a portion of the heel sequence of the first heeled
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primer which portion is of a nucleotide length sufficient to hybridize with
its complementary sequence under the hybridization conditions
specified, (2) a primer comprising a portion of the heel sequence of the
second heeled primer which portion is of a nucleotide length sufficient to
s hybridize -with its complementary sequence under the hybridization
conditions specified, and (3) a mixture of the primers (1 ) and (2), wherein
the total concentration of primers preferably ranges between 0.02 and
200 ng per reaction in the following conditions:
(vii) obtaining single stranded DNA molecule at a temperature
io comprised between 80°C and 95°C;
(viii) adding a thermoresistant DNA polymerase to the single
stranded DNA molecules obtained at step (vii);
(ix) annealing and elongating the single stranded DNA
molecules at a temperature comprised between 65°C and 75°C;
is (x) carrying out steps (vii) and (ix) for a desired number of
cycles.
With regard to the magnesium concentration used at step (d),
these are preferably of (a) 2.5 mM magnesium at steps (d) (i) to (vi) and
(b) 2mM magnesium at steps (d) '(vii) to (x).
2o As for amplification reaction steps (d) (i) to (vi) these are
performed with a respective concentration of primers which ranges from
0.02 to 90 ng, preferably from 10 to 50 ng, most preferably about 30 ng,
then step (d) (vii) to (x) is performed with a respective concentration of
primers that ranges from 50 ng to 300 ng, preferably from 65 ng to 200
2s ng and most preferably about 100 ng.
Thus, when the initial sample contained a small amount of
mRNA species, step (d) (i) to (x) are preferably performed, using a total
amount of primers ranging from 0.02 to 500 ng, preferably from 60 to
300 ng and most preferably about 130 ng.
3o Preferably, step (d) (x) of the second alternative of the third
amplification method described above comprises between 20 and 60
amplification cycles, preferably between 30 and 50 amplification cycles
and most preferably about 40 amplification cycles.
As it is detailed above, when step (d) is performed by carrying
3s out steps (i) to (x), the first set of amplification reactions of steps (i)
to (vi)
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is performed with a smaller amount of primers than when step (d) is
performed by carrying out solely steps (i) to (vi). This lower amount of
primers added at step (i) in this specific situation will permit a reduction
in
the level of mis-hybridizations in the first set of amplification reactions.
s Thus the products obtained at step d) (vi) are fully representative of the
mRNA species initially contained in the sample. According to this variant
of the embodiment, the second set of amplification reactions, namely
steps (d) (vii) to (x) will increase the amount of material initially
amplified
at steps (d) (i) to (vi).
io According to the method above, the amplification reaction steps
(d) (viii) to (x) are preferably performed in the presence of both a
thermoresistant DNA polymerise and a thermoresistant proof reading
enzyme.
Preferably, the amplification reactions steps (d) (viii) to (x) are
is performed in the presence of a concentration of magnesium up to 4
mM, preferably between 1.6 and 2.5 mM and most preferably at a
magnesium concentration of 2.0 mM.
In a preferred embodiment, the respective concentration of
primers used at steps (d) (vii) to (x) ranges from 10 to 500 ng, and most
2o preferably from 30 to 300 ng.
Step e)
With regard to step e) (referred to above) the method may
comprise a further step wherein the DNA molecules obtained at step e)
2s having a length of less than 50 by are discarded from the reaction
mixture.
Step e')
In an advantageous alternative variant of this embodiment, step
3o d) is followed by the following steps
i) incubating the DNA molecules obtained at step e) with at least
one restriction enzyme that specifically recognizes a restriction site
included in the heeled sequence of the first and/or second heeled primer;
and/or
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ii) diluting the product of step i) to obtain a diluted cDNA
solution containing a cDNA concentration which is between about 2 and
100 times inferior to the cDNA concentration of the product of step i);
and
s adding a thermoresistant DNA polymerase to the diluted
sample and performing a further set of amplification reaction cycles
without adding any nucleic acid primer; and/or
iii) confirming the presence of at least one nucleic acid
sequence contained in the population of DNA molecules obtained at step
io i) and/or ii).
In the above variants and especially in the variant wherein step
i) is performed, the primers preferably each comprise at least one rare
restriction site.
These variants may also comprise a further step wherein the
is DNA molecules obtained at step i) having a length of less than 50 by are
discarded from the reaction mixture.
Preferably , the number of amplification reaction cycles
performed in step ii) is comprised between 20 and 40, more preferably
between 25 and 40 and most preferably between 30 and 40.
20 As for step iii), it can comprise anyone of the following methods:
(i) detection of sequences of interest with specific
oligonucleotides probes;
(ii) amplification of sequences of interest with specific
oligonucleotide primers; and
2s (iii) cloning of the DNA molecules obtained in a replication
and/or expression vector.
The conditions for performing step i) to iii) of this preferred
variant are the same as those described for performing steps d) to g) of
the first and second embodiments.
Step f)
One specific variant of this embodiment comprises the
additional step of:
f) confirming the presence of at least one nucleic acid sequence
3s contained in the population of DNA molecules obtained at step e) or e').
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Step f) comprises any one of the following methods:
(i) detection of sequences of interest with specific
oligonucleotide probes;
(ii) amplification of sequences of interest with specific
s oligonucleotide primers;
(iii) cloning of the DNA molecules obtained in a replication
and/or expression vector; or
(iv) in vitro RNA transcription, either for hybridization assays or
for further reverse transcription using unlabeled or labeled substrate
io followed by gene specific PCR or hybridization.
It is important to note that, the resulting cDNA may be
submitted to in vitro transcription, either immediately after step c) if the
appropriate concentration of cDNA is present in the sample or after
is further amplification such as through step d) or through optional steps of
e') described above. In this context, it is essential that one of the primers
comprises a RNA polymerase binding site such as the T7 RNA
polymerase promoter. The RNA generated can then be subjected to
further process steps, for instance either by being labeled and hybridized
2o to DNA arrays or by being reverse transcribed, optionally using a
fluorescent, radioactive or otherwise labeled , substrate, to generate
labeled cDNA strands. The resulting product can then be hybridized to a
DNA array or used in gene-specific PCR experiments. If unlabelled the
products can be attached to a microarray base and be hybridized to
2s labeled oligonucleotides.
It is to be noted that the labeling of any of the reactants used in
this embodiment of the invention, although optional, can be very useful in
that it allows the skilled person to directly attach or hybridize to a DNA
3o array the products of the process of the present invention.
Alternatively, after step e) or e' RNA transcription can be carried
out by first optionally removing low molecular weight DNA, including heel
3s primers, to provide a 'cleaner' environment for subsequent RNA
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polymerase reactions to take place. This 'cleaning up' also allows the
skilled person to change the buffer solution to a buffer that would be
more appropriate for subsequent RNA polymerase reactions.
s It is important to note that the resulting cDNA may be submitted to
in vitro transcription It should be noted that inclusion of the RNA
polymerase promoter in the primer allows synthesis of complementary
RNA, suitable for hybridising to Gene Chips or arrays bearing sense
gene specific oligonucleotides, or for subsequent reverse transcription
io and hybridising of the resultant cDNA to antisense gene specific
oligonucleotides. In contrast, inclusion of the RNA polymerase promoter
in the second heeled primer allows synthesis of sense RNA, suitable for
hybridising to arrays bearing antisense oligonucleotides, or for
subsequent reverse transcription and hybridization of the resultant cDNA
is to GeneChips or arrays bearing sense gene specfic oligonucleotides.
In this context, it is essential that one of the primers comprises a RNA
polymerase binding site such as the T7 RNA polymerase promoter. The
RNA generated can then be subjected to further process steps, for
instance either by being labeled and hybrdized to DNA on arrays or by
2o being reverse transcribed, optionally using a fluorescent, radioactive or
otherwise labeled substrate, to generate labeled cDNA strands. The
resulting product can then be hybridised to a DNA array, or attached to a
support (e.g. glass, nylon, silcon etc) for subsequent hybridisation with
other nucleic acids, or used in gene-specific PCR experiments.
PRIMERS
The specific structural features of the primers used in this
embodiment (first and second heeled primers, primers used in step c)
3o include an increase in GC content as compared to primers of
embodiments I and II.
Thus, the primers used in this embodiment comprise a heel
sequence having a GC content ranging from 60% to 80%, most
preferably of about 75%. This increase in the GC content permits an
3s increase in the stringency of the hybridization conditions used in the
first
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set of amplification cycles of step c), thereby preventing the generation
of nucleic acid bridges inside the amplified cDNA molecules and thus
preventing the synthesis of the high molecular weight cDNA species
observed during step c) of the first and second embodiments.
In a preferred embodiment, the above primers comprise at least
one cleavage site in their heel sequences or at the 3' end of their heel
sequence.
io In another aspect of the third embodiment of the present invention, the
first heeled primer population consists of a population of nucleic acids
comprising, from 5' end to 3' end:
(i) a heel sequence of 15 to 22 nucleotides in length which
is not complementary to the first strand cDNA nor the
is mRNA molecules initially present in the sample;
(ii) An option but preferably present RNA polymerase
promoter site;
(iii) an oligo dT sequence of 15 to 35 nucleotides in length; and
(iv) a variable sequence of 2-4 nucleotides in length that can
2o hybridize to a mRNA molecule at the 5'end of the poly-A tail thereof,
wherein substantially every possible variable sequence combination is
found in said first heeled primer population.
According to this specific embodiment of the method, the
2s variable sequence of 2 to 4 nucleotides is selected among the following
variable nucleotide sequence : 5'-(A or G or C)-N-~_3, wherein N is a
nucleotide selected from A, T, C or G.
Preferably, the first heeled primer includes the sequence of a
rare restriction site which may be located at any position within the heel
3o sequence and preferably at the 5' end or at the 3'end of the heel
sequence of said first heeled primer.
Preferably, the oligo dT sequence has a length comprised
between 20 and 35, more preferably between 25 and 35 and is most
preferably of about 30 nucleotides in length.
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In a preferred variant, the variable sequence of 2 to 4
nucleotides of the first heeled primer is selected among the following
variable nucleotide sequences: 5'-(A or G or C)-N~_3-3', wherein N is a
nucleotide selected from A, T, C or G.
s As already described, the GC content of the heel sequence of
the first heeled primer is comprised between 50 and 80%, more
preferably between 60 and 80% and is most preferably of about 75% .
The high GC content of the heel sequence of the first heeled primer
allows a good annealing of said primer to the corresponding
io complementary sequence, even at the medium stringency hybridization
conditions that are used notably at step d) of the present third cDNA
amplification.
In another aspect of the third embodiment, the second heeled
is primer population consists of a population of nucleic acid sequences
each comprising, from 5'end to 3' end:
(i) a heel sequence of 25 to 75 nucleotides in length which is
not complementary to the mRNA molecules present in the sample or with
the first strand cDNA molecules synthesized at step a); and
20 (ii) a first variable sequence of 15 to 25 nucleotides in length
selected such that substantially every possible sequence combination of
15 to 25 nucleotides is found in said second heeled primer population.
Preferably, the heel sequence of said second heeled primer
comprises the sequence of a rare restriction site, which may be located
2s at any location within the heel sequence, but is preferably located at the
3'end or at the 5'end of the heel sequence of said second heeled primer.
In a specific embodiment, the heel sequence of the second
heeled primer ranges from 25 to 35 nucleotides in length.
In another specific variant, the heel sequence of the second
3o heeled primer ranges from 45 to 75 nucleotides in length and comprises
a RNA polymerase binding site.
In a further specific variant, the heel sequence of the second
heeled primer ranges from 45 and 75 nucleotides in length and
comprises a RNA polymerase binding site located at the 3' end of the
3s heel sequence.
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The first variable sequence of the second heeled primer
population has preferably 15 to 20 nucleotides in length and is most
preferably of about 17 nucleotides in length. The first variable sequence
s of the second heeled primer population is longer the first variable
sequence of the second heeled primer used to perform the first and
second embodiments described above and are thus suitable to stabilize
every second heeled primer of the population to its corresponding
complementary DNA sequence during the annealing and the elongation
io step of the first and second set of amplifications cycles of steps c) and
d).
A longer first variable sequence for stabilizing the primers
belonging to the second heeled primer population was required,
particularly due to the greater length of the heel sequence, which is
is preferably comprised between 25 and 30 nucleotides in length and is
most preferably of about 27 nucleotides in length.
In a first preferred embodiment of the second heeled primer
population, each nucleic acid sequence also comprises a second
variable nucleotide sequence preferably selected according to the criteria
2o set forth in the first embodiment. Preferably, the second variable
sequence of the second heeled primer is chosen from the group of
sequences consisting of 5'-CGAGA-3', 5'-CGACA-3', 5'-CGTAC-3' and
5'-ATGCG-3', such that each of second said variable sequence is found
in said second heeled primer population.
2s In a preferred variant, the heel sequence has 28 nucleotides in
length.
In a preferred variant of the second heeled primer population for
performing the third embodiment, said second heeled primer population
comprises a heel sequence of 25 to 30 nucleotides in length, more
3o preferably about 28 nucleotides in length and having a GC content
comprised between 50 and 70%, more preferably between 60 and 70%
and is most preferably of about 68%.
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In one specific variant of the second alternative of the third
embodiment, the heel sequences of the first heeled primer and the
second heeled primer are identical.
Alternatively, the heel sequences of the first heeled primer and
s the second heeled primer share a sequence of at least 15 consecutive
nucleotides, preferably at least 20 or 25 consecutive nucleotides.
According to this specific embodiment, the cDNA strands
present in the mixture obtained at the end of step (c) of the present
method comprise a sequence in their 5' end of at least 15 nucleotides
io which are complementary to a sequence comprised in their 3' end. In this
context, second cDNA strands of a short nucleic acid length that are
regenerated during step c) have a high tendency to self-anneal and thus
be no longer available for the sets) of amplification reactions of step (d).
Accordingly, the first and second cDNA strands that are amplified when
is carrying out step (d) of the present method are mainly large cDNA
molecules, including cDNA molecules comprising a sequence which is
identical or which is complementary to the full length mRNA species
initially present in the sample.
2o The heel sequences of the first and second heeled primers
preferably comprised the sequence of a rare restriction site located at
the 3'-end or at the 5'end of their respective heel sequence, as well as a
RNA polymerase binding site, preferably located downstream from the
restriction site.
2s In a first variant, the restriction site sequence of the first heeled
primer is identical to the restriction site sequence present in the heel of
the second heeled primer.
In a second variant, the restriction site sequence of the first
heeled primer is different from the restriction site sequence present in
3o the heel of the second heeled primer.
Advantageously, the restriction site sequence included in the
heel sequence of the first heeled primer or the second heeled primer is
selected from the group of so-called 'rare cutters' which comprises for
example Not1, Bsshll, Narl, Mlul, Nrul and Nael.
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KITS
The present invention further relates to kits for the amplification
of the mRNA species present in a sample, said kit being specifically
designed for performing the third cDNA amplification method described
s above.
Thus, another object of the invention consists of a kit for the
amplification of the mRNA species present in a sample, wherein said kit
comprises:
(i) a first heeled primer population; and
io (ii) a second heeled primer population,
the first and second heeled primer populations being defined above.
Said amplification kits may further comprises:
(iii) a first primer consisting of at least a portion of the heel
sequence of the first heeled primer; and
is (iv) a second primer consisting of at least a portion of the heel
sequence of the second heeled primer.
In a specific embodiment of the kit above, the heel sequences
of the first heeled primer and of the second heeled primers are identical
or alternatively share a common sequence of at least 15, preferably at
20 least 20, most preferably at least 25 consecutive nucleotides in length.
In a specific embodiment, said amplification kit may further
comprises a restriction enzyme that recognizes the rare restriction site
sequence present in the heeled sequence of the second heeled primer.
In another specific embodiment, said amplification kit may
2s further comprises a restriction enzyme that recognizes the rare restriction
site sequence present in the heeled sequence of the first heeled primer.
In yet another embodiment, the kit further comprises a suitable
RNA polymerase.
The three mRNA amplification methods of the invention make it
3o possible to amplify large numbers of samples easily and with high
sensitivity.
The ability to analyze subsequently the expression of many
genes of annealed sequences, both at high and low abundance, in
samples taken from as little as a single cell, potentially allows it to be
3s used in high throughput screening systems.
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Various kinds of mRNA containing samples may be used as
starting materials for performing the cDNA amplification methods of the
invention, such as the whole content of a cell cytoplasm or even a
portion of the cell cytoplasm such a portion of cytoplasm of neuronal
s cells and also mRNA molecules extracted from a desired tissue.
cDNA molecules obtained at the end of any one of the three
cDNA amplification methods described above can be used for many
purposes including:
a) cloning and production of cDNA libraries from small amounts
to of tissue;
b) sequencing analysis of gene expression in small tissue
samples;
c) subtracting the amplified product from two different samples
and analysing genes differentially expressed between them such as
is described by Diatchenko et al. (1996, Proc. Natl. Acad. Sci. USA, vol. 93:
6025-6030), and then by cloning and sequencing the sequences
expressed only in one or several tissues;
d) transcribing mRNA using labelled precursors for use on
hybridisation arrays, such as described by Duggan , D. J. et al., (1999,
2o Nature Genetics, vo1.21: S10-S14);
e) transcribing RNA and reverse transcribing in the presence of
labelled precursors for use on hybridising arrays;
f) labelling the cDNA obtained by anyone of the cDNA
amplification methods of the invention during the amplification for use on
2s hybridization arrays;
g) diagnosis of aberrant gene expression in small tissue
samples from humans, animals and plants.
h) analysis of the effects of drugs and other agents (e.g.
infectious agents, carcinogens) on gene expression in vivo and in vitro.
3o In the latter case, small tissue samples or cultured cells may be
used. In the former case, small tissue samples can be taken from living
organisms without any disadvantages since only very small samples are
needed;
i) analysis of gene expression in single cells using anyone of
3s the cDNA amplification methods described above.
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j) amplification of full length RNA samples from single cells and
small samples, for subsequent library making or expression in suitable
expression systems.
Preferred embodiments of the invention will now be illustrated,
s but not limited to, the examples presented hereafter.
EXAMPLE I - Rat brain mRNA amplification using the first
embodiment.
lo mRNA isolated from whole rat brain was reverse transcribed.
cDNA derived from 100 pg total RNA (equivalent to the RNA content of
between 5 and 10 cells) was amplified according to the first embodiment.
After reverse transcription only, gene specific PCR assays were positive
when cDNA derived from more than 10 pg of total RNA was used in each
is assay, as shown in figure 1A. After the first amplification step (c), the
majority of the genes were detected using 2.5% of the amplified product
in each gene specific assay (i.e. each sample contained material derived
from 2.5 pg of the original RNA), with some gene sequences detectable
at lower levels, as shown in figure 1 B. After step (f) a further increase in
2o sensitivity was observed with all the genes assayed being positive using
as little as 0.1 % of the amplified product (i.e. amplified cDNA derived
from 0.1 pg of the initial total mRNA sample), as shown in figure 1 C.
Therefore, using this approach, the expression of up to a 1000 genes
could be assayed using 0.1 % of the final product in each gene specific
2s PCR reaction.
Reverse transcription
Total mRNA was prepared from rat whole brain using the total
mRNA isolation system from Promega according to the manufacturer's
3o instructions. Reverse transcription was performed using thermoscript
reverse transcriptase or MMLV reverse transcriptase according to the
manufacturer's (GIBCO-BRL,Paisley, Scotland) instructions. The
reverse transcription primers used were composed of an anchored oligo-
dT primer with a specific 5' heel sequence absent from the mammalian
3s data bases. In some instances a RNA polymerase (T7) promoter site
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was incorporated at the 3' end of the heel sequence. The primers used
were as shown in SEQ ID N°3:
SEQ ID N°3: CTCTCAAGGATCTTACCGCTTTTTTTTTTTTTTTTT
(A,G,C)(A,G,C,T)
s
Second strand cDNA synthesis was initiated by incubating cDNA
derived from 100 pg of total RNA with 25 pg of a mixed primer population
consisting of (5'-3'): a 5' heel sequence absent from the mammalian data
bases (CTGCATCTATCTAATGCTCC), a stretch of 5 random
io nucleotides (NNNNN, where N represents A, C, G or T) and a variable
pentameric sequence chosen from CGAGA, CGACA, CGTAC and
ATGCG, as shown in SEQ ID N°4, 5, 6 and 7. These primers will bind
at
multiple sites on the first strand cDNA and prime second strand
synthesis from such priming sites. After annealing (7.5 mins at 50°C),
is primer extension was performed for 8 mins at 72°C' using ampliTaq
DNA
polymerase (0.35 units, Applied Biosystems, Warrington, UK) in PCR-1
buffer containing 67 mM TrisHCl (pH 8.3) 4.5 mM MgCl2, 6 mM beta-
mercaptoethanol, 0.16% bovine serum albumin and 0.5 mM dNTPs.
Subsequently 1 ng (each) of the reverse transcription primer heel
2o and second strand primer heel were added in 5 NI of PCR-1 buffer and
the reaction subjected to 10 cycles of 92°C for 0.5 min, 60°C
for 1.5 min
and 72°C extension of 1 min, followed by a final 10 min extension. A
further 10 ng of each heel primer were then added in 20 NI of PCR-1
buffer and subjected to a further 40 cycles (as before). The final product
2s was then diluted to 100 NI with water and samples (2.5 or 5 NI) used for
subsequent gene specific PCR assays, or subjected to a further 40
cycles (of 92oC for 0.5 min, 60°C for 1.5 min and 72°C for 1
min,
followed by a final 10 min extension) in the absence of added primers.
This was performed in a PCR-2 buffer containing 3.5 mM MgCl2, 45 mM
3o Tris HCI pH 8.8 and, 12.5% sucrose, 0.1 mM cresol red, 12 mM beta
mercaptoethanol, 0.5 mM dNTPs (Pharmacia), with 0.6 U AmpIiTaq DNA
polymerase (Applied Biosystems). This product was electrophoresed in
a 2% agarose gel (E-gel, Invitrogen) and the high molecular weight
products isolated from the gel using the Qiagen Gel extraction kit
3s according to the manufacturer's instructions.
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Gene specific PCR was performed on samples (2.5 to 10 NI) of
amplified cDNA in PCR-2 buffer with gene specific primers at 100
ng/reaction. Following an initial 2 min denaturing step (92°C), each
PCR
cycle consisted of 0.5 min denaturing (92°C), 1.5 min annealing
(55°C),
s and 1 min elongation (72°C). with a final extension for 10 min at
72°C.
The PCR products were then separated by electrophoresis in a 2.5%
agarose gel, stained with ethidium bromide and the image recorded.
The gene-specific primers used were as follows: a Tubulin, (accession
number, V01226, SEQ ID N°8 and 9), ~3-actin, (accession number,
io V01217, SEQ ID N°10 and 11), Cyclophilin, (accession number,
M25637, SEQ ID N°12 and 13), Adenosine A1 receptor, (accession
number, Y12519, SEQ ID N°14 and 15), Adenosine A2A receptor,
(accession number, L08102, SEQ ID N°16 and 17), Adenosine A2B
receptor (accession number, M91466, SEQ ID N°18 and 19), Adenosine
is A3 receptor, (accession number, M94152, SEQ ID N°20 and 21), NK1
receptor (accession number, J05097, SEQ ID N°22 and 23), NK2
receptor (accession number, M31838, SEQ ID N°24 and 25), trkA
receptor (accession number, M85214, SEQ ID N°26 and 27), trkB
receptor (accession number, M55291, SEQ ID N°28 and 29),
2o proenkephalin, (accession number, S49491, SEQ ID N°30 and 31),
synaptotagmin 1 (accession number, X52772, SEQ ID N°32 and 33),
synaptotagmin 5 (accession number, X84884, SEQ ID N° 34 and 35),
mammalian degenerin, (accession number, U53211, SEQ ID N°36 and
37), Glutamate decarboxylase (GAD67, accession number, X57573,
2s SEQ ID N°38 and 39), choline acetyltransferase (not in GenBank/EMBL
data bases, see Brice et al., (1989) J. Neursoci. Res., 23, 266-273, SEQ
ID N°40 and 41),
Without wishing to be bound by any particular theory, the inventors
3o believe that the increased sensitivity seen after step (e) is due to the
removal of products formed during step (c) which compete in the gene
specific PCR amplification. These products contain repetitive primer
sequences which arecapable of priming on the amplified cDNA
molecules and thus reduce theefficiency of the gene specific reaction.
3s These products are removed
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during step (f), while the amplified gene sequences which have been
incorporated into the high molecular weight products during step (e) are
retained.
As shown in Figure 1, amplification of cDNA derived from 100
s pg of total RNA permits the detection of specific gene sequences by
PCR at levels lower than those of unamplified cDNA. (A) dilutions of
unamplified cDNA; B) dilutions of the amplified cDNA (step c); C)
dilutions of the amplified cDNA (step e). The scale in (A) indicates the
amount of total RNA from which the cDNA used in each gene specific
io assay was synthesised. In (B) and (C), the scale indicates the amount of
total RNA from which the gene specific assay sample was amplified (i.e.
0.1 pg represents one thousandth of the final product obtained after
amplification of cDNA derived from 100 pg of total RNA). Gene
sequences were detected after amplification (as described in steps (a) to
is ( c ) of the first embodiment of the invention when using amplified
product containing as little 1 pg of the initial RNA. After step (e) a further
increase in sensitivity can be seen with detection at levels as low as 0.05
pg
zo EXAMPLE II: The effect of restriction dictestion on the detection of
specific seauences after rat brain mRNA amplification using the
second and third embodiments.
2s I. mRNA isolated from whole rat brain was reverse transcribed,
and the cDNA derived from 25 pg total mRNA (equivalent to the mRNA
content of between 2 and 5 cells) amplified according to Example 1, with
(A) or without (B) cutting with MIu1 as described in step d), followed by
steps e) to g), figure 2. Each gene specific assay contained amplified
3o product derived from 0.6 pg of total RNA. Note the detection of the
adenosine A1 and A3 receptor after cutting (A) which were not detected
without cutting (B).
II. mRNA isolated from whole rat brain was reverse transcribed,
and the cDNA derived from 25 pg total mRNA (equivalent to the mRNA
3s content of between 2 and 5 cells) amplified according to Example III, with
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(A) or without (B) cutting with MIu1 as described in step g), followed by
steps h) to j), figure 2. Each gene specific assay contained amplified
product derived from 0.6 pg of total RNA. Note the increased frequency
of detection of the low abundance mRNAs (mammalian degenerin
s (MDEG), A2B receptor) as well as those of medium (GAD67, (glutamate
decarboxylase; ChAT, choline acetyltransferase) and high
(Synaptotagmin 1 ) abundance.
cDNA (derived from 25 pg total RNA from rat brain) was prepared
io and subjected to second strand synthesis as described in Example 1,
except the heel of the second strand primers as with SEQ ID N°2,which
contains the Mlul cleavage site in particular a rare restriction site
(ACGCGT) at the 3' end. After amplification to step c) as described in
Example 1 (I) or Example 3 (II)), 10 NI of the diluted product was
is incubated in a total of 20 NI at 37°C for 60 min with 2 units of
Mlul in 6.0
mM Mg2+, according to the manufacturer's instructions (Promega). After
addition of EDTA to chelate the Mg2+ and incubation at 65°C for 5 mins
to inactivate the enzyme, 10 p1 aliquots were reamplified in PCR-2 buffer
containing 0.625 units of AmpIiTaq (and 0.05 units of pfu) DNA
2o polymerases for 40 cycles of 92°C for 0.5 min, 60°C for 1.5
min and
72°C extension of 1 min, followed by a final 10 min extension, in the
absence of added primers (I). In (II), 10 ~,I aliquots were subjected to a
further 40 cycles at 92°C for 1.0 min, 95°C for 0.33 min,
72°C for 3 min,
followed by a final 15 min. extension. The product was diluted to a final
2s volume of 50 NI or 100 NI and samples subjected to gene specific PCR
assays as described in Example 1.
In this second embodiment of the method of the invention, removal
of the heel sequence of the second strand primer was designed to
increase the sensitivity of the gene specific PCR by cutting of the
3o competitor products described in Example I (so that they no longer
compete in any of the subsequent PCR reactions), and also to promote
the detection of gene sequences upstream from the reverse transcription
primer site. The increased sensitivity due to removal of the primer
sequences, is apparent in the increased sensitivity of detection of the
3s gene sequences indicated. It is believed (but the applicants do. not wish
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to be bound by any theory) that short amplified products of gene
sequence generated during step (c) can, after strand separation at
92°C,
be extended, after annealing to longer complementary products.
Removal of the second strand primer heel, and amplification with the
s proof reading DNA polymerase pfu can assist this process. In this way
the amount of amplified material containing sequence upstream from the
reverse transcription primer site can be increased.
As shown in Figure 2, part (I), gene specific PCR after step (f), without
io (B) and with (A ) cutting with the rare cutter restriction enzyme MIu1
shows that cutting increases the detection of low abundance gene
sequences such as the adenosine A1 and A3 receptors.
EXAMPLE III - Amplification of rat brain mRNA using the third
~s embodiment.
In order to increase the sensitivity and specificity of the
amplification process, two heel primers were designed for use at high
stringency which were able to amplify single copies of lambda
2o bacteriophage DNA in the presence of a 1000-fold excess of rat genomic
DNA i.e. they were highly specific for the complementary sequences and
able to amplify single copies (data not shown). Using these primers in
the third embodiment of the method of the present invention, amplified
product derived from as little as 0.01 pg of the initial RNA were positive
2s in the gene specific PCR assays. This amount of RNA represents
approximately 0.1 % of that contained in a single cell.
Reverse transcription: Total mRNA prepared from rat whole brain using
the total mRNA isolation system from Promega according to the
3o manufacturer's instructions. Reverse transcription was performed using
MMLV reverse transcriptase again according to the manufacturer's
(GIBCO-BRL, Paisley, Scotland) instructions. The reverse transcription
primers used were composed of an anchored oligo-dT primer with a
specific 5' heel sequence absent from the mammalian data bases. The
3s primer used SEQ ID N°42 is indicated below:
so
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ACTGCCAGACCGCGCGCCTGAATTTTTTTTTTTTTTTTTTTTTTTTTT
TTTT(A, C, G) (A, C, G, T) .
s Second strand synthesis was initiated by incubating cDNA derived
from 1000 pg of total RNA with 20 ng of second strand primers which
were composed of (5' to 3'): a heel sequence absent from the
mammalian data bases (TGTCCGTTTGCCGGTCGTGGGC) an MIu1
site (ACGCGT), 17 random nucleotides (NNNNNNNNNNNNNNNNN)
Io and three arbitrary bases (G, T) (A, G, T) (A, C, G), as shown in SEQ ID
N°43.
These primers were added to give a final volume of 20 NI of PCR-1
buffer containing 4.5 mM Mg2+, 1 unit of AmpIiTaq DNA polymerase and
0.05 units of pfu DNA polymerase (Stratagene) and annealed and
is extended over 40 cycles under the following conditions: 92°C for 0.5
min,
40°C for 0.5 min (optional) and 72°C for 5 min, followed by a
final 30 min
extension. The repetitive annealing of these primers serves to increase
the probability that all the gene sequences present in the initial cDNA
population are copied into double stranded products.
2o Subsequently 300 ng of the reverse transcription and second strand
heel primers were added with a further 1.25 units of AmpIiTaq DNA
polymerase in the presence of the Taqstart antibody (CIonTech) and
0.25 units of pfu DNA polymerase in 35 p1 of buffer containing 1.5 mM
Mg2+, 67 mM Tris HCI (pH8.3) and 0.17 mM dNTPs. In some cases the
2s amplification products were then subjected to restriction digestion with
MIu1 as described in Example 2, the low molecular weight products
(including the heel of the second strand primers) removed through a
Nanosep 10K column (double stranded DNA less than 100bp in length
pass through the filter of these Nanosep columns) and the larger
3o products either subjected to gene specific PCR, or subjected to a further
40 cycles of 92°C for 1.0 min, 95°C for 0.33 min, 72°C
for 3 min,
followed by a final 15 min extension. Removal of the second strand heel
sequences was designed to both reduce the influence of any competing
products and primers in the gene specific PCR and to permit product
3s priming/repair as described in Embodiment 2.
s1
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The outcome of this procedure was the ability to detect genes by
gene specific PCR (as described in Example 1) at dilutions of the
amplified cDNA derived from as little as 0.01 pg of total RNA, illustrating
s that approximately 100,000 gene specific PCR assays could successfully
be performed after amplification of cDNA derived from 1 ng of total RNA
(i.e. the content of approximately 100 cells). In addition product
priming/product repair was shown to occur with the detection of a gene
sequences 2.4 kb 5' to the reverse transcription priming site (Figure 4).
io
As shown in Figure 4, high stringency amplification cDNA
derived from 1000 pg of total RNA as described in embodiment 3 permits
increased detection of gene sequences. (I) The scale indicates the
amount of total RNA from which the cDNA used in each gene specific
is assay was synthesised (A), or the amount of RNA from which the gene
specific assay sample was amplified (B).
Specific sequences were detected when product amplified from
as little as 0.01 pg of the initial RNA was used in the gene specific PCR.
(II) Gene specific PCR using cDNA amplified from 2.5 pg of total RNA
2o per gene specific assay. Inclusion of steps g) to i) of Amplification
Method 3 also resulted in the detection of NK2 receptor gene sequence
located 2350 by upstream of the polyA splice site (A). This sequence
was not detected if step g) was omitted (B). III Amplification of cDNA
derived from 1 ng total whole brain RNA by Amplification Method 3
2s permits the detection of gene specific sequences in 0.006%of the
product (equivalent to cDNA amplified from 0.06 pg of total RNA).
Control: no amplification.
Furthermore, as shown in (II) of Figure 2, including steps g) to i)
in embodiment 3 (A) increases the detection of low abundance
3o messages such as those encoding the adenosine A2B receptor and the
mammalian degenerin MDEG, when compared to an amplification which
omitted step g) (B). In addition the detection of abundant mRNA species
such as that encoding synaptotagmin 1 was also increased.
s2
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EXAMPLE IV In vitro transcription of RNA from cDNA amplified
according to embodiments 1 and 3
Incorporation of the T7 promoter into the reverse transcription
primer heel was performed so that RNA could be produced for
subsequent analysis by hybridisation methods, for instance on oligo
arrays. The yield of RNA from the amplified cDNA was estimated by
running two parallel transcriptions, one for RNA synthesis and the other
containing 35S-UTP as a substrate, so that the incorporation of the
io radioactivity into RNA could be used used as an index of RNA synthesis.
After amplification of rat liver cDNA derived from 500 pg of total RNA by
Amplification Procedure 1 to step (c), in vitro transcription resulted in a
yield of 12.5 micrograms of RNA. Using Embodiment 3 to step (c), the
yield of RNA from liver cDNA (derived from 500 pg of total RNA) was 34
is micrograms (mean of 5 experiments). Similarly the yield from cDNA
derived from 2500 pg was 90 micrograms (mean of 5 experiments)..
Inclusion of step g) of Embodiment 3 prior to in vitro transcription
increased the yield of RNA 1.7-fold (mean of two experiments). In order
to examine the sequence content of the RNA transcribed from cDNA
2o amplified according to Embodiment 3 (to step c) the RNA was reverse
transcribed using the heel of the second strand heeled primer. Figure 5
illustrates that the cDNA derived from the transcribed RNA contained
abundant gene sequence with actin tubulin and cyclophilin sequences
being detected in aliquots representing 0.0001 % of the RNA so
25 produced. Therefore it appears that the expression of up to 1,000,000
genes may be assessed in amplified samples derived from 2500 pg of
total RNA i.e. RNA derived from approximately 250 cells.
Reverse transcription of liver RNA was performed essentially as
described in Examples 1 and 3, using primers containing a T7 RNA
3o polymerase promoter site. The primers usedwere, SEQ ID N°44,:
CTCTCAAGGATCTTACCGCTAATACGACTCACTATAGGCGCTTTTTT
TTTTTTTTTTTTTTTTTT
(A,G,C)(A,G,C,T) and SEQ ID N°45
53
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GACTGCCAGACCGCGCGCCTGACGCGTAATACGACTCACTATAGG
GTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
s (A, C, G) (A, C, G, T)
Subsequent second strand synthesis and amplification was
carried out as described in the relevant examples. After step (c) of each
Embodiment the amplified cDNA was isolated using a Qiaquick PCR
io purification kit (Qiagen) according to the manufacturer's instructions to
remove primers and other low molecular weight products, and 5 p1
aliquots subjected to in vitro RNA transcription using the T7 Megascript
kit (Ambion) according to the manufacturer's instructions. After DNase
treatment to remove cDNA (DNase 1, 30 min, 37°C), the RNA was
Is isolated using the Rneasy kit (Qiagen). One of the aliquots was
transcribed in the presence of 35S-UTP to determine the yield of RNA.
The RNA was subsequently ethanol precipitated (75% ethanol, 5%
sodium acetate at -20°C for 30 mins), before reverse transcription with
MMLV reverse transcriptase according to the manufacturer's instructions.
2o The reverse transcription primers used were part or the whole of the heel
sequences of the second strand primers, SEQ ID N°1 and 44
Subsequent gene specific PCR using this cDNA as substrate
revealed that as little as 0.0001 % of the product could be used in gene
2s specific PCR and result in the detection of gene sequence. The data
presented show that embodiment 3 with in vitro RNA transcription
generates sufficient RNA of good quality for application to cDNA and
oligonucleotide arrays. This will permit the analysis of the expression of
thousands of genes from tissue samples containing approximately 250
3o cells and with further improvements perhaps from samples as small as
single cells.
As shown in Figure 5, in vitro transcription of RNA from
amplified cDNA contains large amounts of bona fide gene sequence.
3s cDNA derived from 125, 500 and 2500 pg of liver total RNA was
54
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amplified using Amplification Method 3 as far step e) and RNA
transcribed. Cutting with the restriction enzyme (step g) was omitted so
that the RNA so produced would contain the heel sequence of the
second strand primer. This heel primer was then used to prime reverse
s transcription of the RNA, and the resulting cDNA analysed for the
presence of 3 gene sequences. Note that all 3 gene sequences were
detected even after 106 fold dilution of the product.
Example V. Amplification of rat spinal cord cDNA derived from 1 na
io total RNA (eguivalent to approximately 100 cells) using the third
embodiment.
In example IV it was shown that antisense RNA could be in vitro
transcribed from cDNA amplified by embodiment 3, this RNA could be
is applied to gene chips bearing sense probes, or reverse transcribed and
applied to microarrays bearing antisense probes. However many
microarrays bear sense probes (i.e. they recognise antisense DNA), but
are not suitable for the hybridization of labelled RNA samples. In order
to maximise the utility of embodiment 3, sense RNA was also transcribed
2o from the amplified cDNA in vitro, reverse transcribed and the gene
sequence content assessed by gene specific PCR.
Reverse transcription
Reverse transcription (RT) was performed in 10 NI containing;
2s 1x first-strand buffer, 200 Units MMLV reverse transcriptase (BRL), and
0.5 ng first strand primer for 60 min at 37°C. The first strand primer
(SEQ ID N°46),ACTGCCAGACCGCGCGCCTGAACGCG
TAATACGACTCACTATAGGGTTTTTTTTTTTTTfTTTTTT(A, C,
G) (A, C, G, T), contained (5' to 3') a 26 base sequence absent from the
3o mammalian data bases capable of hybridising to its complement at
72°C
in the presence of 2mM Mgz+ with an Mlul site at its 3' end, the T7 RNA
polymerase promoter sequence, and an anchored stretch of oligodTs for
hybridising to the 5' end of the polyA sequence of mRNA.
ss
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Second strand synthesis
Second strand synthesis was performed by adding an excess of
second strand primer (1 ng) (to increase chances of annealing to every
first strand sequence) in 4 microlitres of buffer giving a final Mg2+
s concentration of 3.5mM.
After heating to 80°C, 5 units of Taq (Applied Biosystems,
Warrington, UK) was added with 0.25 units of the proofreading enzyme
pfu (Stratagene). Adding the Taq at high temperature ("hot start")
prevents the enzyme copying mishybridised sequences in the mixture,
io such mishybridization tending to occur at the low temperatures
encountered when setting up the reaction.
Primer annealing occurred at 50°C (7.5 mins decreasing by 10
secs per cycle) and extension at 72°C for 2.5mins. The temperature was
cycled between 50°C and 72°C 40 times.
is Although not wishing to be bound by theory, is the inventors
believe that under these conditions each first strand cDNA will be
annealed in multiple positions by the second strand primer. Each cycle
permits further annealing by the primer. However, unlike normal PCR,
the second strands are not dissociated from the first strand by melting in
2o each cycle, consequently each primer has an equal chance of being
extended to the 5' end of the first strand (which bears one of the heels),
thus increasing the efficiency of subsequent PCR. It is envisaged that
extension of primers at the 3' end of the first strand will displace those
nearer the 5' end producing multiple copies of each second strand. The
2s second strand primer contained (from 5' to 3'): a sequence absent from
the mammalian data bases which is capable of hybridising to its
complement and 72oC in the presence of 2mM Mg2+ and standard PCR
buffers, an Mlul site (ACGCG), the T3 RNA polymerase promoter and a
random sequence of 15 bases, SEQ ID N°47:
3o AAAACTGCCAGACCGCGCGCCTGAACGCGTCGTATTAACCCTCACT
AAAGGGN15
Amplification reactions
Subsequent PCR was performed by adding 4 microliters in
3s AmpIiTaq buffer (Applied Biosystems, Warrington, UK) containing1.25
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mM dNTPs and 33ng of primers (the sequence absent from the
mammalian data bases which is capable of hybridising to its complement
and 72°C in the presence of 2mM Mg2+ with an Mlul site) to give a final
Mg2+ concentration of 2.6 mM.
s In this example, this primer was common to both the first and
second strand primers). After heating to 80°C, 5 units of Taq (Applied
Biosystems, Warrington, UK) was added with 0.25 unit of the
proofreading enzyme pfu (Startagene).
The reaction was then subjected to 20 cycles of denaturation
io (94°C, 20 secs), and annealing with extension (72°C, 5mins).
19
microlitres of AmpIiTaq buffer were then added (at 80°C) containing and
0.2 mM dNTPS, 100 ng of primers and giving a final Mg2+ concentration
of 2.1 mM. 5 units of Taq (Applied Biosystems, Warrington, UK) was
then added with 0.25 units of the proofreading enzyme pfu (Stratagene).
is The reaction was then cycled 40 times as described above, with a final
extension at 72°C for 30 min.
After amplification small MW primers and products were removed
by passage through a Qiaquick PCR purification kit (Qiagen). The
amplified cDNA was then cut with Mlul and recleaned using the same kit
2o same prior to subsequent gene specific PCR assays or in vitro
transcription. Gene specific PCR was performed as previously described.
In vitro transcription of RNA was performed using the Ambion Megascript
Kit according to the manufacturers instructions.
After DNase treatment, some of the resulting RNA was reverse
2s transcribed for gene specific PCR. Figure 6 shows the size distribution
of the RNA produced from both the T3 and T7 RNA polymerase
promoters (i.e. most between 200 and 600 by with detectable higher
molecular weight material). 10% of the product obtained after
amplification of cDNA derived from 1 ng of total RNA was in vitro
3o transcribed with T7 polymerase or T3 polymerase and 30% of each RNA
applied to a gel. The estimated yields from the two RNA polymerases
were 0.5 and 1.5 micrograms respectively. Figure 7A shows that the
amplified cDNA contained both rare (A2A receptor) and abundant (e.g.
tubulin) gene sequences detectable by gene specific PCR. I:
ss amplification with a second strand primer lacking the T3 promoter, II
s7
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amplification with a second strand primer bearing the T3 promoter.
Samples were diluted up to 1/3,000 prior to gene specific PCR. Figure
7B shows that the in vitro transcribed sense RNA generated using the T3
RNA polymerase (after reverse transcription to cDNA) also contains
s abundant gene sequence..
Example VI. Single cell expression analysis using microarrays after
cDNA amplification of striatal cholinergic neuron al mRNA usinct
embodiment 3.
io
In order to assess single cell gene expression, mRNA was
amplified by the third embodiment using the primers and conditions
described in Example V. T3 RNA polymerase was used to generate
sense RNA which was then reverse transcribed using fluorescently
is labelled dCTP (Cy3 or Cy5) for application to glass microarrays bearing
sense DNA probes
Harvesting of single cell mRNA
Striatal cholinergic neurons were identified on the basis of their
2o size and electrophysiological characteristics in 300 ~m coronal slices
from 14-28 day-old male Sprague Dawley rats containing the striatum
were viewed with a Zeiss Axioskop microscope (Carl Zeiss Ltd., Welwyn
Garden City, U.K.) fitted with a x64 water-immersion objective lens.
Light in the infrared range (>740nm) was used in conjunction with
2s a contrast-enhancing Newvicon camera (Hamamatsu, Hamamatsu City,
Japan) to resolve individual neurones within slices (Lee et al., 1998).
The physiological saline bathing the slices contained (mM) 125
NaCI, 25 NaHC03, 10 glucose, 2.5 KCI, 1.25 NaH2P04, 2 CaCl2, 1
MgCl2 and was bubbled with 95%/5% 02/C02. The electrode buffer
3o contained 120 K gluconate, 10 NaCI, 2 MgCl2, 0.5 EGTA, 10 HEPES, 1-
4 mM Na2ATP, 0.3 Na2GTP, pH adjusted to 7.2 with KOH. 0.5 Ng/ml
glycogen (Boehringer) and RNase inhibitor (Pharmacia, 0.1 units/NI)
were included to facilitate harvesting of RNA from the cells. This buffer
also contained 10 fg each of bacterial sequences derived from the trp,
3s thr and lys codons of E. coli. These mRNAs had polyA sequences
s8
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attached to the 3' end so that they could be amplified by XTPEA. All
solutions were made up in diethylpyrocarbonate (DEPC) treated water.
Borosilicate recording electrodes were baked (2h, 250°C) before
being
pulled to a resistance of between 3 and 5 M$. Electrophysiological
s signals were detected using an Axopatch-1 D patch-clamp amplifier
(Axon Instruments, CA, USA) and were recorded onto digital audiotape.
Following formation of the whole cell configuration, series resistance was
partially compensated using the amplifier, and cellular conductance
continuously monitored via the injection of hyperpolarising current or
io voltage. Membrane signals were filtered at 1 kHz and were digitized at
SkHz through a Digidata 1200A/D converter using pClamp 6.0 software
(Axon Instruments Inc, CA, USA).
Extraction of Neuronal contents and amplification
is The cytoplasm from large cells (>30Nm in one dimension) was
aspirated under visual control into a patch-clamp recording electrode
until approximately 40% of the somatic cytoplasm had been collected.
Usually the nucleus was sucked onto the end of the electrode until an
electrical seal (>0.5G$) was formed prior to withdrawal of the electrode
2o to prevent contamination from the slice. Since withdrawal of the nucleus
from the cells caused structural damage, outside-out patches were used
to seal the electrodes if the cells were to be subsequently examined
immunohistochemically. The contents of the electrode were forced into
a microtube and reverse transcribed, amplified, low molecular weight
2s components removed and all of the product in vitro transcribed as
described for Example V. After Dnase treatment the RNA was reverse
transcribed using Cy3 or Cy5 labelled dCTP prior to application to the
microarrays.
3o Microarray Synthesis
Custom synthesised amine-modified oligonucleotide probes
(probes) were purified in desalting columns to remove amine
contaminants. The probes were prepared to a final concentration of 10-
25 nmole/ml in 1X Surmodics Printing Buffer, containing 150 mM sodium
3s phosphate, pH 8.5 (SurModics Inc, USA). The probe solution was printed
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on 3D-Link Activated Slides (SurModics Inc, USA), and stored overnight
in a saturated NaCI chamber. Printed slides were stored at room
temperature. The microarrays contained probes capabale of recognising
the bacterial sequences which were included in the patch electrode
s buffer. These served to ensure that successful amplification had
occurred. In addition 3 probes from the Dengue virus genome were
included as negative controls. The arrays contained a total of 510
oligonucleotide probes, recognising 141 different transcripts, each
transcript being recognised by 3 or more separate probes.
1o
Microarray hybridisation
Slides were exposed to 15 ml SurModics Blocking Solution (50
mM ethanolamine, 0.1 M Tris, pH 9) with 0.1 % SDS at 50°C for 15
minutes. Slides were rinsed twice with water, and washed with 15 ml 4X
is SSC / 0.1 % SDS prewarmed to 50°C for 40 minutes on a shaker. Slides
were washed with water, and centrifuged at 800 rpm for 3 minutes.
Labelled cDNA (target) hybridisation mixture was heated for 2 minutes in
a boiling water bath, spun briefly to cool, and 2.5 I of target added per
cm2 of coverslip. Slides were placed in a humidified incubator overnight.
2o Slides were removed from the incubation chamber and successively
washed with 4X SSC for 30 seconds, 2X SSC / 0.1 % SDS for 5 minutes,
0.2X SSC for 1 minute and 0.1X SSC for 1 minute. Slides were spun to
dry and scanned.
Zs Gene expression analysis of single cells after cDNA amplification
using microarrays.
cDNA amplification by embodiment 3 was used to assess the
expression of a large number of genes in 4 striatal cholinergic neurons,
the aim being the detection of both low and high abundance transcripts.
3o In any analysis of gene expression at the single cell level problems are
encountered with low abundance transcripts and with the non detection
of some mRNAs in subpopulations of cells. This has been discussed in
Surmeier et al (1996) J. Neurosci. 16, 6579-6591 and Richardson et al.
(2000) J. Neurochem 74, 839-846.
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Currently it is accepted that the number of cells in which a
transcript is detected is related to the abundance of the transcript i.e. the
more often a transcript is detected in individual cells the more abundant
is the mRNA. Thus in any study of an apparently homogeneous
s population of cells, some low abundance transcripts may be detected in
only a subpopulation of cells. For example, many GABAA receptor
subunit mRNAs were detected in less than 100% of the cholinergic
neurons tested by Yan and Surmeier (1997), suggesting that these
transcripts were expressed either at low abundance in all the cells, or
io only in a specific subpopulation of cells. In the former case, more
sensitive techniques will reveal a higher proportion of cells as positive for
given transcripts, whereas in the latter there will be little change in the
of cells positive for a given transcript.
Table 1 shows some of the genes detected i.e. those whose
is expression in these cells had been previously characterised, and that the
bacterial positive controls were detected but not the viral negative
controls. All the housekeeping mRNAs are expected to be expressed in
all cholinergic neurons. The neuronal markers dynorphin, enkephalin
and PPTA are markers for non cholinergic neurons in the striatum, and
20 lipoprotein lipase for endothelial cells.
Table 1 also shows that the use of embodiment 3 increased the
number of cells (compared to previous estimates) in which the Voltage
sensitive Na channel a6, trkC and NK3R mRNAs were detected,
showing the ability of this method to detect low abundance transcripts in
2s single cells.
In addition a number of mRNAs not previously suspected to be
expressed in these cells were detected including somatostatin (SST),
mAChR3 and 5, SUR2 and the D3 and D4 receptors, again showing the
high sensitivity of this method.
3o In contrast, many of the GABA receptor subunit mRNAs were only
detected in a proportion of the cells, suggesting that subpopulations of
these cells may exist which express different complements of GABA
receptor subunits, as suggested by Yan and Surmeier (1997). Other
references in table 1 are: Yan & Surmeier (1996), Yan et al., (1997) and
3s Tallaksen-Greene et al (1998).
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Table 1: List of mRNAs detected in 4 single cholinergic neurons
using embodiment 3 followed by hybridization to microarrays. The
percentage of cells expected (e.g. housekeeping mRNAs are expected
s to be expressed in all cells) or previously shown by other methods, is
shown in the third column (% positive cells) with the appropriate
reference in the second column. The percentage of cholinergic cells in
which the corresponding mRNAs were detected after embodiment 3 and
microarray analysis is shown in column 4 (% positive cells by
io embodiment III).
62
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~ N ~ ~ ~ ~ ~ ~ ~ ~ ~
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d > > > > ~ ~J > > > ~ tntnr~
>
-p .a = f~flJ(n(n(n (!~fnfn!O-a- -p
ot3xSo~SotSoS O ~tS~tSottotSm o cacua~
a m
U O ~ ~ = c c C C C Q~ C C C C t = ~ ~ c
_ N N v f0f0t0IOf0 f~ (UfCt0V U U_U
r f0
J J
.a
G1
a ~ a m ~
a ~ ~ a o
s ~ s s s .o.o.o.o~~
~ N ~ p
.
c
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a a0U O u1 U
> > > o ~ ~ ~ ~ ~ a a a a ~ a ~ ~ o
a
v~ cncn ~ ~ a ~ ~ ~ C~ ~ E E a a a a o
E
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
~ ~Of7 ~ ~ ~ N ~ O N N ~ N ~ ~ ~ r O
O
O O O ~ O N ~ ~ N ~! O O O O
r r r r r
c c
3 3
0 0
c c
c c
.-.~..~.
0 0 0 0 0 0 0
N N ~
N -' N N N N
.
t0(aN ~ N N IC N
N O ~ N ~ N
N
~ r-r e-C C C C c c c
v ~ ~r~r _
N N ~ ~ N N ~ N
N N N N f3(CN ~C (0N N I9 O
U U
U U V U U V = X X X N X
_ LIJLIJW W
~
C
Q Q ~ ~ C
p p O
.
d a :
.
c c c c
_ _ _ _
~E~E~E~E a~ d c ~
~ Y
0 0 0 o Y O c~cn W o ~ ~n~ a
D D D D Z Z Z 7 t3~it3 ti ~ m ?~~ : 7 D
ti
66
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
1
SEQUENCE LISTING
<110> WARNER-LAMBERT
<120> A method for amplifying low abondance nucleic acid
sequences and means for performing said method.
<130> 5973PCT
<140>
<141>
<160> 50
<170> PatentIn Ver. 2.1
<210> 1
<211> 20
<212> DNA
2o <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 1
ctgcatctat ctaatgctcc 20
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 2
4o ctgcatctat ctagtacgcg t 21
<210> 3
<211> 38
<212> DNA
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
2
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 3
ctctcaagga tcttaccgct tttttttttt tttttrvn 3 g
to
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 4
ctgcatctat ctaatgctcc nnnnncgaga 30
<210> 5
2s <211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 5
ctgcatctat ctaatgctcc nnnnncgaca 30
3s
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
3
<400> 6
ctgcatctat ctaatgctcc nnnnncgtac 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 7
ctgcatctat ctaatgctcc nnnnnatgcg 30
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 8
cactggtacg tgggtgagg 19
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 9
tttgacatga tacagggact gc 22
<210> 10
<211> 18
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
4
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 10
catccatgcc ctgagtcc 18
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 11
acacctcaaa ccactcccag 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 12
actgccaaga ctgagtggct 20
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
<400> 13
aatggtttga tgggtaaaat gc 22
5
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
i5 <400> 14
actctgctga gcctggatgt 20
<210> 15
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 15
accagggaca ccttgcttc 19
<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 16
tctgaccaac aaagctggc 19
<210> 17
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
6
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 17
tggaaggaaa ggcagtagtc a 21
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 18
ggggacagca actcagaaaa 20
30
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 19
cagctctcca agtttccacc 20
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
7
oligonucleotide
<400> 20
cagacttcgc ccttccttc 19
<210> 21
<211> 21
<212> DNA
to <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 21
tcaattcact ccctgtgttc c 21
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 22
3o ctggaaagag gagccttgtg 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
~120>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 23
ctgagacgga aaggaacagc 20
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
8
<210> 24
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 24
agaggatgcg cacagtcac 19
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 25
tgatgggaaa gaggttctgg 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 26
cgttctgggc taggagtctg 20
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence
4s <220>
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
9
<223> Description of Artificial Sequence:
oligonucleotide
<400> 27
ttgctattat gatggatgct gg 22
<210> 28
<211> 22
i o <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 28
ccacgaaagg tctcatttta gg 22
25
<210> 29
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 29
gagcttccct gtccctcag 19
<210> 30
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 30
agttgccctc gtggtctg 18
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
<210> 31
<211> 22
<212> DNA
5 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
l0
<400> 31
tgtcagaagg gatgaggtaa ca 22
i5 <210> 32
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 32
aggggctttc ctatctaagg g 21
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 33
gttggcagtg ttgcaagaga 20
<210> 34
<211> 19
<212> DNA
<213> Artificial Sequence
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
11
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 34
aagcacctga ccccagatc 19
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 35
ccagactttc ccaacttttc c 21
<210> 36
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 36
attttctccg tgcggtttc 19
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 37
cggtcacaaa caacacaagg 20
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
12
<210> 38
<211> 22
s <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 38
atcttgcttc agtagccttt gc 22
20
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 39
tgtcttcaaa aacacttgtg gg 22
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 40
tactaagctc tgttcccatc cc 22
<210> 41
<211> 19
<212> DNA
<213> Artificial Sequence
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
13
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 41
acccaggttg cttccaaac 19
i o <210> 42
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 42
2o actgccagac cgcgcgcctg aatttttttt tttttttttt tttttttttt ttvn 54
<210> 43
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 43
tgtccgtttg ccggtcgtgg gcacgcgtnn nnnnnnnnnn nnnrmkdv 48
<210> 44
<211> 67
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
4s <400> 44
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
14
ctctcaagga tcttaccgct aatacgactc actataggcg cttttttttt tttttttttt 60
tttttvn 67
<210> 45
<211> 78
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 45
gactgccaga ccgcgcgcct gacgcgtaat acgactcact atagggtttt tttttttrtt 60
ttttlttttt ttttnvn 78
<210> 46
<211> 68
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 46
actgccagac cgcgcgcctg aacgcgtaat acgactcact atagggtttt tttttttttt 60
3o ttttttvn 68
<210> 47
<211> 68
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 47
aaaactgcca gaccgcgcgc ctgaacgcgt cgtattaacc ctcactaaag ggnnnnnnnn 60
nnrmnnnn 68
CA 02378070 2002-O1-08
WO 01/06004 PCT/EP00/06887
<210> 48
<211> 67
<212> DNA
5 <213> Artificial Sequence
to
20
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 48
ctctcaagga tcttaccgct aatacgactc actataggcg ctttritttt tttttttttt 60
tttttvn 67
<210> 49
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 49
ctctcaagga tcttaccgct trtttttrtt ttttttvn 3 8
<210> SO
<211> 68
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> SO
ctctcaagac gcgtgatctc taatacgact cactataggc gctttttttt tttttttttt 60
4o ttttttvn 68
