Note: Descriptions are shown in the official language in which they were submitted.
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SAMPLING METHOD AND APPARATUS FOR AMPLIFICATION REACTION ANALYSIS
RELATED APPLICATIONS
This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional
Application
Nos. 60/428,038, filed November 21, 2002; 60/439,982, filed January 14, 2003;
and 60/440,010,
filed January 14, 2003, the entirety of each is hereby incorporated by
reference.
FIELD OF THE INVENTION
Aspects of the invention relate to sampling methods and apparatuses for
nucleic acid
amplification reactions. Such sampling methods find use in deriving
quantitative information
from nucleic acid amplification analyses, for example, transcriptional
profiling analysis.
BACKGROUND
Nucleic acid probe technology has developed rapidly in recent years as
researchers have
discovered its value for detection of various diseases, organisms or genetic
features which are
present in small quantities in a human or animal test sample.
A targeted nucleic acid sequence in an organism or cell may be only a very
small portion
of the entire DNA molecule so that it is very difficult to detect its presence
using most labeled
DNA probes. Much research has been carried out to find ways to detect only a
few molecules of
a targeted nucleic acid.
A significant advance in the art is described in U.S. Patent Nos. 4,683,195;
4,683,202;
and 4,965,188. These patents describe amplification and detection methods
wherein primers are
hybridized to the strands of a targeted nucleic acid (considered the
templates) in the presence of a
nucleotide polymerization agent (such as a DNA polymerase) and
deoxyribonucleoside
triphosphates. Under specified conditions, the result is the formation of
primer extension
products as nucleotides are added along the templates from the 3'-end of the
primers. These
products are then denatured and used as templates for more of the same primers
in another
extension reaction. When this cycle of denaturation, hybridization and primer
extension is
carried out a number of times (for example 25 to 30 cycles), the process which
is known as
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"polymerase chain reaction" expanentially increases the original amount of
targeted nucleic acid
so that it is readily detected.
Once the targeted nucleic acid has been sufficiently amplified (that is, many
times more
copies of the molecule have been made), various detection procedures can be
used to detect it.
The patents noted above, for example, describe the use of insolubilized or
detectably labeled
probes and gel electrophoresis as representative detection methods.
A wide range of times and temperatures for amplification methods are generally
described, with the specific combination of time and temperature largely
dependent upon the
type of DNA polymerase used, the complexity of the mixture of nucleic acids
including the
targeted nucleic acid, the length and specificity of the primers, the length
of the targeted nucleic
acid, pH and several other reaction conditions and components.
Amplification reactions have been used for a number of applications, for
example, in
transcription profiling. Transcription profiling promises to impact upon the
process of target
identification and validation in accelerating the pace of drug discovery, as
well as disease
diagnosis and prognosis. This method compares expression of genes in a
specific situation: for
example, between diseased and normal cells, between control and drug-treated
cells or between
cells responding to treatment and those resistant to it. The information
generated by this
approach may directly identify specific genes to be targeted by a therapy,
and, importantly,
reveals biochemical pathways involved in disease and treatment. In brief, it
not only provides
biochemical targets, but at the same time, a way to assess the quality of
these targets. Moreover,
in combination with cell-based screening, transcription profiling is
positioned to dramatically
change the field of drug discovery. Historically, screening for a potential
drug was successfully
performed using phenotypic change as a marker in functional cellular system.
For example,
growth of tumor cells in culture was monitored to identify anticancer drugs.
Similarly, bacterial
viability was used in assays aimed at identifying antibiotic compounds. Such
screens were
typically conducted without prior knowledge of the targeted biochemical
pathway. In fact, the
identified effective compounds revealed such pathways and pointed out the true
molecular target,
enabling subsequent rational design of the next generations of drugs.
Modern tools of transcription profiling can be used to design novel screening
methods
that will utilize gene expression in place of phenotypic changes to assess the
effectiveness of a
drug. For example, such methods are described in U.S. Patent Nos. 5,262,311;
5,665,547;
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5,599,672; 5,580,726; 6,045,988 and 5,994,076, as well as in Luehrsen et al.
(1997,
Biotechniques, 22:168-74), and in Liang and Pardee (1998, Mol Biotechnol.
10:261-7). This
approach will be invaluable for drug discovery in the field of central nervous
system (CNS)
disorders such as dementia, mild cognitive impairment, depression, etc., where
phenotypic
screening is inapplicable, but a desired transcription profile can be readily
established and linked
to particular disorders. Once again, the identified effective compounds will
reveal the
underlying molecular processes. In addition, this method can be instrumental
for the
development of improved versions of existent drugs, which act at several
biochemical targets at
the same time to generate the desired pharmacological effect. In such case the
change in the
transcriptional response may be a better marker for drug action than selection
based on
optimization of binding to multiple targets.
A number of advanced methods of transcription profiling are based on
technology using
DNA microarrays, for example, as reviewed in Greenberg, 2001 Neurology 57:755-
61; Wu,
2001, J Pathol. 195:53-65; Dhiman et al., 2001, Vaccine 20:22-30; Bier et al.,
2001 Fresenius J
Anal Chem. 371:151-6; Mills et al., 2001, Nat Cell Bioh. 3:E175-8; and as
described in U.S.
Patent Nos. 5,593,839; 5,837,832; 5,856,101; 6,203,989; 6,271,957; and
6,287,778. DNA
microarray analysis is a method which provides simultaneous comparison of the
expression of
several thousand genes in a given sample by assessing the hybridization of
labeled
pohynucleotide samples, obtained by reverse transcription of mRNAs, to the DNA
molecules
attached to the surface of the test array.
One of the most sought after benefits believed possible with the sensitivity
of nucleic
acid amplification technology is the reliable quantitation of the amount of
template present in a
sample before amplification. Such a method fords direct application in, for
example,
transcription profiling. The high sensitivity and fidelity of the
amplification reactions makes it
possible to extrapolate the original template abundance from the amount of
amplification
products generated. However, the kinetics of amplification vary with respect
to template and
stage of the amplification process, making it difficult to fully realize the
quantitative potential of
nucleic acid amplification procedures.
In order to obtain data that reliably reflect the amount of original template,
it is necessary
to collect quantitative data at a point in which every target sequence is in
the exponential phase
of amplification (since it is only in this phase that amplification is
extremely reproducible and
accurately reflects the abundance of template molecules prior to
amplification). Analysis of
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reactions during exponential phase at a given cycle number should
tlieoretically provide several
orders of magnitude of dynamic range. However, low abundance targets will
often be below the
limit of detection at a set cycle number, while abundant targets will be past
the exponential
phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-
point relative
PCR. In order to extend this range, replicate reactions may be performed for a
greater or lesser
number of cycles, so that all of the samples can be analyzed in the
exponential phase.
Holland et al. (1991, Proc. Natl. Acad. Sci. U.S.A. 88: 7276-7280), U.S.
Patent No.
5,210,015 and others have disclosed fluorescence-based approaches to provide
real time
measurements of amplification products during PCR. Such approaches have either
employed
intercalating dyes (such as ethidium bromide) to indicate the amount of double-
stranded DNA
present or they have employed probes containing fluorescence-quencher pairs
(also referred to as
the "Taq-Man" approach) where the probe is cleaved during amplification to
release a
fluorescent molecule the concentration of which is proportional to the amount
of double-stranded
DNA present. During amplification, the probe is digested by the nuclease
activity of a
polymerase when hybridized to the target sequence to cause the fluorescent
molecule to be
separated from the quencher molecule, thereby causing fluorescence from the
reporter molecule
to appear.
The Taq-Man approach uses an oligonucleotide probe containing a reporter
molecule -
quencher molecule pair that specifically anneals to a region of a target
polynucleotide
"downstream", i.e. in the direction of extension of primer binding sites. The
reporter molecule
and quencher molecule are positioned on the probe sufficiently close to each
other such that
whenever the reporter molecule is excited, the energy of the excited state
nonradiatively transfers
to the quencher molecule where it either dissipates nonradiatively or is
emitted at a different
emission frequency than that of the reporter molecule. During strand extension
by a DNA
polymerase, the probe anneals to the template where it is digested by the 5'
to 3' exonuclease
activity of the polymerase. As a result of the probe being digested, the
reporter molecule is
effectively separated from the quencher molecule such that the quencher
molecule is no longer
close enough to the reporter molecule to quench the reporter molecule's
fluorescence. Thus, as
more and more probes are digested during amplification, the number of reporter
molecules in
solution increases, thus resulting in an increasing number of unquenched
reporter molecules
which produce a stronger and stronger fluorescent signal.
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The other most commonly used real time PCR approach uses the so-called
"molecular
beacons" technology. This approach is also based upon the presence of a
quencher-fluorophore
pair on an oligonucleotide probe. In the beacon approach, a probe is designed
with a stem-loop
structure, and the two ends of the molecule are labeled with a fluorophore and
a quencher of that
fluorophore, respectively. In the absence of target polynucleotide, the
complementary sequences
on either end of the molecule permit stem formation, bringing the labeled ends
of the molecule
together, so that fluorescence from the fluorophore is quenched. In the
presence of the target
polynucleotide, which bears sequence complementary to the loop and part of the
stem structure
of the beacon probe, the intermolecular hybridization of the probe to the
target is energetically
favored over intramolecular stem-loop formation, resulting in the separation
of the fluorophore
and the quencher, so that fluorescent signal is emitted upon excitation of the
fluorophore. The
more target present, the more probe hybridizes to it, and the more fluorophore
is freed from
quenching, providing a read out of the amplification process in real time.
Capillary electrophoresis has been used to quantitatively detect gene
expression. Rajevic
at el. (2001, Pflugers Arch. 442(6 Suppl 1):R190-2) discloses a method for
detecting differential
expression of oncogenes by using seven pairs of primers for detecting the
differences in
expression of a number of oncogenes simultaneously. Sense primers were 5' end-
labelled with a
fluorescent dye and multiplex fluorescent RT-PCR results were analyzed by
capillary
electrophoresis on an ABI-PRISM 310 Genetic Analyzer. Borson et al. (1998,
Biotechniques
25:130-7) describes a strategy for dependable quantitation of low-abundance
mRNA transcripts
based on quantitative competitive reverse transcription PCR (QC-RT-PCR)
coupled to capillary
electrophoresis (CE) for rapid separation and detection of products. George et
al., (1997, J.
Chromatogr. B. Biomed. Sci. Appl. 695:93-102) describes the application of a
capillary
electrophoresis system (ABI 310) to the identification of fluorescent
differential display-
generated EST patterns. Odin et al. (1999, J. Chromatogr. B. Biomed. Sci.
Appl. 734:47-53)
describes an automated capillary gel electrophoresis with multicolor detection
for separation and
quantification of PCR-amplified cDNA.
SUMMARY OF THE INVENTION
Real time amplification methods are provided that monitor the abundance of one
or more
amplification products at multiple points during the amplification regimen. A
sampling method
withdraws or extrudes aliquots from the amplification reaction mixture during
the amplification
regimen. Quantitative analysis can then be prefonned on the sampled nucleic
acid amplification
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reactions. Quantitative information garnered during an amplification regimen
can be used to
develop a detailed amplification profile that in turn permits the reliable
determination of original
template abundance.
The invention encompasses a method of analyzing a nucleic acid amplification
comprising:
providing a nucleic acid amplification reaction mixture comprising a plurality
of different
amplification templates;
subjecting the reaction mixture to an amplification regimen;
dispensing or withdrawing an aliquot from the reaction mixture at plural
stages during the
amplification regimen;
separating and detecting nucleic acids in the aliquot;
determining the quantity of a plurality of separated nucleic acid species in
the aliquot;
and
for each separated nucleic acid species from each stage, correlating the
quantity of the
species with the stage at which the aliquot comprising the species was
dispensed, wherein the
correlating generates an amplification profile of the nucleic acid
amplification.
In one embodiment, the plurality of different amplification templates
comprises at least
three different amplification templates.
In another embodiment, the plurality of different amplification templates
comprises at
least five different amplification templates.
In another embodiment, the plurality of different amplification templates
comprises at
least ten different amplification templates.
In another embodiment, the plurality of different amplification templates
comprises at
least 20 different amplification templates.
In another embodiment, the plurality of different amplification templates
comprises at
least 50 different amplification templates.
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In another embodiment, the plurality of different amplification templates
comprises at
least 100 different amplification templates.
In another embodiment, the plurality of different amplification templates
comprises at
least 200 different amplification templates.
In another embodiment, a plurality of amplification reaction mixtures is
subjected to the
method. W another embodiment, the plurality of amplification reaction mixtures
is subjected to
the method simultaneously.
In another embodiment, the method generates an amplification profile for a
plurality of
amplified nucleic acid species.
In another embodiment, the amplification profile provides quantitative
information
regarding the abundance of a nucleic acid species present in the nucleic acid
amplification
reaction mixture at the start of the amplification regimen.
In another embodiment, the amplification profile is a transcriptional profile.
In another embodiment, the nucleic acid amplification regimen comprises
thermal
cycling.
In another embodiment, the nucleic acid amplification regimen comprises
isothermal
cycling.
In another embodiment, the nucleic acid amplification regimen comprises PCR.
In another embodiment, the nucleic acid amplification regimen comprises a
method
selected from the group consisting of ligase-mediated amplification, NASBA,
and rolling circle
amplification.
In another embodiment, the aliquot is dispensed into a receptacle having a
plurality of
aliquot-receiving sites. In one embodiment, the receptacle is a multiwell
plate. In another
embodiment, the receptacle comprises a plurality of CE capillaries.
In another embodiment, the aliquot is dispensed into or onto a receptacle
capable of
holding a plurality of aliquots without mixing among the aliquots.
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In another embodiment, the amplification regimen is cyclic. In another
embodiment, the
dispensing or withdrawing is performed after each of a plurality of cycles. In
another
embodiment, the dispensing or withdrawing is performed after every cycle in
the regimen.
In another embodiment, the separating comprises electrophoresis.
In another embodiment, the separating comprises capillary electrophoresis.
In another embodiment, the separating comprises liquid chromatography.
In another embodiment, the detecting comprises detection of one or more
fluorescent
labels.
In another embodiment, the detecting comprises mass spectrometry.
In another embodiment, the amplification regimen is performed in a container,
and the
aliquot dispensing is performed by withdrawing the aliquot from the container.
In another
embodiment, the container is a well or a test tube.
In another embodiment, the amplification regimen is performed in a container,
and the
dispensing is performed by extruding the aliquot from the container.
In another embodiment, the amplification regimen is performed in a container
with
openings at one or both ends. In another embodiment, the container is a
capillary tube.
The invention further encompasses a method of analyzing the expression of a
plurality of
RNA transcripts between first and second gene expressing entities, the method
comprising
providing a first nucleic acid amplification reaction mixture, the mixture
comprising a
plurality of different amplification templates, wherein the amplification
templates comprise
reverse transcription products from a plurality of RNA transcripts from a
first gene expressing
entity;
providing a second nucleic acid amplification reaction mixture, the mixture
comprising a
plurality of different amplification templates, wherein the amplification
templates comprise
reverse transcription products from a plurality of RNA transcripts from a
second gene expressing
entity;
subjecting the reaction mixtures to an amplification regimen;
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dispensing or withdrawing an aliquot from the first and the second reaction
mixtures at
plural stages during the amplification regimen;
separating and detecting nucleic acids in the aliquot;
determining the quantity of a plurality of separated nucleic acid species in
the aliquot;
for each separated nucleic acid species from each stage, correlating the
quantity of the
species with the stage at which the aliquot comprising the species was
dispensed, thereby
generating a transcriptional profile of the plurality of RNA transcripts
expressed by the first and
the second gene expressing entities; and
comparing the transcriptional profile from the first gene expressing entity
with the
transcriptional profile from the second gene expressing entity.
In one embodiment, the plurality of RNA transcripts comprises at least three
different
RNA transcripts.
In another embodiment, the plurality of RNA transcripts comprises at least
five different
RNA transcripts.
In another embodiment, the plurality of RNA transcripts comprises at least ten
different
RNA transcripts.
In another embodiment, the plurality of RNA transcripts comprises at least 20
different
RNA transcripts.
In another embodiment, the plurality of RNA transcripts comprises at least 50
different
RNA transcripts.
In another embodiment, the plurality of RNA transcripts comprises at least 100
different
RNA transcripts.
In another embodiment, the plurality of RNA transcripts comprises at least 200
different
RNA transcripts.
In another embodiment, the amplification regimen is cyclic. In another
embodiment, the
nucleic acid amplification regimen comprises thermal cycling. In another
embodiment, the
nucleic acid amplification regimen comprises isothermal cycling. In another
embodiment, the
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nucleic acid amplification regimen comprises PCR. In another embodiment, the
nucleic acid
amplification regimen comprises a method selected from the group consisting of
ligase-mediated
amplification, NASBA, and rolling circle amplification.
In another embodiment, the aliquot is dispensed into a receptacle having a
plurality of
aliquot-receiving sites. In another embodiment, the receptacle is a multiwell
plate. In another
embodiment, the receptacle comprises a plurality of CE capillaries.
In another embodiment, the aliquot is dispensed into or onto a receptacle
capable of
holding a plurality of aliquots without mixing among the aliquots.
In another embodiment, the dispensing or withdrawing is performed after a
plurality of
cycles.
In another embodiment, the dispensing or withdrawing is performed after every
cycle in
the regimen.
In another embodiment, the separating comprises electrophoresis.
In another embodiment, the separating comprises capillary electrophoresis.
In another embodiment, the separating comprises liquid chromatography.
In another embodiment, the detecting comprises detection of one or more
fluorescent
labels.
In another embodiment, the detecting comprises mass spectrometry.
In another embodiment, the amplification regimen is performed in a container,
and the
aliquot dispensing is performed by withdrawing the sample from the container.
In another embodiment, the container is a well or a test tube.
In another embodiment, the amplification regimen is performed in a container,
and
wherein the dispensing is performed by extruding the aliquot from the
container.
In another embodiment, the container is a capillary tube.
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In accordance with one aspect of the invention, a polymerase chain reaction
(PCR)
apparatus is provided. A solution holder separately holds plural samples of
reaction mixture. A
heat exchanging structure cyclically controls, for specified durations, the
temperature of the
plural samples of reaction mixture among plural temperatures. A loading
apparatus may be
provided to load samples into the solution holder. An aliquot dispensing
mechanism dispenses
or withdraws, from each of a set (all or any subset) of the plural samples
held by the solution
holder, plural aliquots of a given sample to respective separate aliquot
holders or directly to
another instrument or instrument module.
In accordance with another aspect of the invention, a nucleic acid
amplification apparatus
is provided. A solution holder separately holds plural samples of reaction
mixture. A reaction
system is provided to cause amplification of certain nucleic acids in the
reaction mixture of each
sample. A loading apparatus may be provided to load samples into the solution
holder. An
aliquot dispensing mechanism dispenses, from each of a set of the plural
samples held by the
solution holder, plural aliquots of a given sample to respective separate
aliquot holders.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a block diagram of an amplification profiling system.
Fig. 2 is a flow chart of a process for performing amplification profiling.
Fig. 3 is a block diagram of an amplification profiling system comprising a
PCR
apparatus with an aliquot dispensing apparatus.
Fig. 4 shows a cross-sectional view of one tube and part of a heat exchanging
structure of
a PCR apparatus, along with one embodiment of an aliquot dispensing apparatus.
Fig. 5 shows tubes provided in a reaction chamber, coupled to another
embodiment of an
aliquot dispensing apparatus.
Fig. 6 is a flow chart of a process for dispensing aliquots with the aliquot
dispensing
apparatus shown in Fig. 4.
Fig. 7 is partial cross-sectional view of a capillary PCR device.
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Figs. 8 and 9 show respective different embodiments of aliquot dispensing
apparatuses
for use with the capillary PCR device.
Fig. 10 is a flow chart of a process for dispensing aliquots with the aliquot
dispensing
apparatus shown in Fig. 8.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, the term "automatic" refers to a process or act where plural
actions are
taken without human intervention; i.e., a machine performs the actions under
the control of a
computer. In the context of the dispensing of aliquots from samples in a
nucleic acid
amplification apparatus (e.g., a PCR device), such aliquots are dispensed
automatically when
plural aliquots are dispensed from samples (containing reaction mixtures
undergoing a reaction
in an amplification apparatus) into respective aliquot holders without human
intervention.
As used herein, the term "sample" refers to a biological material which is
isolated from
its natural environment and containing a polynucleotide. A "sample" according
to the invention
may consist of purified or isolated polynucleotide, or it may comprise a
biological sample such
as a tissue sample, a biological fluid sample, or a cell sample comprising a
polynucleotide. A
biological fluid includes blood, plasma, sputum, urine, cerebrospinal fluid,
lavages, and
leukophoresis samples. A sample of the present invention can comprise any
plant, animal,
bacterial or viral material containing a polynucleotide.
As used herein, a "polynucleotide molecule derived from a specific sample" may
be a
polynucleotide isolated from a specific sample, or it may be a polynucleotide
synthesized from a
specific sample, e.g., through the technologies of reverse transcription (RT)
or polymerase chain
reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence based
amplification
(NASBA), strand displacement amplification (SDA), and any other nucleic acid
amplification
technologies known in the art.
As used herein, the term "amplification profile" or the equivalent terms
"amplification
curve" and "amplification plot" mean a mathematical curve representing the
signal from a
detectable label incorporated into a nucleic acid sequence of interest at two
or more steps in an
amplification regimen, plotted as a function of the cycle number or stage at
which the samples
were withdrawn or extruded. The amplification profile is preferably generated
by plotting the
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fluorescence of each band detected after capillary electrophoresis separation
of nucleic acids in
individual reaction samples. Most commercially available fluorescence
detectors are interfaced
with software permitting the generation of curves based on the signal
detected.
As used herein, the term "aliquot" refers to a sample volume taken from a
prepared
reaction mixture. The volume of an.aliquot can vary, but will generally be
constant within a
given experimental run. An aliquot will be less than the volume of the entire
reaction mixture.
Where there are X aliquots to be withdrawn during an amplification regimen,
the volume of an
aliquot will be less than or equal to 1lX times the reaction volume.
As used herein, the term "dispense" means dispense, transfer, withdraw,
extnide or
remove.
As used herein, the term "reaction chamber" refers to a fluid chamber for
locating
reactants undergoing or about to undergo a reaction (e.g., an amplification
reaction or an
extraction process). A~ "reaction chamber" may be comprised of any suitable
material that
exhibits minimal non-specific adsorptivity or is treated to exhibit minimal
non-specific
adsorptivity, for example, including, but not limited to, glass, plastic,
nylon, ceramic, or
combinations thereof.
As used herein, the term "amplified product" refers to polynucleotides which
are copies
of all or a portion of a particular polynucleotide sequence and/or its
complementary sequence,
which correspond in nucleotide sequence to a template polynucleotide sequence
and its
complementary sequence. An "amplified product," according to the invention,
may be DNA or
RNA, and it may be double-stranded or single-stranded.
As used herein, the terms "synthesis" and "amplification" are used
interchangeably to
refer to a reaction for generating a copy of a pauticular polynucleotide
sequence or for increasing
the copy number or amount of a particular polynucleotide sequence. It may be
accomplished,
without limitation, by the in vitro methods of polymerase chain reaction
(PCR), ligase chain
reaction (LCR), nucleic acid sequence based amplification (NSBA), strand
displacement
amplification, or any other method known in the art. For example,
polynucleotide amplification
can be a process using a polymerase and a pair of oligonucleotide primers for
producing any
particular polynucleotide sequence, i.e., the target polynucleotide sequence
or target
polynucleotide, in an amount which is greater than that initially present.
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As used herein, a "target polynucleotide" is a polynucleotide sequence whose
abundance
in a biological sample is to be analyzed. A target polynucleotide may be
isolated or amplified
before its expression level is analyzed. For example, a target polynucleotide
may be a sequence
that lies between the hybridization regions of two members of a pair of
oligonucleotide primers
which are used to amplify it. A target polynucleotide may be RNA or DNA, for
example, it may
be mRNA or cDNA, a coding region of a gene or a portion thereof. A target
polynucleotide
sequence generally exists as part of a larger "template" sequence; however, in
some cases, a
target sequence and the template are the same. Although "template sequence"
generally refers to
the polynucleotide sequence initially present prior to amplification, the
products from an
amplification reaction may also be used as template sequence in subsequent
amplification
reactions. A "target polynucleotide" or a "template sequence" may be a normal
polynucleotide
(e.g., wild type) or a mutant polynucleotide that is or includes a particular
sequence.
As used herein, an "oligonucleotide primer" refers to a polynucleotide
molecule (i.e.,
DNA or RNA) capable of annealing to a polynucleotide template and providing a
3' end to
produce an extension product which is complementary to the polynucleotide
template. The
conditions for initiation and extension usually include the presence of four
different
deoxyribonucleoside triphosphates and a polymerization-inducing agent such as
DNA
polymerase or reverse transcriptase, in a suitable buffer ("buffer" includes
substituents which are
cofactors, or which affect pH, ionic strength, etc.) and at a suitable
temperature. The primer
according to the invention may be single- or double-stranded. The primer is
single-stranded for
maximum efficiency in amplification, and the primer and its complement form a
double-stranded
polynucleotide. But it may be double-stranded. "Primers" in specific
embodiments of the
methods described are less than or equal to 100 nucleotides in length, e.g.,
less than or equal to
90, or 80, or 70, or 60, or 50, or 40, or 30, or 20, or 15, or equal to 10
nucleotides in length.
As used herein, a "polynucleotide" generally refers to any polyribonucleotide
or poly-
deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or
DNA.
"Polynucleotides" include, without limitation, single- and double-stranded
polynucleotides. As
used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as
described above, that
contain one or more modified bases. Thus, DNAs or RNAs with baclcbones
modified for
stability or for other reasons are "polynucleotides". The term
"polynucleotides" as it is used
herein embraces such chemically, enzymatically or metabolically modified forms
of
polynucleotides, as well as the chemical forms of DNA and RNA characteristic
of viruses and
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cells, including for example, simple and complex cells. A polynucleotide
useful for the present
invention may be an isolated or purified polynucleotide or it may be an
amplified polynucleotide
in an amplification reaction.
As used herein, "isolated" or "purified" when used in reference to a
polynucleotide
means that a naturally occurring sequence has been removed from its normal
cellular (e.g.,
chromosomal) environment or is synthesized in a non-natural environment (e.g.,
artificially
synthesized). Thus, an "isolated" or "purified" sequence may be in a cell-free
solution or placed
in a different cellular environment. The term "purified" does not imply that
the sequence is the
only nucleotide present, but that it is essentially free (about 90-95%, up to
99-100% pure) of
non-nucleotide or polynucleotide material naturally associated with it, and
thus is distinguished
from isolated chromosomes.
As used herein, the term "cDNA" refers to complementary or copy polynucleotide
produced from an RNA template by the action of RNA-dependent DNA polymerase
(e.g.,
reverse transcriptase). A "cDNA clone" refers to a duplex DNA sequence
complementary to an
RNA molecule of interest, carried in a cloning vector.
As used herein, "genomic DNA" refers to chromosomal DNA, as opposed to
complementary DNA copied from an RNA transcript. "Genomic DNA", as used
herein, may be
all of the DNA present in a single cell, or may be a portion of the DNA in a
single cell.
The term "expression" refers to the production of a protein or nucleotide
sequence in a
cell or in a cell-free system, and includes transcription into an RNA product,
post-transcriptional
modification and/or translation into a protein product or polypeptide from a
DNA encoding that
product, as well as possible post-translational modifications.
As used herein, the term "gene expressing entities" refers to a cell, a
tissue, or an
organism that expresses one or more genes as RNA transcripts. The term also
encompasses
entities that are not comprised by a cell, a tissue or an organism, but that
nonetheless produce
RNA transcripts from a nucleic acid template, for example, an in vitro
transcription reaction.
As used herein, the term "expression profile" or "transcriptional profile"
refers to a
representation of the quantitative (i.e., abundance) and qualitative
expression of one or more
genes in a sample. Preferably the transcriptional profile describes the
activity of multiple (i.e., at
least 3, preferably at least 5, 10, 15, 20, 30, 50, 100, 200, 500, 1000,
10,000 or more) genes or
CA 02509769 2005-05-19
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transcription units in a sample. A transcriptional profile for a biological
sample can be
assembled from the nucleic acid amplification profiles from one or more
amplification regimens.
As used herein, the term "comparing the transcriptional profile" refers to
comparing the
differential expression of one or more polynucleotides in two or more samples.
Comparison can
be between the overall pattern of expression, including the presence, absence
and/or abundance
of individual amplicons or sets of amplicons. Comparison can be manual or
automated.
As used herein, the term "abundance" refers to the amount (e.g., measured in
~.g, ~,mol or
copy number) of a target polynucleotide in a sample. The "abundance" of a
polynucleotide may
be measured by methods well known in the art (e.g., by UV absorption, by
comparing band
intensity on a gel with a reference of known length and amount), for example,
as described in
Basic Methods in Molecular Biolo~y, (19i~6, Davis et al., Elsevier, NY); and
Current Protocols
in Molecular Biolo~y (1997, Ausubel et al., John Wiley & Sons, Inc.). One way
of measuring
the abundance of a polynucleotide in the present invention is to measure the
fluorescence
intensity emitted by such polynucleotide, and compare it with the fluorescence
intensity emitted
by a reference polynucleotide, i.e., a polynucleotide with a known amount.
As used herein, the term "sampling device" refers to a mechanism that
withdraws or
extrudes an aliquot from an amplification during the amplification regimen.
Sampling devices in
the embodiments herein are adapted to minimize contamination of the
amplification reaction(s),
by, for example, using pipeting tips or needles that are either disposed of
after a single sample is
withdrawn, or by incorporating one or more steps of washing the needle or tip
after each sample
is withdrawn. Alternatively, the sampling device can contact the capillary to
be used for
capillary electrophoresis directly with the amplification reaction in order to
load an aliquot into
the capillary. Alternatively, the sample device can include a fluidic line
(e.g. a tube) connected
to a controllable valve which will open at a particular cycle or point in the
amplification regimen.
Sampling devices known in the art include, for example, the multipurpose
Robbins Scientific
Hydra 96 pipettor, which is adapted to sampling to or from 96 well plates.
This and others can
be readily adapted for use according to the methods of the invention.
As used herein, the term "robotic arm" means a device, preferably controlled
by a
microprocessor, that physically transfers samples, tubes, or plates containing
samples from one
location to another. Each location can be a unit in a modular apparatus. An
example of a robotic
16
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arm useful according to the invention is the Mitsubishi RV-E2 Robotic Arm.
Software for the
control of robotic arms is generally available from the manufacturer of the
arm.
As used herein, the term "multiwell plate" refers to a receptacle comprising
multiple
(e.g., at least two, but often 5, 8, 12, 24, 36, 96, or 384) discrete sites
for the deposition and
holding of a liquid sample. The sites can be depressions or wells formed in or
on a piece of
plastic or similar material, and are preferably arranged in a regular pattern,
e.g., in a grid (a 96
well plate, for example, will comprise a rectangular grid of 8 wells in one
dimension and 12
wells in the other. It is noted that aliquots of amplification reactions can
also be deposited on a
flat surface (i.e., one without depressions or wells) as long as they are
deposited such that there is
no mixing among the deposited aliquots.
As used herein, the term "amplification templates" refers to a nucleic acid
that can act as
a template for the enzymatic polymerization of a complementary strand.
Amplification
templates can comprise DNA, RNA and PNA, and can be double or single stranded.
As used herein, the term "amplification regimen" means a process of
specifically
amplifying the abundance of a nucleic acid sequence of interest. Amplification
regimens are
most often "cyclic," i.e., they are comprised of repeated steps of primer
annealing and
polymerization, usually in conjunction with repeated steps of thermal
denaturation of template
nucleic acids. A cyclic amplification regimen will preferably comprise at
least two, and
preferably at least 5, 10, 15, 20, 25, 30, 35 or more iterative cycles of
thermal denaturation,
oligonucleotide primer annealing to template molecules, and nucleic acid
polynerase extension
of the annealed primers. Conditions and times necessary for each of these
steps are well known
in the art. Amplification achieved using an amplification regimen is
preferably exponential, but
can alternatively be linear. Other amplification regimens are non-cyclic, or
continuous. Non-
cyclic amplifications proceed to completion once initiated, and most often
involve templates
with RNA polymerase recognition sites and the action of RNA polymerase and
reverse
transcriptase.
As used herein, the term "thermal cycled amplification regimen" refers to an
amplification regimen comprising a plurality of cycles of thermal
denaturation, primer annealing
and primer extension or polymerization.
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As used herein, the term "isothermal" when applied to an amplification regimen
means
that the amplification, once initiated, proceeds at a single temperature,
without need for cycles
involving thermal denaturation of template nucleic acids.
As used herein, the phrase "dispensing an aliquot from the reaction mixture at
plural
stages" refers to the withdrawal or extrusion of an aliquot at least twice,
and preferably at least 3,
4, 5, 10, 15, 20, 30 or more times during an amplification regimen. A "stage"
will refer to a
point after a given number of cycles, or, where the amplification regimen is
non-cyclic, will refer
to a selected time after the initiation of the regimen.
As used herein, the term "extrude" means that an aliquot is forced out of one
end or
orifice of a reaction vessel by pressure, e.g., air pressure, applied on
another end or orifice of the
vessel.
As used herein, the term "quantitative information regarding the abundance of
a nucleic
acid species" refers to information about the amount of a nucleic acid
species. The quantitative
information can be relative (e.g., fold difference over the amount of that
nucleic acid in another
sample), or absolute.
Description of the preferred embodiments
In one aspect, the present invention is directed to systems, apparatus,
methods, and any
one or more subparts thereof, for concurrently quantitatively monitoring and
analyzing the
amplification of plural (numerous, in the illustrated embodiment) of species
of nucleic acid
sequences.
In accordance with one aspect of the invention, a method is provided for
quantitatively
monitoring the amplification of nucleic acid sequences. In a given act of the
method, a nucleic
acid amplification reaction mixture is provided. The mixture comprises a
plurality of nucleic
acid species. An amplification regimen is performed on the mixture, causing
plural nucleic acid
species to be amplified concurrently. An aliquot of the reaction mixture is
dispensed at intervals
preceding completion of the amplification regimen. The nucleic acid species in
the aliquot are
separated and detected. For respective ones of plural separated species, the
quantity of those
separated nucleic acid species in the aliquot is concurrently determined.
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This method facilitates high throughput quantitative expression analysis on a
plurality of
nucleic acid species (e.g., transcripts, genes) - numerous (dozens, hundreds,
thousands, etc.) in
certain illustrated embodiments.
The amplification regimen may be performed on plural independent nucleic acid
amplification mixtures. The plural independent amplification mixtures may be
present on a
multi-well container. In the illustrated embodiments, the amplification
regimen comprises
thermal cycling, e.g., PCR.
The dispensing may be performed following one or more cycles in the
amplification
regimen. For example, the dispensing may be performed following each cycle in
the
amplification regimen. The separating may be performed by capillary
electrophoresis. In the
illustrated embodiment, the plural separated species are amplified from RNA
transcripts of a
plurality of genes.
In one aspect, an aliquot dispensing apparatus described herein can be used in
a method
of monitoring the amplification of a nucleic acid sequence, preferably a
plurality of sequences.
In such a method, samples or aliquots dispensed during a nucleic acid
amplification regimen
(e.g., after one or more cycles, preferably up to and including after each
cycle) by such an aliquot
dispensing apparatus are loaded into a separation apparatus, preferably into
capillaries for
capillary electrophoresis. The nucleic acids in the loaded samples are
separated, e.g., by size
and/or charge, and the separated species are detected, thereby generating an
amplification
profile. When the amplified nucleic acids represent transcribed RNAs, e.g.,
when expressed
RNA is reverse-transcribed and then amplified, the amplification profile
provides a
transcriptional profile for the original sample. Whereas the non-linearity of
amplification at late
stages of the ampliftcation process normally precludes the ability to
accurately quantitate the
amount of a given transcript in a nucleic acid sample by measuring amplimer
abundance after
multiple cycles, the transcriptional profile generated in this manner provides
quantitative as well
as qualitative data that do permit such determination. The detection of
amplimer abundance at
various cycles during the amplification provides a real time representation of
how the
amplification proceeded for each species amplified and detected in a given
reaction. Because
non-linearity in the amplification process can be accounted for in such a real
time profile, the
profile permits the efficient quantitative determination of the amount of RNA
corresponding to a
given amplimer in an original sample. This is but one example of the
advantages provided by a
real time transcriptional profile generated by such a method.
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Other advantages provided by the real time profiling performed in such a
manner include,
for example; the ability to follow the amplification profiles for multiple
transcripts in a single
sample. Because the size separation by, for example, CE, can resolve species
differing by as
little as one nucleotide, the sample withdrawn from an amplification reaction
can have multiple
differently sized amplimers, each representing a different transcript in the
original sample.
When this is considered along with the simultaneous amplification of multiple
samples, as in
amplification performed in mufti-well plates or in parallel in multiple tubes
or capillaries, the
amount of information obtainable increases dramatically.
The dispensing apparatus described herein can similarly be used in any method
calling
for the withdrawal of samples at one or more times during an amplification
reaction.
Fig. 1 is a block diagram of a system fox amplification profiling. An RNAIDNA
aimplification apparatus 12 is provided to produce amplified product of
nucleic acid molecules
(RNA or DNA). Amplification apparatus 12 comprises a reaction system,
described more fully
hereinbelow, to cause amplification of nucleic acids in the reaction mixture
of respective ones of
plural samples held by a solution holder 13.
An aliquot dispensing apparatus 14 is coupled to amplification apparatus 12,
and
dispenses, from each sample of a set of plural samples held by solution holder
13, plural aliquots
of a given sample to respective aliquot holders in an aliquot holding
structure 16. Aliquot
holding structure 16 may comprise one of a set of microtitre trays.
Alternatively, the aliquot
holders may comprise sample holders of another instrument or of an instrument
module.
A separation and quantitative analysis system 20 is provided which is coupled
to a data
processing for allowing processing, display, andlor storage of data produced
by system 20.
A process control mechanism 18, e.g., a microprocessor, is coupled to
apparatuses 12, 14,
and 20 to control operation of the same. Process control mechanism may be
programmed to
control each action performed by these apparatuses without human intervention.
Optionally, the
program may allow for any desired degree of human intervention. For example,
process control
mechanism 18 may be provided with a computer interface (not shown) that allows
a user to
make adjustments to the amplification and dispensing processes performed by
apparatuses 12
and 14 (and optionally also to the separation and quantitative analysis
performed by system 20)
by either changing the program altogether or by influencing the process by
interjecting additional
acts or modifications to acts to be performed by such apparatuses.
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In the illustrated embodiment, aliquot dispensing apparatus 14 and process
control
mechanism 1~ collectively operate to automatically dispense, from each sample
of a set of plural
samples held by solution holder 13, plural aliquots of a given sample to
respective separate
aliquot holders in aliquot holding structure 16. More specifically, the plural
aliquots are
dispensed at respective different times during an amplification regimen.
Accordingly, aliquot
dispensing apparatus is provided with one or more automated actuation
mechanisms 15 (e.g.,
computer actuable arms, robotic arms), and process control mechanism comprises
a dispense
control process object 19 for controlling the operation of such automated
actuation mechanisms
15.
The illustrated amplification apparatus 12 may perform an amplification
regimen that is
cyclic or that is non-cyclic, or continuous. Moreover, the apparatus may
perform a thermal
amplification regimen (e.g., PCR), or it may perform an amplification regimen
not involving a
thermal approach (e.g., ligase chain reaction (LCR)). More information is
provided hereinbelow
regarding approaches for amplification.
Separation and quantitative analysis system 20 may comprise any suitable
device or
system that analyzes plural samples and separates, from respective ones of the
samples (aliquots,
in the illustrated embodiment) individual nucleic acid molecules based on
physical properties of
the molecules (e.g., charge, length, mass). By way of example, system 20 may
comprise a CE
(capillary electrophoresis) device, a liquid chromotography mass spectrometry
(LC-MS)
apparatus, or a high performance liquid chromotography (HPLC) apparatus.
Fig. 2 is a flow chart of a process for performing amplification profiling
using the
apparatus shown in Fig. 1. In an initial act 50, samples are loaded into
solution holder 13. In act
52, an amplification regimen is started. Then, at act 54, at designated points
in time during the
amplification regimen, aliquots are dispensed into respective different
aliquot holders.
In act 56, a given set of aliquots, corresponding to a particular point in the
amplification
regimen, is provided for input to separation system 20. At act 58, the
separation and quantitative
analysis is performed by system 20.
Fig. 3 is a schematic diagram of an amplification profiling system -- for
concurrently
quantitatively monitoring and analyzing the amplification of numerous species
of nucleic acid
sequences. The species may specifically be amplified from RNA transcripts of a
plurality of
genes. The system comprises a thermal cycling amplification machine - e.g., a
PCR apparatus
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30. Mechanisms 32 are provided for temperature sensing and control. A heat
exchanging
structure 34 is provided, which is coupled to one or more reaction chambers 36
which holds one
or more structures 35 carrying one or more sets (e.g., arrays) of samples. The
samples may be
held, e.g., by wells, tubes, or capillaries.
An aliquot dispensing apparatus 38 is provided which is controllable to
automatically, on
demand from a process control 42, dispense aliquots from the samples in
structures) 35.
Dispensing apparatus 38 may comprise any mechanisms known in the art or
commercially
available for automatically acquiring aliquots from respective samples and
placing such aliquots
into an aliquot holding structure 40. Process control 42 may comprise, e.g., a
computer and a
computer user interface allowing a human operator to intercede in the process
or program the
process. Loading/unloading apparatus 44 is provided for loading and unloading
the samples for
a given amplification regimen.
In the illustrated embodiment, aliquot dispensing apparatus is provided with
one or more
automated actuation mechanisms 39 (e.g., computer actuable amps, robotic
arms), and process
control mechanism 42 comprises a dispense control process object 43 for
controlling the
operation of such automated actuation mechanisms 39.
Solution holder 13 may comprise tubes (or other vessels) provided in recesses
of one or
more blocks made of any heat-conducting substance (if the amplification
apparatus is a thermal
amplification apparatus), e.g., metal (specifically, in the embodiment
illustrated in Figure 3, it
comprises aluminum). Alternatively, solution holder 13 may comprise any vessel
of any
material, such as wells etched in Silica.
The solution holder 13 may comprise plural capillary tubes having closed ends.
Alternatively, it may comprise plural capillary tubes having open ends - -
where the solution is
held inside the capillaries by some external force, e.g., by pressure.
?5 The plural samples held by solution holder 13 may comprise 96 samples, or
any multiple
of 96 samples, e.g., 192, 384, etc.
Analysis system 48, which may comprise an EC device, receives a set of
aliquots from
structures) 40. Aliquot structures) 40 may be made compatible with the
analysis system 48 to
facilitate an easy interface and input into the analysis system.
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Data processing/storage system 46 stores and allows processing of data
produced by
analysis system 48.
Various components of the illustrated thermal amplification apparatus 30 may
comprise
mechanisms known in the art or available in off the-shelf devices, e.g., PCR
devices. By way of
example, but not for purposes of limitation, device 30 (or one or more parts
thereof) may be
made in accordance with one or more of US Patent Nos. 5,038,852 and 5,827,480.
The illustrated analysis system may comprise mechanisms known in the art or
available
in off the-shelf devices, e.g., EC devices. By way of example, but not for
purposes of limitation,
device 26 (or one or more parts thereof) may be made in accordance with one or
more of US
Patent Nos. 6,217,731 and 6,001,230.
In operation, samples are loaded into the reaction chambers) 36. An
amplification
regimen is started. At designated points during the amplification regimen, the
aliquots are
dispensed (automatically, in the illustrated embodiment) for subsequent
analysis by analysis
system. The aliquots are provided to the analysis system. Such dispensed
samples may be
analyzed right away or they may be set aside for batch processing once all the
sets of
intermediate (mid-amplification regimen) aliquots are obtained and after the
regimen is
complete. The samples are then analyzed.
PCR may be performed with automatic sampling (by dispensing apparatus 20)
after each
PCR cycle, after each set of cycles, or at given points as defined by a user
during the PCR
amplification regimen yet before completion of the regimen. The resulting
aliquots may be
dispensed into a sample tray. The sample trays may be stacked (e.g., manually)
and analyzed by
an analysis system (e.g., an EC device).
Apparatus 30 dispenses in a sample collecting tray (or plate) an aliquot of
the reaction
mixture after each amplification (temperature) cycle or after a predetermined
number of such
cycles. The aliquot dispensing apparatus 38 may comprise an automatically
controllable
mechanism for withdrawing an aliquot from the reaction mixture by pipeting
(e.g.,
autosampling) or by applying pressure to one end of the reaction vessel (where
the samples are
carried by a capillary, tube, or other kind of vessel which has an inlet and
outlet for liquid
movement).
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In the illustrated embodiment, the cycled temperatures may comprise two or
three
incubation temperatures. Should there be two incubation temperatures, the
temperatures may be
94 degrees Celsius and 50-65 degrees Celsius. Should there be three incubation
temperatures,
they may be in a range of 45-99 degrees Celsius. More specifically, they nay
be (for
denaturing) 80-99 degrees Celsius, (for annealing) 45-65 degrees Celsius, and
(for extension) 60-
75 degrees Celsius.
Fig. 4 shows one embodiment of an aliquot dispensing apparatus for use with a
tube-type
solution holder. In Fig. 4, a cross-sectional view of one tube 61 (among an
array of tubes, not
shown in the figure) is provided. A pipettor apparatus 72 is provided to,
under automated
control by process control 42, extract and dispense into respective separate
aliquot holders plural
aliquots from the sample within each such tube. Each separate aliquot is
obtained at a different
point within the amplification regimen.
As shown in Fig. 4, a given tube 61 is set into a recess of a reaction chamber
(a heat
exchanging structure in the illustrated embodiment). The illustrated heat
exchanging structure
62 may comprise an aluminum block; alternatively, a hot air oven or water bath
structure may be
provided. Tube wall 63 is made of a suitable impermeable material, and is
configured to have a
good fit in the reaction chamber and have thin walls to promote the efficient
transfer of heat
energy between the heating block and the reaction mixture.
Tube 61 comprises a reaction mixture 66. Oil or wax 68 may be provided on top
of the
reaction mixture to prevent evaporation of the mixture. In addition or in the
alternative, a cap 64
may be provided. A mechanism (not shown) may be provided to heat the cover so
as to prevent
condensation and reentry of liquids into the solution, which could corrupt the
mixture and
adversely affect the reliability of the results.
Pipettor apparatus 72 obtains aliquots from respective ones of the W bes 61
and dispenses
them into respective ones of wells 76 of a microtitre tray 74. Pipettor may
comprise one or a
small set of pipettes 69, and an actuator for controlling the position of
pipettes) 69 to remove a
portion of each mixture 66 and dispense the portion (aliquot) into a
corresponding well 76.
Alternatively, pipettor may comprise an array of pipettes 69, for concurrently
removing aliquots
and, upon relocation of the array of pipettes over tray 74, dispensing the
aliquots.
Each pipette 69 may be provided with a detachable pipette tip 70. Each pipette
tip 70
may be detached for disposal and replacement - to prevent contamination of the
reaction mixture
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when subsequent aliquots are taken. Alternatively, a cleansing mechanism (not
shown) may be
used.
Fig. 6 is a flow chart of a process involving loading of tubes in the
amplification
apparatus and dispensing of aliquots using the apparatus shown in Fig. 4. In
act 110, the tubes
are loaded. The samples are covered in act 112. This may involve covering the
surface of the
sample with wax or oil and/or covering with (heated) cap 64 the tube.
In act 114, a temperature controlled incubation occurs. This is repeated for
each of the
"M" cycles of the thermal amplification apparatus. Thereafter, for a given
cycle, in act 116, the
pipette is aspirated while in the sample. If there is wax or oil at the
surface of the sample, air is
expelled from pipette while pipette tip 70 is traversing the wax or oil, so as
to prevent the uptake
of the wax or oil. Once tip 70 is past the covering liquid, it can be
aspirated to take the aliquot.
After this occurs and the pipette tip 70 is removed, the protective cover is
restored- e.g., by
reclosing cap 64. At act 118, the pipette tip is cleansed or disposed of.
Aspiration and cleansing
is repeated (or performed concurrently) for all of the "N" samples. If the
pipette tips are not
disponsed of, they may be cleaned by any known means, e.g., using a cleansing
solution or using
a process such as ultrasonic cleaning.
Fig. 5 shows an aliquot dispensing apparatus 60', which uses another approach
to aliquot
dispensing in a thermal amplification system as shown in Fig. 3. An array of
sample tubes 80
(e.g., 94 tubes) is provided in a reaction chamber 82. Each tube includes a
sealed comlection to a
multi-way valve 90. In the illustrated embodiment, each valve comprises a
control input 92, a
cleaning port 94, and a capillary tube port. A capillary tube 96 is connected
to each capillary
tube port.
The other end of each capillary tube 96 terminates at a pipette tip or
capillary portion for
positioned for dispensing into a corresponding well 84 of an aliquot holding
structure 86 (a
microtitre tray in the embodiment).
An aspiration/dispensing structure 98 is provided for controlling the
aspiration (or
extraction) of an aliquot from a given tube 80 and dispensing of the same into
a corresponding
well of aliquot holding structure 86.
In operation, the tubes are loaded. The samples are then covered. Again, this
may
involve covering the surface of the sample with wax or oil and/or covering
with a (heated) cap
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the tube. A temperature controlled incubation then occurs. This is repeated
for each of the
cycles of the thermal amplification apparatus. Thereafter, for a given cycle,
valve 90 is opened
and structure 98 is operated to aspirated or extract an aliquot from the
sample.
Fig. 7 is a schematic view of a cross section of portions of a capillary PCR
device 130.
The illustrated PCR device 130 comprises (among other elements, some shown and
others not
shown) plural reaction chambers 144, 146 (comprising thermostatted baths in
the illustrated
embodiment). The chambers 144, 146 are separated by an insulation barrier 134.
An opening is
provided in the barrier along with a seal 136. One capillary tube 132 (of
plural tubes not
specifically shown) passes through the sealed opening. In the drawing, samples
142 are in
reaction chamber 146. Specifically, capillary tube 132 comprises two end seal
liquid portions
138a and 138b at either end of the sample grouping. Between the end seal
liquid portions, a
plurality of sample portions are provided separated by slug flow elements.
Each slug flow element may comprise, e.g., air or immiscible fluid. The
portions of the
sample between the slugs may be set and uniform amounts of reaction mixture -
such amounts
corresponding to the desired volume of aliquots to be taken.
A multiway valve 148 is provided, to which both ends of capillary tube 132 are
connected. Valve 148 facilitates the sealing of the ends of capillary tube
132, while allowing the
seal to be broken for the purpose of aspirating or extracting a given aliquot.
Valve 148 has a port
for inputting sealing liquid and another port for receiving cleanser. Two
capillary tube
exit/entrance portions 152 and 153 are provided, to allow the loading of the
reaction mixture into
the capillary tube (or the exiting of the same from the capillary tube).
In operation, first the valve 148 opens both the comiections to both ends of
capillary tube
132 to the respective entrance/exit ports 152 and 154. At this point, end seal
liquid portions
138a, 138b, samples 142, and slug flow elements 140 are input - in the
appropriate sequence;
and pressure is applied to move these elements into the appropriate reaction
chamber (chamber
146 as illustrated in Fig. 7). Then, a thermal cycle is carried out. When
aliquots are to be taken
fox a given cycle, the train of elements is moved toward a chosen "dispensing"
one of ports 152
and 154. Using dispensing mechanism 150, end seal liquid 138a is expelled out
of the end seal
liquid port, while the subsequent first aliquot size volume element 142 is
dispensed into a
corresponding well of an aliquot holder. After this occurs, the end seal
liquid 138a can be
restored allowing input from the end seal liquid port, ports 152 and 154 are
closed off, and the
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elements are moved to the appropriate location for another thermal cycle.
Meanwhile the
cleanser port is used to cleanse the exit and entrance ports 152 and 154,
while such ports are
sealed off from capillary tube 132.
Fig. 8 is block diagram of an aliquot dispensing apparatus using a
centrifugation
approach to extract an aliquot for dispensing into a well 176 of an aliquot
holder. The figure
shows a portion of a capillary tube 168 of a PCR device. Tube 168 is connected
to valve 174.
An aliquot exit capillary 170 is connected to valve 174. Aliquot exit
capillary has an end 172
that can be sealed by operation of valve 174. A centrifugation structure 178
is coupled to aliquot
exit capillary 170.
Fig. 10 is a flow chart illustrating a process of aliquot dispensing using the
apparatus
shown in Fig. 8. In act 200, an aliquot is pushed into an exit capillary. In
act 202, the seal liquid
at the end of the element chain is disposed of through the seal liquid port.
In act 204, the valve is
operated to seal the end of the exit capillary. Then, in act 206,
centrifugation structure 178 is
operated to expell an aliquot into well 176.
Fig. 9 is a block diagram of an aliquot dispensing apparatus using a
dispensing/aspiration
structure to extract an aliquot for dispensing into a well 176 of an aliquot
holder. The figure
shows a capillary tube of a PCR device coupled at its ends through valve 182
to ports 184 and
186 of dispensing/aspirating structure 188. Dispensing/aspiration structure
controlls with
pressure variations the dispensing into aliquot holder 192 and the aspiration
to obtain samples
?0 from sample source 190.
In another aspect, the present invention is directed to sampling methods for
quantitatively
monitoring and analyzing the amplification of polynucleotides. While the most
frequently used
nucleic acid amplification method is thermal cycling PCR, the methods
disclosed herein find
application not only in PCR, but also in any nucleic acid amplification
protocol, most
;5 particularly, but not limited to, those that involve repeated cycles of
nucleic acid synthesis.
Continuous methods, such as the RNA polymeraselreverse transcriptase mediated
methods (e.g.,
3SR or NASBA, see below) can also benefit from the sampling methods described
herein, by,
for example, removing samples at given times during the amplification process.
A basic PCR amplification can be broken down into three phrases: (1)
exponential phase:
0 exact doubling of product is accumulated at every cycle, assuming 100%
reaction efficiency.
The reaction is very specific and precise; (2) Linear (high variability)
phase: the reaction
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components are being consumed, the reaction is slowing and the products are
starting to degrade;
(3) plateau (end-point) phase: the reaction has stopped, no more products are
being made and if
left long enough, the PCR products will begin to degrade. The problem with
detection in the
plateau phase of PCR is that the quantitation is affected so as to no longer
reflect the amount of
the starting nucleic acid template.
"Real-time PCR" analysis detects specific nucleic acid amplification products
as they
accumulate in real-time. Real-time PCR provides advantages over traditional
end-point PCR by
allowing for the detection of PCR amplification during the early phases of the
reaction.
Nucleic acid amplification profiling involves the measurement of amplification
products
present at various stages during an amplification regimen. Because it can
identify the limits of
the exponential, linear and plateau phases of an amplification reaction,
knowledge of the
abundance of amplification product at various stages of the amplification
process permits one to
reliably extrapolate the abundance of the original template in a biological
sample. While an
amplification profile of a single nucleic acid template or a small set of such
templates can be
1 S generated through use of the TaqManTM or "molecular beacons" -type real
time approaches,
these methods are rather limited in the number of targets that can be followed
in a single
reaction. A major limitation is that each different species must be labeled
with a differentially
detectable fluorophore.
U.S. Patent Application with Serial No. 60/372,045 describes a real-time PCR
method
using capillary electrophoresis for analysis (the entirety of which is
incorporated herein by
reference). The Patent application provides a method for monitoring the
amplification of a
nucleic acid sequence of interest, the method comprising: (a) contacting a
nucleic acid sample
with a first and a second oligonucleotide primer, wherein the first
oligonucleotide primer
specifically hybridizes with a nucleic acid molecule comprising the nucleic
acid sequence of -
interest, and the second oligonucleotide primer specifically hybridizes with
the complementary
strand of the nucleic acid sequence of interest, wherein the primer extension
product of one
oligonucleotide primer, when separated from its complement, can serve as a
template for the
synthesis of the extension product of the other primer, and wherein at least
one of the first and
the second primers is labeled and preferably, labeled with a detectable
marker; (b) subjecting the
mixture resulting from step (a) to an amplification regimen, the regimen
comprising at least two
cycles of nucleic acid strand separation, oligonucleotide primer annealing,
and polymerise
extension of annealed primers; and (c) removing an aliquot of the mixture,
separating nucleic
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acid molecules in the aliquot, and detecting incorporation of the at least one
detectable marker,
wherein the removing is performed during the cycling regimen of step (b), and
wherein the
detection permits the monitoring of the amplification in real time. Data
analysis, including
standard curve generation and copy number calculation, can be performed
automatically.
The sampling method disclosed herein permits the removal or extrusion of
samples from
an amplification reaction at various cycles during the amplification process.
By withdrawing or
extruding samples of the reaction mixture at various cycles of the
amplification regimen and
detecting the size and amount of various amplified species present, the amount
of numerous
amplified products can be monitored at each phase of the amplification,
thereby identifying the
limits of the exponential, linear and plateau phases for a target sequence in
a reaction mixture.
This approach can be optimally applied to amplification methods that permit
the multiplex
amplification of greater numbers of target sequences in a single reaction
vessel. By highlighting
the exponential phase for the amplification of each different template species
present in a
biological sample, this approach permits the accurate extrapolation of the
amounts of numerous
templates present in a biological sample. Thus, the sampling method, alone or
particularly in
combination with methods that increase the multiplex ability of amplification
reactions, provides
a dramatic increase in the amount of quantitative template information one can
obtain from a
single amplification reaction. When the initial biological sample contains
mRNA and the
amplification process amplifies the mRNA or a DNA copy of it, the
amplification profile is
useful to quantitate the abundance of the mRNA species in the original sample.
The profile
generated by such a sampling protocol is a transcriptional profile, which, as
discussed above, is
extremely useful for a number of approaches related to drug development.
In the practice of cyclic nucleic acid amplification, the experimentally
defined parameter
"Ct" refers to the cycle number at which the signal generated from a
quantitative amplification
reaction first rises above a "threshold", i.e., where there is the first
reliable detection of
amplification of a target nucleic acid sequence. "Reliable" means that the
signal reflects a
detectable level of amplified product during amplification. Ct generally
correlates with starting
quantity of an unknown amount of a target nucleic acid, i.e., lower amounts of
target result in
later Ct. Ct is linked to the initial copy number or concentration of starting
nucleic acid by a
simple mathematical equation:
Log(copy number) = aCt+ b , where a and b are constants.
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Therefore, by measuring Ct for the fragments of the same gene sequence
originating from
two different samples, the original relative concentration of this gene
sequence in these salnples
can be easily evaluated.
Sampling methods and devices
The method described herein facilitates the sampling of nucleic acid
amplification
reaction mixtures necessary for amplification profiling. Sampling may occur at
any time during
or after an amplification reaction. In one embodiment, an aliquot of the
reaction is withdrawn or
extruded from the tube or reaction vessel at the end of each PCR cycle. In
another embodiment,
an aliquot of the reaction is withdrawn or extruded from the tube or reaction
vessel at the end of
every several PCR cycle, e.g., every two cycles, every three cycles, every
four cycles. In another
embodiment, an aliquot of the reaction is withdrawn or extruded from the tube
or reaction vessel
at the end of a series of pre-determined cycles. While a uniform sample
interval will most often
be desired, there is no requirement that sampling be performed at uniform
intervals. As just one
example, the sampling routine may involve sampling after every cycle for the
first five cycles,
and then sampling after every other cycle.
As discussed ~ above, amplification methods that are continuous, rather than
cyclic can
also benefit from the sampling methods described herein. In such cases,
samples can be
withdrawn at given times during the amplification process, for example, ever y
minute, every two
minutes, every three minutes, etc.
Sampling or removal of an aliquot from an amplification reaction can be
performed in
any of several different general formats. First, an aliquot can be withdrawn
from a reaction
vessel (test tube, capillary or well in a multiwell plate) by reaching into
the vessel with a
pipetting device or capillary tube, preferably using an automated device. The
method of
sampling used for this approach will preferably be adapted to minimize
contamination of the
cycling reaction(s), by, for example, using pipetting tips or needles that are
either disposed of
after a single aliquot is withdrawn, or by incorporating one or more steps of
washing the needle
or tip after each aliquot is withdrawn.
Alternatively, the sampling can be done by a device which can contact a
capillary to be
used for capillary electrophoresis directly with the amplification reaction in
order to load an
aliquot into the capillary.
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As another alternative, the sampling can be done using a device which includes
a fluidic
line (e.g. a tube) connected to a controllable valve which will open at
particular cycle. Sampling
devices known in the art include, for example, the multipurpose Robbins
Scientific Hydra 96
pipettor, which is adapted to sampling to or from 96 well plates. This device
and others like it
can be readily adapted for use according to the methods of the invention.
In one embodiment, the sampling and detection are performed concurrently, such
that a
curve representing product abundance as a function of amplification time,
e.g., measure by
minutes or by PCR cycles, is generated during or soon after the amplification
regimen.
For this and other aspects of the invention, it is preferred, although not
necessary that the
cycling be performed in a,microtiter or multiwell plate format. This format,
which uses plates
comprising multiple reaction wells, not only increases the throughput of the
assay process, but is
also well adapted for automated sampling steps due to the modular nature of
the plates and the
uniform grid layout of the wells on the plates. Common microtiter plate
designs useful
according to the invention have, for example 12, 24, 48, 96, 384 or more
wells, although any
number of wells that physically fit on the plate and accommodate the desired
reaction volume
(usually 10-100 p,l) can be used according to the invention. Generally, the 96
or 384 well plate
format is preferred.
An automated sampling process can be readily executed as a programmed routine
and
avoids both human error in sampling (i.e., error in aliquot size and tracking
of sample identity)
and the possibility of contamination from the person sampling. Robotic
samplers capable of
withdrawing aliquots from thermal cyclers are available in the art. For
example, the Mitsubishi
RV-E2 Robotic Arm can be used in conjunction with a SciCloneTM Liquid Handler
or a Robbins
Scientific Hydra 96 pipettor.
The robotic sampler in the embodiments described herein can be integrated with
the
thermal cycler, or the sampler and cycler can be modular in design. When the
cycler and
sampler are integrated, thermal cycling and sampling occur in the same
location, with samples
being withdrawn at programmed intervals by a robotic sampler. When the cycler
and sampler
are modular in design, the cycler and sampler are separate modules. In one
embodiment, the
assay plate or other container is physically moved, e.g., by a robotic arm,
from the cycler to the
sampler and back to the cycler.
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The volume of an aliquot removed at the sampling step can vary, depending, for
example,
upon the total volume of the amplification reaction, the sensitivity of
product detection, and the
type of separation used. Amplification volumes can vary from several
microliters to several
hundred microliters (e.g., 5 ~,1, 10 ~1, 20 ~1, 40 ~1, 60 ~Zl, ~0 ~,1, 100
~,1, 120 pl, 150 ~.1, or 200 ~l
or more), preferably in the range of 10-150 ~.1, more preferably in the range
of 10-100 ~,1.
Aliquot volumes can vary from 0.1 to 30 % of the reaction mixture.
In accordance with one aspect of the invention, a method is provided for
quantitatively
monitoring the amplification of nucleic acid sequences. In a given performance
of the method, a
nucleic acid amplification reaction mixture is provided. The mixture comprises
a plurality of
nucleic acid species. An amplification regimen is performed on the mixture,
causing plural
nucleic acid species to be amplified concurrently. An aliquot of the reaction
mixture is
dispensed at intervals preceding completion of the amplification regimen. The
nucleic acid
species in the aliquot are separated and detected. For respective ones of
plural separated species,
the quantity of those separated nucleic acid species in the aliquot is
concurrently determined.
This method facilitates high throughput quantitative expression analysis on a
plurality of
nucleic acid species (e.g., transcripts, genes) - numerous (dozens, hundreds,
thousands, etc.) in
certain illustrated embodiments.
The amplification regimen may be performed on plural independent nucleic acid
amplification mixtures. The plural independent amplification mixtures may be
present on a
?0 mufti-well container. In the illustrated embodiments, the amplification
regimen comprises
thermal cycling, e.g., PCR.
The dispensing may be performed following one or more cycles in the
amplification
regimen. For example, the dispensing may be performed following each cycle in
the
amplification regimen. The separating may be performed by capillary
electrophoresis. In the
?5 illustrated embodiment, the plural separated species are amplified from RNA
transcripts of a
plurality of genes.
In one aspect, a dispensing apparatus described herein can be used in a method
of
monitoring the amplification of a nucleic acid sequence, preferably a
plurality of sequences. In
such a method, aliquots dispensed during a nucleic acid amplification regimen
(e.g., after one or
~0 more cycles, preferably up to and including after each cycle) by such a
dispensing apparatus are
loaded into a separation apparatus, preferably into capillaries for capillary
electrophoresis. The
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nucleic acids in the loaded samples are separated, e.g., by size and/or
charge, and the separated
species are detected, thereby generating an amplification profile. When the
amplified nucleic
acids represent transcribed RNAs, e.g., when expressed RNA is reverse-
transcribed and then
amplified, the amplification profile provides a transcriptional profile for
the original sample.
Whereas the non-linearity of amplification at late stages of the amplification
process normally
precludes the ability to accurately quantitate the amount of a given
transcript in a nucleic acid
sample by measuring amplicon abundance after multiple cycles, the
transcriptional profile
generated in this manner provides quantitative as well as qualitative data
that do permit such
determination. The detection of amplicon abundance at various cycles during
the amplification
provides a real time representation of how the amplification proceeded for
each species
amplified and detected in a given reaction. Because non-linearity in the
amplification process
can be accounted for in such a real time profile, the profile permits the
efficient quantitative
determination of the amount of RNA corresponding to a given amplicon in an
original sample.
This is but one example of the advantages provided by a real time
transcriptional profile
generated by such a method.
Other advantages provided by the real time profiling performed in such a
manner include,
for example, the ability to follow the amplification profiles for multiple
amplicons, representing,
for example, multiple transcripts in a single sample. Because the size
separation by, for
example, CE, can resolve species differing by as little as one nucleotide, the
sample withdrawn
from an amplification reaction can have multiple differently sized amplicons,
each representing a
different transcript in the original sample. When this is considered along
with the simultaneous
amplification of multiple samples, as in amplification performed in mufti-well
plates or in
parallel in multiple tubes or capillaries, the amount of information
obtainable increases
dramatically.
~5 The sampling and analysis methods described herein are particularly well
suited for the
comparative analysis of gene expression. That is, the methods described permit
one to generate
a transcriptional profile for a given cell or tissue and to compare that
profile with the profile from
another cell or tissue to determine differences in the gene expression
patterns. Such differences
are useful for diagnostic purposes where, for example, a given pattern of
expression is elaborated
in a particular disease condition. In that instance, one would compare
transcriptional profiles of
a sample from an individual suspected of having a particular disease condition
with the
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transcriptional profile from one or more individuals known to have that
disease condition.
Similarities between the patterns of expression would confirm the diagnosis.
Comparative analysis of transcriptional profiles is also useful for the
identification and/or
validation of genes involved in disease. This approach is similar to the use
of microarray
hybridization methods, but has the added power provided by the ability to
obtain quantitative
data. In this approach, samples from healthy and diseased individuals are used
to generate
transcriptional profiles of multiple transcripts. The profiles can be
generated using primers that
hybridize to known genes, or, alternatively, can be generated using random or,
preferably, semi-
random primers. Semi-random primers are primers that have variation introduced
within the 3'-
terminal 1, 2 or 3 nucleotides. W one aspect, one can use a set of reverse-
transcription primers
with variation introduced at the 3' terminal l, 2 or 3 nucleotides (primers of
this design are used
in the art for the method of "differential display," described by Liang &
Pardee (1998, Mol.
Biotechnol. 10:261-7)). A further aspect of this primer design involves the
addition of an
invariant tag sequence 5' of the variable region. Amplification can then be
preformed using one
or more arbitrary upstream amplification primers (generally about 10-14
nucleotides, but can be
longer) with a downstream amplification primer that hybridizes to the
complement of the
invariant region of the reverse transcription primer. One or more of the
primers can be labeled,
for example, with one or more fluorophores. In this manner, a set of
transcripts is amplified;
when combined with the sampling approach described herein, the amplification
generates a
transcriptional profile for a subset of the transcripts present in the
original sample. This profile
can then be compared to those of other samples produced with the same
combination of primers.
Differences in the profiles obtained from different samples amplified using
the same set
of reverse-transcription and amplification primers can be used for diagnostic
or prognostic
purposes, for predicting the response of an individual to a drug, and for drug
target identification
and drug screening. The differences observed between samples from healthy and
diseased
individuals can be indicative of genes related to the disease state.
Differences observed between
samples from cells treated with or without a drug or other influence can be
used to screen for
drug effects on a target gene, or for example, on the pattern of genes
expressed in a given disease
state. Additional applications for the transcriptional profiles permitted by
the sampling methods
described herein will be apparent to the skilled artisan.
Differences in the overall pattern of expression, for example, transcripts
present in one
sample but not in another, as well as quantitative differences in expression
of individual
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transcripts from one sample to the next are determined in the manner described
above. Whereas
classical differential display is notoriously error-prone when one observes
differences in
abundance of an amplicon, rather than the discrete presence or absence of the
amplicon
(primarily because detection occurs only after multiple amplification cycles),
the sampling
method described herein permits meaningful distinctions based on differences
in amplicon
abundance.
In the transcriptional profiling approach using arbitrary or randomized
primers, the
identity of the amplified transcripts will generally not be known. However,
amplified products
that vary in representation between one sample and another can, if desired, be
isolated and
sequenced to identify the transcript (the isolated sequence can be used as a
probe to isolate the
full length transcript sequence using methods well known in the art).
To summarize, the transcriptional profiles generated using the sampling
methods
described herein can represent the transcriptional profile of individual known
transcription units
or genes, multiple known transcription units or genes, or multiple unknown
transcription units or
genes.
It is preferred that sample or aliquot dispensing is performed by an automated
apparatus.
"Automated" can refer to an apparatus that follows a programmed routine from
start to finish
without user input during the process, an apparatus that requires user input
for each repetition of
dispensing, or any combination thereof. Preferably the apparatus does not
require user input
after the initiation of a dispensing routine.
Amplification methods
Any nucleic acid amplification method can benefit from an automated sampling
method
as described herein. Of particular interest are amplification methods that
involve repeated cycles
of nucleic acid synthesis or polymerization, a number of which are known to
those skilled in the
art.
The most commonly used amplification method is thermal cycling PCR, originally
described by Mullis and Faloona (1987, Meth. Enzymol. 155:335-350). In thermal
cycling PCR,
two oligonucleotide primers, a template and a thermostable nucleic acid
polymerase are
generally used for each template sequence to be amplified. In the general PCR
scheme, one of
CA 02509769 2005-05-19
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the oligonucleotide primers anneals to a template nucleic acid strand. The
annealed primer is
extended by the thermostable template-dependent nucleic acid polymerase, and
that
polymerization product has a sequence complementary to the second primer such
that the
polymerization product can serve as template for the extension of the second
primer. The
polymerization product is thermally denatured to separate the strands, and the
pair of primers is
annealed to the respective strands and extended. Because each extension
product serves as the
template fox subsequent extension reactions, the target sequence is
exponentially amplified.
Numerous variations on the general principle of thermal cycling PCR have been
described and are known to those of skill in the art. When amplification
profiling is to be
performed, e.g., in order to derive quantitative information regarding the
abundance of template
in a biological sample, sampling can be performed after various cycles in the
process. Ideally, an
aliquot is withdrawn or extruded from the amplification reaction mixture after
each cycle in the
amplification regimen. Sampling can be performed after any desired cycles,
e.g., after every
other cycle, every third cycle, every fourth cycle, etc., but the most
detailed and accurate
information regarding the amplification profile will be obtained when sampling
is performed
after each cycle in the regimen. This is particularly so when more than one
target amplification
product is monitored in a single amplification reaction or set of
amplification reactions. This is
so because the kinetics of amplification of different target sequences can
differ dramatically with
the sequence and initial abundance of the different target sequences. Sampling
at every cycle
will permit the generation of a complete amplification profile for each target
in a single reaction
mixture regardless of the kinetics of amplification for the individual
targets.
Another method of nucleic acid amplification that can benefit from the
sampling methods
described herein is isothermal, Self Sustained Sequence Replication (3SR;
Gingeras et al., 1990,
Annales de Biologie Clinique, 48(7): 498-501; Guatelli et al., 1990, Proc.
Natl. Acad. Sci.
U.S.A., 87: 1874). The contents of these articles are herein incorporated by
reference. 3SR is
an outgrowth of the transcription-based amplification system (TAS), which
capitalizes on the
high promoter sequence specificity and reiterative properties of bacteriophage
DNA-dependent
RNA polymerases to decrease the number of amplification cycles necessary to
achieve high
amplification levels (Kwoh et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 83:
1173-1177).
In 3SR, each priming oligonucleotide contains a bacteriophage RNA polymerase
binding
sequence and the preferred transcriptional initiation sequence, e.g., the T7
RNA polymerase
binding sequence (TAATACGACTCACTATA [SEQ ID NQ: 1]) and the preferred T7
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polymerase transcriptional initiation site. The remaining sequence of each
primer is
complementary to the target sequence on the molecule to be amplified.
Exemplary 3SR conditions are described herein as follows. The 3SR
amplification
reaction is carried out in 100 pl and contains the target RNA, 40 mM Tris-HCI,
ph 8.1, 20 mM
MgCl2, 2 mM spermidine-HCl, SmM dithiothreitol, 80 pg/ml BSA, 1 mM dATP, 1 mM
dGTP,
1 mM dTTP, 4 mMATP, 4 mM CTP, 1 mM GTP, 4 mM dTTP, 4 mM ATP, 4 mM CTP, 4 mM
GTP, 4 mMUTP, and a suitable amount of oligonucleotide primer (250 ng of a 57-
mer; this
amount is scaled up or down, proportionally, depending upon the length of the
primer sequence).
Three to six attomoles of the nucleic acid target for the 3SR reactions is
used. As a control for
background, a 3SR reaction without any target is run in parallel. The reaction
mixture is heated
to 100°C for 1 minute, and then rapidly chilled to 42°C. After 1
minute, 10 units (usually in a
volume of approximately 2 ~zl) of reverse transcriptase, (e.g. avian
myoblastosis virus reverse
transcriptase, AMV-RT; Life Technologies/Gibco-BRL) is added. The reaction is
incubated for
10 minutes, at 42°C and then heated to 100°C. for 1 minute. (If
a 3SR reaction is performed
using a single-stranded template, the reaction mixture is heated instead to
65°C for 1 minute.)
Reactions are then cooled to 37°C for 2 minutes prior to the addition
of 4.6 pl of a 3SR enzyme
mix, which contains 1.6 ul of AMV-RT at 18.5 units/ul, 1.0 pl T7 RNA
polymerase (both e.g.
from Stratagene; La Jolla, CA) at 100 units/ul, and 2.0 ul E. Coli RNase H at
4 units/~l (e.g.
from Gibco/Life Technologies; Gaithersburg, MD). It is well within the
knowledge of one of
skill in the art to adjust enzyme volumes as needed to account for variations
in the specific
activities of enzymes drawn from different production lots or supplied by
different
manufacturers. Variations can also be made to the units of the enzymes as
necessary. The
reaction is incubated at 37°C for 1 hour and stopped by freezing.
Sampling can be performed at any stage of the 3SR reaction. Because 3SR
proceeds
continuously at a single temperature, there are not individual cycles at which
aliquots will be
withdrawn. In this instance, sampling can be performed at set times during the
amplification
incubation period, for example, every minute, every two minutes, every three
minutes, etc.
Nucleic acids in the aliquots withdrawn or extruded are separated and nucleic
acids detected,
thereby permitting the generation of an amplification profile, from which the
abundance of target
in the initial sample can be determined.
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3SR is also referred to by some as Nucleic Acid Sequence Based Amplification,
or
NASBA (see for example, Compton, 1991, Nature, 350: 91-92; I~ievits et al.,
1991, J. Virol
Meth. 35: 273-286, each of which is incorporated herein by reference).
Another method of nucleic acid amplification that is of use according to the
invention is
the DNA ligase amplification reaction (LAR), which has been described as
permitting the
exponential increase of specific short sequences through the activities of any
one of several
bacterial DNA ligases (Wu and Wallace, 1989, Genomics, 4: 560; Barany, 1991,
Proc. Natl.
Acad. Sci. USA 88: 189, each of which is incorporated herein by reference).
This technique is
based upon the ligation of oligonucleotide probes. The probes are designed to
exactly match two
adjacent sequences of a specific target nucleic acid. The amplification
reaction is repeated in
three steps in the presence of excess probe: (1) heat denaturation of double-
stranded nucleic
acid, (2) annealing of probes to target nucleic acid, and (3) joining of the
probes by thennostable
DNA ligase. The reaction is generally repeated for 20-30 cycles. The sampling
methods
disclosed herein permit the generation of a detailed amplification profile. As
with any cyclic
amplification protocol, sampling can be performed after any cycle, but
preferably after each
cycle.
Rolling circle amplification (RCA) is an alternative amplification technology
that may
prove to have as large an impact as PCR. This technique draws on the DNA
replication
mechanism of some viruses. In RCA, similar to the replication technique used
by many viruses,
a polymerase enzyme reads off of a single promoter around a circle of DNA -
continuously
rolling out linear, concatenated copies of the circle. In such linear RCA, the
reaction can run for
three days, producing millions of copies of the small circle sequence. An
exponential variant has
been developed in which a second promoter displaces the double strands at each
repeat and
initiates hyperbranching in the DNA replication, creating as many as 1012
copies per hour.
Another amplification method that can benefit from the sampling methods
disclosed
herein is strand-displacement amplification (SDA; Walker et al., 1992, Nucleic
Acids Res., 20:
1691-1696; Spargo et al., 1993, Mol. Cellular Probes 7: 395-404, each of which
is incorporated
herein by reference). SDA uses two types of primers and two enzymes (DNA
polymerase and a
restriction endonuclease) to exponentially produce single- stranded amplieons
asynchronously.
A variant of the basic method in which sets of the amplification primers were
anchored to
distinct zones on a chip reduces primer- primer interactions. This so-called
"anchored SDA"
approach permits multiplex DNA or RNA amplification without decreasing
amplification
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efficiency (Westin et al., 2000, Nature Biotechnology 18: 199- 204,
incorporated herein by
reference. SDA can benefit from the sampling methods disclosed herein, as they
permit the
generation of a detailed amplification profile.
One of the limitations of thermal cycled PCR and, for that matter, any method
that
requires a specific primer for each different target sequence in a reaction is
that the concentration
of primers tends to introduce artifacts to the reactions. Primer-primer
interactions often result in
the incorporation of primers into complexes independent of template. A common
example is the
so-called "primer-dimer" encountered in thermal cycling PCR. When the 3' end
of one primer
hybridizes to a site within another primer, the polymerase enzyme can extend
the primer to
generate a template-independent product. Because the concentration of
amplification primers is
generally in excess of specific target sequences, a large number of primer
dimer artifacts can be
generated when there is the necessary complementarity between two or more
primers. It follows
that the higher the number of different primers in an amplification reaction,
the greater the
chance that one will find a region of complementarity in another and result in
primer dimer-type
artifacts. As one attempts to multiplex additional target sequence
amplifications into a single
reaction mixture, the chances for this type of artifact increase dramatically.
Sampling methods as disclosed herein can help in avoiding or at least
minimizing the
effects of artifacts induced by the presence of multiple primers. First, when
sampling is used, the
nucleic acid products in the samples can be separated by size and detected,
and primer dimer-
type artifacts can be excluded by their small size. Another approach is to
limit the number of
different primers in the amplification reaction. One approach to this is
described in U.S. patent
application No. 60/372,045, which is incorporated herein by reference. The
application
describes a number of approaches that permit the detection of multiple
different amplification
products in a single reaction mixture. Several of those methods reduce the
number of different
primers necessary for quantitative multiplex PCR by incorporating downstream
primers
comprising a common tag sequence into each of a number of different reverse-
transcription
products. The first round of amplification additionally incorporates one or
more upstream
primers comprising a different common tag sequence. Subsequent amplification
is then
performed with a single pair of amplification primers that recognize the
common tag sequences.
The sizes of the various amplicons is selected such that subsequent separation
of the products
can distinguish the various species amplified in the reaction. This approach
has the benefit that a
smaller number of primers is present in the amplification reaction. An
additional benefit is that
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differences in the annealing efficiency for the primers are minimized by the
reduction in the
number of different amplification primers used. Differences in primer
annealing efficiency,
caused by, for example, different G+C content of the primers, is known to
influence the
reliability of multiplex quantitative amplification approaches.
Figure 4 shows a schematic diagram of an amplification approach wherein two
different
targets of different size are amplified with a single pair of amplification
primers that anneal to
common tag sequences incorporated by primer extension. Tl is a downstream tag
sequence
common to all amplicons, and T2 is an upstream tag sequence common to all
amplicons (Tl' and
T2' denote their respective complements). The tag sequences are incorporated
as part of the
sequence of primer extension primers that are annealed to the target sequence
and extended.
Each primer extension primer has a region complementary to a sequence on the
target sequence
or the complement of the target sequence (designated a, b, c and d and a', b',
c' and d' in the
figure), and a tag sequence. Two rounds of primer extension primer annealing
and extension
generates a set of molecules each comprising both an upstream and a downstream
tag sequence
or its complement. At this point, primer extension primers are removed and
amplification is
performed using amplification primers corresponding to the tag sequences T1
and T2, one of
which primers is fluorescently labeled. Subsequent size separation, (e.g., by
capillary
electrophoresis) and detection distinguishes the targets by size and
determines the abundance of
the individual targets. In this way, two different target sequences are
amplified with a single pair
of amplification primers. This approach can be scaled up to include numerous
differently-sized
target sequences that are then resolved by size and detected. Further
multiplexing can occur
without dramatically increasing the number of amplification primers by using a
set of optically
distinguishable fluorescent labels on additional amplification primers.
Amplification reaction devices
Devices for performing the amplification reactions must be capable of
achieving and
maintaining reaction temperatures required for the amplification reaction in
one or more tubes,
capillaries or multiwell containers. Any device capable of achieving and
maintaining the
temperature or temperatures necessary for an amplification reaction can be
used. As a non-
limiting example, convenient devices include thermal cyclers commonly used for
PCR
, amplification. Thermal cyclers are widely available commercially.
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Amplification reaction devices such as thermal cyclers can be interfaced with
a sampling
device, for example, in a modular design, or the sampling device can be
integral with the
amplification reaction device. In one aspect, the amplification reactions
themselves are
performed in capillary or other tubes that are open at one or both ends, and
sampling is
performed by applying pressure or a (vacuum) to one end of the tube such that
a portion of the
reaction mixture is extruded from the tube and collected by a sampling device.
Thermal cyclers
capable of accepting tubes, including capillary tubes, are commercially
available and include, for
example the LightcyclerTM (Roche Molecular Biochemicals, Indianapolis, IN),
the
RapidCyclerTM (Idaho Technology, Salt Lake City, UT) and the BioOven III
Thermocycler (St.
John Associates, Beltsville, MD).
Separation and detection methods
Any of a number of different nucleic acid separation methods can be used in
the methods
disclosed herein. For example, various adaptations of electrophoresis and
liquid chromatography
are well suited for separating nucleic acid species in a sample from an
amplification reaction.
Electrophoretic separation is preferably performed as capillary
electrophoresis (CE), due
to the small sample sizes necessary and the speed and resolution achievable.
Another benefit of
CE is that there exist a variety of off the-shelf CE devices that are
interfaced with fluorescence
detectors, for example, high throughput CE equipment is available
commercially, for example,
the HTS9610 High Throughput Analysis System and SCE 9610 fully automated 96-
capillary
electrophoresis genetic analysis system from Spectrumedix Corporation (State
College, PA).
Others include the PACE 5000 series from Beckman Instruments Inc (Fullerton,
CA) and the
ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Each of
these
devices comprises a fluorescence detector that monitors the emission of light
by molecules in the
sample near the end of the CE column. The standard fluorescence detectors can
distinguish
numerous different wavelengths of fluorescence emission, providing the ability
to detect
multiple fluorescently labeled species in a single CE run from an
amplification sample.
CE devices capable of running 96 samples at a time mesh nicely with, for
example,
thermal cyclers or other amplification devices that run multiple samples
simultaneously. CE
devices that provide automated sample loading, electrophoresis and detection
for multiple
samples in parallel are described in U.S. Patent Nos. 6,217,731 and 6,001,230.
As an alternative
to fluorescence detection, a CE device can be interfaced with a mass
spectrometry device for
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detection of the various nucleic acid species in an amplification reaction by
molecular mass
(CE1MS). Mass spectrometry devices capable of such detection are commercially
available.
Liquid chromatography (LC) is another option for the separation of nucleic
acids in
samples withdrawn or extruded from an amplification reaction. Commonly, LC is
coupled with
mass spectrometry (LC/MS), such that the mass of HPLC-separated species is
determined by
mass spectrometry. LC/MS systems are commercially available, for example, from
Agilent
Technologies (e.g., the 1100 SeriesTM LC/MS) and from Applied Biosystems
(e.g., the API
3000TM or API 4000TM LC/MS systems), among others.
All patents, patent applications, and published references cited herein are
hereby
incorporated by reference in their entirety. While this invention has been
particularly shown and
described with references to preferred embodiments thereof, it will be
understood by those
skilled in the art that various changes in form and details may be made
therein without departing
from the scope of the invention encompassed by the appended claims.
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PATENTIN.ST25.tXt
SEQUENCE LISTING
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<120> Sampling Method and Apparatus for Amplification Reaction
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<130> 19781/2068
<150> US 60/428,038
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<150> US 60/439,982
<151> 2003-01-14
<150> US 60/440,010
<151> 2003-01-14
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<223> Consensus T7 RNA polymerase binding sequence
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taatacgact cactata
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