U.S. patent application number 10/845996 was filed with the patent office on 2005-02-24 for single molecule amplification and detection of dna length.
This patent application is currently assigned to Caliper Life Sciences, Inc.. Invention is credited to Baker, Jill M., Chow, Andrea W., Knapp, Michael R., Kopf-Sill, Anne R., Spaid, Michael.
Application Number | 20050042639 10/845996 |
Document ID | / |
Family ID | 34969685 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050042639 |
Kind Code |
A1 |
Knapp, Michael R. ; et
al. |
February 24, 2005 |
Single molecule amplification and detection of DNA length
Abstract
Methods and systems for performing single molecule amplification
for detection, quantification and statistical analysis of nucleic
acids are provided. Methods and systems are provided for
determining and quantifying lengths of nucleic acids of
interest.
Inventors: |
Knapp, Michael R.; (Palo
Alto, CA) ; Baker, Jill M.; (Redwood City, CA)
; Chow, Andrea W.; (Los Altos, CA) ; Kopf-Sill,
Anne R.; (Portola Valley, CA) ; Spaid, Michael;
(Mountain View, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Life Sciences, Inc.
Mountain View
CA
|
Family ID: |
34969685 |
Appl. No.: |
10/845996 |
Filed: |
May 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10845996 |
May 14, 2004 |
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10741162 |
Dec 19, 2003 |
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60462384 |
Apr 11, 2003 |
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60436098 |
Dec 20, 2002 |
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Current U.S.
Class: |
435/6.12 ;
435/6.1 |
Current CPC
Class: |
C12Q 1/6851 20130101;
B01L 7/52 20130101; C12Q 1/6818 20130101; B01L 2300/1827 20130101;
C12Q 1/6818 20130101; C12Q 1/6837 20130101; C12Q 1/6851 20130101;
C12Q 1/686 20130101; C12Q 1/6851 20130101; C12Q 1/6851 20130101;
B01L 2200/16 20130101; C12Q 1/686 20130101; G01N 21/6428 20130101;
B01L 2300/0867 20130101; B01L 2200/10 20130101; B01L 2400/0487
20130101; B01L 2300/0864 20130101; B01L 2400/0415 20130101; B01L
2300/1833 20130101; C12Q 2545/113 20130101; C12Q 1/6837 20130101;
C12Q 2565/629 20130101; C12Q 2565/629 20130101; C12Q 2565/629
20130101; C12Q 2531/113 20130101; C12Q 2565/629 20130101; C12Q
2565/629 20130101; C12Q 2565/102 20130101; C12Q 2531/113 20130101;
B01L 7/525 20130101; C12Q 1/6851 20130101; B01L 2300/0816 20130101;
B01L 2400/0406 20130101; C12Q 2527/143 20130101; C12Q 2537/143
20130101; C12Q 2565/102 20130101; C12Q 2537/143 20130101; C12Q
1/6869 20130101; C12Q 1/6869 20130101; B01L 3/5027 20130101; G01N
2021/6441 20130101; B01L 2300/1822 20130101; B01L 3/502761
20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Goverment Interests
[0002] Part of the technology for this application was developed
under NIST-ATP grant 70NANB8H4000. The Government may have certain
rights in this invention.
Claims
What is claimed is:
1. A method of determining whether a nucleic acid of interest in a
sample comprises at least a given length, the method comprising:
contacting the nucleic acid of interest in a reaction mixture with
two or more different probes, wherein the probes each comprise a
detectable marker; flowing the nucleic acid of interest into a
detection region; and, detecting one or more detectable marker
signals from the probes; wherein coincident detection of signals
from two or more of the different probes indicates the nucleic acid
of interest is not fragmented between the probes, thereby
determining the nucleic acid of interest in the sample has at least
a given length.
2. The method of claim 1, wherein the reaction mixture comprises a
single copy of the nucleic acid of interest.
3. The method of claim 1, wherein detecting a signal from only one
of the different probes indicates the nucleic acid of interest is
fragmented.
4. The method of claim 1, wherein determining the given length
comprises determining the integrity of the nucleic acid of
interest.
5. The method of claim 1, wherein the two or more probes each
comprise detectable markers with different signals.
6. The method of claim 1, wherein at least one of the probes
comprise a fluorescent resonant energy transfer (FRET) detectable
marker or a molecular beacon (MB) marker.
7. The method of claim 1, further comprising correlating one or
more lengths of one or more nucleic acids of interest to a disease
state by identifying a ratio or quantitative threshold of lengths
associated with the disease state.
8. The method of claim 1, further comprising: contacting the
nucleic acid of interest with a first primer pair; contacting the
nucleic acid of interest with a second primer pair comprising at
least one primer complimentary to a sequence of the nucleic acid of
interest or its compliment outside of a sequence defined by the
first primer pair; and, amplifying the nucleic acid of interest in
the reaction mixture contained in a microchannel or microchamber
with primer extensions initiated at primers to produce first
amplicons defined by the first primer pair or second amplicons
defined by the second primer pair; wherein at least a first probe
is complimentary to a sequence of the first amplicons and at least
a second probe is complimentary to a sequence of the second
amplicons; whereby a sensitivity of said detecting is
increased.
9. The method of claim 8, wherein one primer pair comprises control
primers defining amplicons of 100 base pairs or less in length and
another primer pair comprises test primers defining longer
amplicons ranging in length from about 100 base pairs to about 3000
base pairs.
10. The method of claim 8, wherein a region of the nucleic acid of
interest defined by the first primer pair does not overlap with a
region of the nucleic acid of interest defined by the second primer
pair.
11. The method of claim 8, wherein at least one of the probes is
complimentary to an amplicon sequence defined by one primer pair
but not complimentary to an amplicon sequence defined by another
primer pair.
12. The method of claim 8, further comprising: contacting the
nucleic acid of interest with one or more additional primer pairs
comprising at least one primer complimentary to a sequence of the
nucleic acid of interest or its compliment outside a sequence
defined by the first primer pair or second primer pair; whereby one
or more additional amplicons are produced, and whereby coincident
detection of signals from a probe specific for the one or more
additional amplicons and the first or second probes indicates the
nucleic acid of interest is not fragmented between sequences
complimentary to probes providing the signals.
13. The method of claim 8, wherein said amplifying comprises: a
polymerase chain reaction (PCR), reverse-transcriptase PCR
(RT-PCR), ligase chain reaction (LCR), a Q-.beta. replicase or
RNA/transcription mediated techniques.
14. The method of claim 1, further comprising: aliquotting the
sample into at least 25 reaction mixtures comprising 2 or fewer
copies of the nucleic acid of interest each; individually
subjecting the sample aliquots to the contacting and the detecting;
and, individually counting a number of aliquots resulting in
detection of a signal from one probe or individually counting a
number of aliquots resulting in detection of signals from two or
more probes.
15. The method of claim 14, wherein the at least 25 reaction
mixtures comprise one or more reaction mixtures comprising single
copies of the nucleic acid of interest.
16. The method of claim 14, wherein the at least 25 reaction
mixtures comprise one or more reaction mixtures comprising zero
copies of the nucleic acid of interest.
17. The method of claim 14, further comprising evaluating the
number of one probe signals and two probe signals to determine a
proportion of nucleic acids of interest having different
lengths.
18. The method of claim 17, further comprising correlating a
disease state with the proportion.
19. The method of claim 1, further comprising quantifying the
nucleic acid of interest.
20. The method of claim 19, wherein said quantifying comprises
counting a number of signals from one or more of the different
probes.
21. The method of claim 19, wherein said quantifying comprises
detecting a volume, width, height, length, area, shape, or ratio,
of the one or more signals.
22. The method of claim 19, wherein said quantifying comprises
comparison of a probe detectable marker signal to an internal
standard signal.
23. The method of claim 19, wherein said quantifying comprises
comparison of signals from two or more reaction mixtures comprising
different degrees of amplification.
24. The method of claim 23, wherein the two or more reaction
mixtures comprise different amplification due to: flowing through a
thermocycler at different flow rates, flowing different distances
into a thermocycler, remaining in a thermocycler for different
amounts of time, or experiencing different numbers of amplification
cycles.
25. The method of claim 1, further comprising diluting the sample
to obtain one or more reaction mixtures containing a single copy of
the nucleic acid of interest.
26. The method of claim 25, wherein the nucleic acid of interest is
diluted to a concentration of about 1 molecule per nanoliter or
less.
27. The method of claim 1, wherein the sample comprises a sample
selected from the group consisting of: whole blood, serum, plasma,
stool, urine, vaginal secretions, ejaculatory fluid, a cervical
swab, synovial fluid, a biopsy, cerebrospinal fluid, amniotic
fluid, sputum, saliva, lymph, tears, sweat, and urine.
28. A method of differentiating lengths of a nucleic acid of
interest in a sample, the method comprising: a) contacting the
nucleic acid of interest with a first primer pair; b) contacting
the nucleic acid of interest with a second primer pair comprising
at least one primer complimentary to a sequence of the nucleic acid
of interest or its compliment outside of a sequence defined by the
first primer pair; c) amplifying the nucleic acid of interest in a
reaction mixture comprising a single copy of the nucleic acid of
interest to produce first amplicons defined by the first primer
pair or second amplicons defined by the second primer pair; d)
contacting the reaction mixture with a first probe complimentary to
a sequence of the first amplicons or with a second probe
complimentary to a sequence of the second amplicons, which probes
comprise signals from detectable markers; and, e) detecting one or
more of the signals; wherein detection of a signal from only one of
the probes indicates a fragmented nucleic acid of interest or
detecting signals from both probes indicates a nucleic acid that
has a given length, thereby differentiating the length of the
nucleic acid of interest.
29. The method of claim 28, wherein the reaction mixture is
contained in a microchannel or microchamber.
30. A method of differentiating lengths of nucleic acids of
interest in a sample, the method comprising: a) contacting a
nucleic acid of interest with a first primer pair; b) contacting
the nucleic acid of interest with a second primer pair comprising
at least one primer complimentary to a sequence of the nucleic acid
of interest or its compliment outside of a sequence defined by the
first primer pair; c) amplifying the nucleic acid of interest in a
reaction mixture contained in a microchannel or microchamber with
primer extensions initiated at the primers to produce first
amplicons defined by the first primer pair or second amplicons
defined by the second primer pair; d) contacting the reaction
mixture with a first probe complimentary to a sequence of the first
amplicons or with a second probe complimentary to a sequence of the
second amplicons, which probes comprise signals from detectable
markers; and, e) detecting one or more signals; wherein detection
of a signal from only one of the probes indicates a fragmented
nucleic acid of interest or detecting signals from both probes
indicates a nucleic acid that has a given length, thereby
differentiating the length of the nucleic acid of interest.
31. The method of claim 1, wherein the reaction mixture comprises a
single copy of the nucleic acid of interest.
32. A method of quantifying a nucleic acid of interest in a sample,
the method comprising: amplifying the nucleic acid of interest
through a plurality of amplification cycles; detecting signals
associated with amplicons produced for two or more of the
amplification cycles; preparing a sample curve of a signal
parameter versus a number of amplification cycles; and, comparing
one or more identifiable points from the sample curve to a standard
curve of identifiable points versus concentration, thereby
quantifying the nucleic acid of interest.
33. The method of claim 32, wherein the identifiable points
comprise: points of inflection, points having a certain slope,
points having a certain absolute signal amplitude, or points having
a certain fraction of a maximum signal amplitude.
34. A method of quantifying a nucleic acid of interest in a sample,
the method comprising: amplifying the nucleic acid of interest
through a plurality of amplification cycles in a reaction mixture
defining two or more different amplicons of the nucleic acid of
interest; detecting from homogenous reaction mixtures different
signals associated with each of the different amplicons after at
least two of the plurality of amplification cycles; preparing
sample curves of the different signals versus numbers of
amplification cycles; and, comparing one or more identifiable
points from the sample curves to one or more standard curves of
identifiable points versus nucleic acid concentration, thereby
quantifying one or more sequences of the nucleic acid of interest
associated with one or more of the amplicons.
35. The method of claim 34, wherein said detecting comprises
detecting one or more signals from a low copy or single copy
reaction mixture.
36. The method of claim 35, wherein coincident detection of two or
more of the different signals indicates a nucleic acid of a given
length, or the detection of only one of the different signals
indicates a fragmented nucleic acid.
37. The method of claim 34, wherein the identifiable points
comprise: points of inflection, points having a certain slope,
points having a certain absolute signal amplitude, or points having
a certain fraction of a maximum signal amplitude.
38. The method of claim 34, wherein the number of amplification
cycles is controlled by: flowing an amplification reaction through
a thermocycler at different flow rates, flowing an amplification
reaction different distances into a thermocycler, an amplification
reaction remaining in a thermocycler for different amounts of time,
or an amplification reaction experiencing different numbers of
amplification cycles.
39. A method of quantifying a nucleic acid of interest in a sample,
the method comprising: amplifying a plurality of nucleic acid of
interest standard materials through a number of amplification
cycles; detecting signals associated with standard amplicons
produced for standard materials having different known
concentrations of the nucleic acid; amplifying the sample nucleic
acid of interest the number of amplification cycles; detecting a
signal associated with sample amplicons produced for the sample
nucleic acid of interest; and, comparing one or more standard
amplicon signals to the sample amplicon signal to determine a
concentration value for the nucleic acid of interest in the sample,
thereby quantifying the nucleic acid of interest.
40. The method of claim 39, wherein said comparing comprises
comparison of signal parameters selected from the group consisting
of: a shape of a signal peak, points of inflection on a signal
peak, slopes of signal peaks, signal peak amplitude, signal peak
areas, and signal peak widths at half height.
41. The method of claim 39, further comprising: repeating said
amplifying, detecting, and comparing steps one or more times, but
with different numbers of amplification cycles, thereby determining
additional concentration values for the sample nucleic acid of
interest; and, Statistically evaluating the concentration values,
thereby providing a more precise or more accurate concentration
value result for the nucleic acid of interest in the sample.
42. A system for differentiating the lengths of nucleic acids of
interest in a sample, the system comprising: a microfluidic device
comprising an amplification microchannel or microchamber containing
a reaction mixture under conditions that provide one or more
amplicons of the nucleic acid of interest; a detector integral with
or proximal to the microfluidic device, which detector is
configured to detect the amplicons as one or more signals from a
homogenous mixture; and, a software system that interprets one or
more coincidently detected signals to indicate lengths of one or
more individual nucleic acid molecules of interest, thereby
differentiating the lengths of the nucleic acids of interest.
43. The system of claim 42, wherein the sample comprises: a nucleic
acid with single nucleotide polymorphism (SNP), a cancer associated
nucleic acid, a nucleic acid from an infective agent, whole blood,
serum, plasma, stool, urine, a vaginal secretion, cervical swab,
ejaculatory fluid, synovial fluid, a biopsy, cerebrospinal fluid,
amniotic fluid, or a forensic nucleic acid.
44. The system of claim 42, wherein the reaction mixture comprises:
the nucleic acid of interest, a first primer pair, a second primer
pair comprising at least one primer complimentary to a sequence of
the nucleic acid of interest outside a sequence defined by the
first primer pair, and a polymerase that can synthesize amplicons
defined by the primer pairs.
45. The system of claim 44, wherein one primer pair comprises
control primers defining amplicons of 100 base pairs or less in
length and another primer pair comprises test primers defining
longer amplicons ranging in length from about 100 base pairs to
about 3000 base pairs.
46. The system of claim 44, wherein a region of the nucleic acid of
interest defined by the first primer pair does not overlap with a
region of the nucleic acid of interest defined by the second primer
pair.
47. The system of claim 42, wherein the amplification microchannel
or microchamber comprises: electrodes to apply a heating current to
the microchannel, a resistive heating element, a Joule-Thompson
device, or a Peltier device.
48. The system of claim 42, wherein the amplification microchannel
or microchamber is configured to thermocycle the reaction mixture
to produce amplicons of the nucleic acid of interest in a volume
sufficiently small to substantially separate amplification products
of a single nucleic acid of interest molecule from other nucleic
acid of interest molecules in the sample or from additional nucleic
acids in the sample.
49. The system of claim 42, wherein the amplicons are detected
without resolution in a size selective media or affinity media.
50. The system of claim 42, wherein the system software interprets
a volume, width, height, length, area, shape, or ratio, of the
signals detected by the detector to indicate: a number of copies of
the nucleic acid of interest in the sample, a number of the nucleic
acids of interest having a given length, or a proportion of nucleic
acids of interest having different lengths.
51. The system of claim 42, further comprising one or more nucleic
acid probes comprising one or more detectable markers and a
sequence complimentary to one or more of the amplicons, wherein the
detectable markers provide a signal detectable by the detector.
52. The system of claim 51, wherein the detector comprises: a
fluorometer, a charge coupled device, a laser, an enzyme, or an
enzyme substrate, a photo multiplier tube, a spectrophotometer,
scanning detector, microscope, or a galvo-scanner.
53. The system of claim 52, wherein the fluorometer can
simultaneously detect emissions at two or more frequencies.
54. The system of claim 51, wherein the detector can independently
detect signals from two or more detectable markers with different
signals.
55. The system of claim 51, wherein at least one of the probes is
complimentary to an amplicon sequence defined by one primer pair
but not complimentary to an amplicon sequence defined by another
primer pair.
56. The system of claim 51, wherein at least one of the probes is
complimentary to a first amplicon sequence and to a second amplicon
sequence.
57. The system of claim 51, wherein two or more probes each
comprise different signals.
58. The system of claim 57, wherein the different signals comprise
different fluorescent emissions.
59. The system of claim 51, wherein at least one of the probes
comprise a fluorescent resonant energy transfer (FRET) detectable
marker or a molecular beacon (MB) marker.
60. The system of claim 59, wherein the FRET detectable marker
comprises a quencher removable from the FRET probe by nuclease
activity.
61. The system of claim 42, wherein the system is a high throughput
system.
62. The system of claim 42, further comprising a dilution
module.
63. The system of claim 62, wherein the dilution module is
configured to dilute the sample to a concentration which provides
one or more single copy reaction mixtures for nucleic acids of
interest in the amplification microchannel or microchamber.
64. The system of claim 62, wherein the dilution module comprises
serial multiwell plate dilutions or a dilution channel in a
microfluidic device.
65. The system of claim 62, further comprising system instructions
that direct the dilution module to aliquot the sample into a
plurality of aliquots, including a plurality of zero copy aliquots
comprising no copies of the nucleic acid of interest, and one or
more single copy aliquots comprising a single copy of the nucleic
acid of interest.
66. The system of claim 42, further comprising a computer in
communication with the detector.
67. The system of claim 42, wherein the microfluidic device further
comprises multiple amplification channels.
68. The system of claim 42, further comprising a sample storage
module that stores the sample before preparation of the reaction
mixture, or a sample retrieval module that retrieves the sample
from the storage module before preparation of the reaction
mixture.
69. The system of claim 42, further comprising a capture
oligonucleotide bound to a solid support to capture nucleotides of
interest before or during preparation of the reaction mixture or to
capture amplicons for detection.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a Continuation in Part of
Non-Provisional patent application U.S. Ser. No. 10/741,162,
"SINGLE MOLECULE AMPLIFICATION AND DETECTION OF DNA IN A
MICROFLUIDIC FORMAT", filed Dec. 12, 2003 by Knapp, et al., which
is a regular utility application corresponding to prior filed
Provisional Patent Application U.S. Ser. No. 60/518,431 "SINGLE
MOLECULE AMPLIFICATION AND DETECTION OF DNA IN A MICROFLUIDIC
FORMAT" by Knapp et al., filed Nov. 6, 2003, Provisional Patent
Application U.S. Ser. No. 60/462,384 "SINGLE MOLECULE AMPLIFICATION
AND DETECTION OF DNA IN A MICROFLUIDIC FORMAT" by Knapp et al.,
filed Apr. 11, 2003, and Provisional Patent Application U.S. Ser.
No. 60/436,098 "SINGLE MOLECULE AMPLIFICATION AND DETECTION OF DNA
IN A MICROFLUIDIC FORMAT" by Knapp et al., filed Dec. 20, 2002. The
subject application claims priority to and benefit to each of these
prior applications, which are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[0003] The invention is in the field of single molecule detection,
e.g., by amplification of single molecules from complex mixtures,
e.g., for disease diagnosis, detection of pathogens, environmental
contaminants, or the like. Amplifications can be conducted in high
throughput systems, e.g., microfluidic systems, to provide an
ability to detect rare molecules in complex samples that are
aliquotted into low copy number reaction mixtures, whereby a rare
copy nucleic acid of interest is detected, e.g., by amplifying
large numbers of aliquots of the complex samples. The methods and
systems can determine whether an individual nucleic acids of
interest has a given length.
BACKGROUND OF THE INVENTION
[0004] The detection of nucleic acids is central to medicine,
forensic science, industrial processing, crop and animal breeding,
and many other fields. The ability to detect disease conditions
(e.g., cancer), infectious organisms (e.g., HIV), genetic lineage,
genetic markers, and the like, is ubiquitous technology for disease
diagnosis and prognosis, marker assisted selection, correct
identification of crime scene features, the ability to propagate
industrial organisms and many other techniques. Determination of
the integrity of a nucleic acid of interest can be relevant to the
pathology of an infection or cancer.
[0005] One of the most powerful and basic technologies for nucleic
acid detection is nucleic acid amplification. That is, in many
typical formats, such as the polymerase chain reaction (PCR),
reverse-transcriptase PCR (RT-PCR), ligase chain reaction (LCR),
and Q-.beta. replicase and other RNA/transcription mediated
techniques (e.g., NASBA), amplification of a nucleic acid of
interest precedes detection of the nucleic acid of interest,
because it is easier to detect many copies of a nucleic acid than
it is to detect a single copy.
[0006] PCR, RT-PCR and LCR are in particularly broad use, in many
different fields. Details regarding the use of these and other
amplification methods can be found in any of a variety of standard
texts, including, e.g.,: Sambrook et al., Molecular Cloning--A
Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook"); Current
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 2002)
("Ausubel")) and PCR Protocols A Guide to Methods and Applications
(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990)
(Innis). Many available biology texts have extended discussions
regarding PCR and related amplification methods.
[0007] More recently, a number of high throughput approaches to
performing PCR and other amplification reactions have been
developed, e.g., involving amplification reactions in microfluidic
devices, as well as methods for detecting and analyzing amplified
nucleic acids in or on the devices. Details regarding such
technology is found in the technical and patent literature, e.g.,
Kopp et al. (1998) "Chemical Amplification: Continuous Flow PCR on
a Chip" Science, 280 (5366):1046; U.S. Pat. No.6,444,461 to Knapp,
et al. (Sep. 3, 2002) MICROFLUIDIC DEVICES AND METHODS FOR
SEPARATION; U.S. Pat. No. 6,406,893 to Knapp, et al. (Jun. 18,
2002) MICROFLUIDIC METHODS FOR NON-THERMAL NUCLEIC ACID
MANIPULATIONS; U.S. Pat. No. 6,391,622 to Knapp, et al. (May 21,
2002) CLOSED-LOOP BIOCHEMICAL ANALYZERS; U.S. Pat. No. 6,303,343 to
Kopf-Sill (Oct. 16, 2001) INEFFICIENT FAST PCR; U.S. Pat. No.
6,171,850 to Nagle, et al. (Jan. 9, 2001) INTEGRATED DEVICES AND
SYSTEMS FOR PERFORMING TEMPERATURE CONTROLLED REACTIONS AND
ANALYSES; U.S. Pat. No. 5,939,291 to Loewy, et al. (Aug. 17, 1999)
MICROFLUIDIC METHOD FOR NUCLEIC ACID AMPLIFICATION; U.S. Pat. No.
5,955,029 to Wilding, et al. (Sep. 21, 1999) MESOSCALE
POLYNUCLEOTIDE AMPLIFICATION DEVICE AND METHOD; U.S. Pat. No.
5,965,410 to Chow, et al. (Oct. 12, 1999) ELECTRICAL CURRENT FOR
CONTROLLING FLUID PARAMETERS IN MICROCHANNELS, and many others.
[0008] Despite the wide-spread use of amplification technologies
and the adaptation of these technologies to truly high throughput
systems, certain technical difficulties persist in amplifying and
detecting nucleic acids, particularly rare copy nucleic acids. This
is particularly true where the amplification reagents amplify a
high copy nucleic acid in a given sample in addition to the rare
nucleic acid and the two nucleic acids differ by only one or a few
nucleotides in the same sample. For example, if a set of primers
hybridizes to a high copy nucleic acid, as well as to a low copy
nucleic acid in a given sample, the geometric amplification of the
high copy nucleic acid proportionately dominates the amplification
reaction and it is difficult or impossible to identify the low copy
nucleic acid in any resulting population of amplified nucleic
acids. Thus, low copy number alleles of a gene can be very
difficult to detect, e.g., where a primer set cannot easily be
identified that only amplifies the rare nucleic acid (and the
practitioner will realize that perfect reagent specificity is rare
or non-existent in practice). Amplification of the higher copy
number nucleic acids in the sample swamps out any signal from the
low copy nucleic acid. In spite of such difficulties,
identification of rare copy nucleic acids can be critical to
identifying disease or infection in the early stages, as well as in
many other applications.
[0009] It is worth noting that these problems simply have not been
addressed by the prior art. While a few authors have described
single copy amplification as a theoretical exercise (e.g., Mullis
et al (1986) Cold Spring Harbor Symp. Quant. Biol. 51:263-273; Li
et al. (1988) Nature 335:414-417; Saiki et al (1988) Science
239:487-491, and Zhang et al (1992) Proc. Natl. Acad. Sci. USA
89:5847-5851), and others have described stochastic PCR
amplification of single DNA template molecules followed by CE
analysis of products in a microscale device (Lagally et al. (2001)
Anal. Chem. 73:565-570), none of these prior approaches are
suitable for detection of rare copy nucleic acids in samples. That
is, none of these approaches are suitable to high throughput
automation and the devices in the prior art cannot be adapted to
practicably detect rare copy nucleic acids. For example, the device
of Lagally et al., id., flowed sample to be amplified into
chambers, stopped flow of the system, ran the amplification
reaction, manually reconfigured the device to flow amplification
products out of the chambers, ran the amplification products out of
the chambers for one reaction at a time, and detected the product.
This cumbersome process results in few amplification reactions
being made and analyzed in any useful time period and required
almost continuous user intervention to make the system operate. In
addition, none of the prior methods unambiguously determine the
length of a nucleic acid of interest from a homogenous mixture,
e.g., without additional steps of size selective
chromatography.
[0010] Another difficulty with amplification methods that is
completely unaddressed in the prior art is that it can be quite
difficult to perform quantitative analysis on rare nucleic acids.
The problems noted above for detection apply to quantitative
analysis as well, with the additional problem that quantification
is impacted by the presence of high copy number nucleic acids in
the sample, even if the rare nucleic acid can be amplified. This is
because, even if the amplification is sufficiently specific for
detection of the rare nucleic acid, the high copy number of other
amplified nucleic acids still has competitive effects on the
amplification reaction for reaction components during the
amplification reaction. Thus, it is not generally possible to
assess accurately the concentration of rare nucleic acids in a
sample, particularly where the components of the system have not
previously been characterized or purified (it is, of course,
somewhat simpler to assess amplification products quantitatively if
the materials selected for amplification are already
characterized). While amplification of materials that have already
been fully characterized is of academic interest, this approach is
of little practical value if it cannot be adapted to
characterization of unknown materials. For example, the inability
to quantify rare nucleic acids limits, e.g., the ability to
diagnose disease, to establish disease prognosis and to perform
accurate statistical assessments of the nucleic acid of
interest.
[0011] Sizing of nucleic acids of interest is an area where
identification of rare sequences can be of particular interest. For
example, in disease states, such as certain cancers and conditions
caused by deletion mutations, the length of a nucleic acid of
interest in a complex mixture of other nucleic acids can be
diagnostic. In U.S. Pat. No. 6,586,177 to Shuber (Jul. 1, 2003)
METHODS FOR DISEASE DETECTION, clinical samples are amplified in a
multiwell format with two or more primer pairs followed by agarose
gel electrophoresis of the amplification reactions to visualize the
amplicons. Shuber suggests the assay can be useful to determine
proportions of degraded DNA from cells after apoptosis relative to
full length DNA from cancer cells in certain clinical samples.
However, this method can be nonspecific, slow and labor intensive,
fail to confirm separated amplicons were amplified from the same
nucleic acid strands (i.e., ambiguous), fail to distinguish between
long nucleic acids with marginally different lengths, fail to
determine length directly from a homogenous mixture, and fail to
detect nucleic acids of interest against a background of other
nucleic acids in many complex clinical samples.
[0012] In view of the above, a need exists for robust high
throughput methods of identifying and quantifying rare nucleic
acids of interest in a sample. It would be desirable to have
methods and systems that efficiently confirm the length, quantity
and proportions of nucleic acids of interest with high resolution
and accuracy. The present invention provides these and other
features that will be apparent upon review of the following.
SUMMARY OF THE INVENTION
[0013] The present invention relates to the surprising discovery
that single molecule amplification can be used for the detection
and statistical characterization of rare nucleic acids of interest
in a sample, e.g., for disease diagnosis (e.g., cancer diagnosis),
detection of pathogens, detection of rare environmental nucleic
acids, and the like. For example, many individual amplification
reactions can be performed on reaction mixtures derived from a
sample comprising a rare nucleic acid of interest, where each
reaction mixture has few (e.g., 1) or no copies of the rare a
nucleic acid of interest, e.g., until the nucleic acid of interest
is identified in a reaction mixture. Additional nucleic acids in
the sample can also be amplified in low copy number reactions and
statistical methods can be used to determine the relative ratio of
the nucleic acid of interest to the additional nucleic acid, e.g.,
to determine relative or absolute concentration of the nucleic acid
of interest including proportions of full length and fragmented
forms. Desirably, most or all of the steps in the methods herein
can be performed in a continuous flow format to greatly speed the
rate of the overall method. Alternately, one or more of the steps
can be performed in a stopped flow mode, e.g., where the detector
is configured to simultaneously scan multiple amplification regions
at once (simultaneous detection provides for increased throughput
in these embodiments).
[0014] High throughput amplification systems such as those embodied
in high throughput microfluidic systems are particularly well
adapted to performing these methods, which can be used to detect
nucleic acids of interest that are present at exceedingly low
concentrations in a sample to be analyzed, e.g., by performing many
low copy number amplification reactions until the nucleic acid of
interest is detected, and/or until enough copies of the nucleic
acid of interest are detected that reliable statistical evaluations
can be performed. In a related aspect, the invention also provides
new ways of determining whether and how many copies of an initial
nucleic acid are present in a reaction mixture (or whether the
initial nucleic acid is present in a reaction mixture) by
considering how far signal from the initial nucleic acid disperses
during amplification and comparing the dispersion to expected
dispersion arising from thermal diffusivity and/or Taylor Aris
dispersion, or related phenomena (or even simply by comparison of
the observed dispersion to empirically observed control reactions).
This can include monitoring the shape (amplitude, signal width,
and/or other signal shape features) of a signal generated from an
aliquot comprising the nucleic acid of interest to a predicted or
empirically observed signal shape. These shape features of the
signal are extremely reproducible, which provides an ability to
distinguish signals of interest from background random signal
fluctuations. Both the number of molecules in an aliquot and the
ability to distinguish signals of interest from background signal
fluctuations can be performed by this class of methods. Systems and
kits adapted to performing the various methods herein are also a
feature of the invention. The nucleic acids that are quantified can
be known (e.g., controls) or unknown in composition. They can
include experimental nucleic acids (the nucleic acids of primary
interest in the experiment at issue) or can be other unknown
nucleic acids (e.g., uncharacterized genomic and/or cDNA from a
biological sample of interest).
[0015] Accordingly, in a first aspect, methods of detecting a
nucleic acid of interest are provided. In the methods, a sample
comprising the nucleic acid of interest and one or more additional
nucleic acid is aliquotted into a plurality of reaction mixtures.
At least two of the reaction mixtures are single copy reaction
mixtures, each comprising a single copy of the nucleic acid of
interest. The plurality of reaction mixtures additionally comprise
at least one additional reaction mixture comprising at least one
copy of the additional nucleic acid. The plurality of reaction
mixtures are subjected to one or more amplification reaction (in
this context, the amplification reaction may or may not amplify the
nucleic acid of interest, i.e., if the reaction has zero copies of
the nucleic acid of interest, it will not be amplified; if it has
one or more copy it will). The nucleic acid of interest is detected
in one or more of the single copy reaction mixtures. Statistical
inferences and mathematical relationships can be determined based
on the plurality of results from such analyses. For example, the
absolute numbers and/or relative proportions of full length and
fragmented nucleic acids of interest in a complex sample can be
determined.
[0016] In a closely related aspect, the invention includes methods
of detecting a low copy nucleic acid of interest in a sample that
has one or more higher copy additional nucleic acid that is
different from the low copy nucleic acid. The method includes
aliquotting the sample into a plurality of reaction mixtures. The
mixtures can include a plurality (e.g., about 5, or more, about 10
or more, about 50 or more, about 100 or more, about 150 or more, or
about 500 or more) of zero copy reaction mixtures that include zero
copies of the nucleic acid of interest and at least one single copy
reaction mixture comprising a single copy of the nucleic acid of
interest. The zero and single copy reaction mixtures are subjected
to an amplification reaction (whether an amplification actually
occurs or not). The nucleic acid of interest is then detected in
the single copy reaction mixture (this includes the possibility
that the nucleic acid of interest is detected in one or in multiple
individual single copy reactions). The proportion of zero copy and
single copy reaction mixtures can be incorporated into mathematical
formulas, along with, e.g., sample dilution data, to determine the
concentration of the nucleic acid of interest in a sample, and/or
the proportions of the nucleic acid in full length or fragmented
forms.
[0017] In an additional related class of embodiments, related
methods of quantifying a nucleic acid of interest in a sample are
provided. In the methods, the sample can be aliquotted into at
least 25 reaction mixtures comprising 2 or fewer copies of the
nucleic acid of interest each (and generally 1 or fewer). The
reaction mixtures can be subjected to one or more amplification
reactions. The nucleic acid of interest is then detected in a
plurality of the reaction mixtures. In a number of embodiments,
statistical evaluations of the nucleic acid of interest are
performed based upon the detection of the nucleic acid of interest
in the plurality of reaction mixtures. In one class of embodiments,
at least 50 or more, at least 75 or more, or at least 100 or more
reaction mixtures, comprising the 2 or fewer copies, are subjected
to the one or more amplification reactions.
[0018] In an additional class of related embodiments, methods of
detecting a low copy nucleic acid of interest are provided. In the
methods, a sample comprising the low copy nucleic acid of interest
is aliquotted into a plurality of reaction mixtures. A plurality of
the reaction mixtures contain zero copies of the nucleic acid of
interest and at least one of the reaction mixtures comprises at
least one copy of the nucleic acid of interest. A plurality of the
plurality of zero copy reaction mixtures is subjected to one or
more amplification reaction in a microfluidic device comprising at
least one microchamber or microchannel. The nucleic acid of
interest is determined not to be present in the zero copy reaction
mixtures. At least one additional zero copy reaction mixture and
the reaction mixture comprising the nucleic acid of interest are
subjected to one or more amplification reaction. The nucleic acid
of interest is detected in the reaction mixture comprising the
nucleic acid of interest. Put another way, the reaction mixtures
are amplified and checked for the presence of the nucleic acid of
interest in the microfluidic device, at least until the nucleic
acid is detected. For a low copy number nucleic acid, this can
require a large number of amplification reactions be performed on
the zero copy reaction mixtures until the nucleic acid of interest
is found.
[0019] The invention also provides methods for quantifying a
nucleic acid of interest in a sample, e.g., by taking
diffusion/dispersion into consideration. In the methods, a sample
comprising a copy of the nucleic acid of interest, or a complement
thereof, is aliquotted into at least one reaction mixture. The
reaction mixture is subjected to at least one amplification
reaction, thereby amplifying the copy of the nucleic acid of
interest. A shape, volume, width, length, height, area, or the
like, in which the nucleic acid of interest, or a signal
corresponding thereto, is present is detected. The shape, volume,
width, height, length, or area is interpreted to indicate a number
of copies of the nucleic acid of interest in the reaction mixture
or sample, thereby quantifying the nucleic acid of interest in the
sample. Because these shape features of the signal are extremely
reproducible, it is straightforward to distinguish signals of
interest from background random signal fluctuations. In a related
aspect, knowledge of diffusion/dispersion and the reproducibility
of these phenomena can be used to reliably distinguish the signal
of a one or more target molecule(s) from random baseline system
fluctuations. In any case, this interpretation can be performed in
any of a variety of ways, e.g., by comparing the shape, volume,
width, height, length and/or other signal shape features to
predicted values taking thermal diffusivity and/or Taylor-Aris
dispersion into account and/or by back calculation from empirically
observed values for known reactions performed in the system. It is
worth noting that this method is particularly relevant to
continuous flow systems, where materials disperse during flow. In
quantitation or proportioning of fragmented and unfragmented forms
of a nucleic acid of interest, signals can be read in parallel from
homogenous mixtures, e.g., by using probes emitting signals at
different wavelengths. Quantitation of the two or more forms
(lengths) of the nucleic acid can separately be based on
interpretation of the same or different signal parameters.
[0020] In yet another class of embodiments, high throughput stopped
flow methods of detecting rare nucleic acids are provided. For
example, methods of detecting a nucleic acid of interest are
provided, in which a sample comprising the nucleic acid of interest
is aliquotted into a plurality of reaction mixtures. At least two
of the reaction mixtures are single copy reaction mixtures, each
comprising a single copy of the nucleic acid of interest. The
reaction mixtures are flowed throughout a network of microchannels
and subjected to one or more amplification reaction under stopped
flow conditions in the network of microchannels. The nucleic acid
of interest is detected in the single copy reaction mixtures under
the stopped flow conditions. Desirably, the detection step can
include detection of multiple reaction products simultaneously. For
example, a CCD array or appropriate image processor can be used to
scan an entire chip (or sub-regions thereof) for "clouds" of signal
from amplified products. That is, an entire channel or network of
channels can be scanned simultaneously (e.g., at two or more
frequencies to detect two or more probes) after amplification and
any or all regions where signal arising from amplification can be
detected simultaneously (or in more than one pass of the
scanner/detector, if desired). Where size detection employs two or
more probes with different signals, system software can compare
signal locations for coincident (typically indicating a nucleic
acid of interest has a given length) and non-coincident signals
(typically indicating a nucleic acid of interest is
fragmented).
[0021] It will be appreciated that the above methods overlap with
one another and that many of the above methods can be performed in
combination with one another. Similarly, any or all of the above
methods can be practiced in a continuous flow format to improve
throughput of the relevant method, and/or can use stopped flow in
combination with image analysis of multiple regions of (or an
entire) microchannel network.
[0022] For any or all of the methods herein, the reaction mixture
can comprise the nucleic acid of interest and one or a plurality of
additional nucleic acids (typically sample native nucleic acids not
of interest or control nucleic acids), with the relevant method
including detecting the nucleic acid of interest and/or the
plurality of additional nucleic acids in the reaction mixture. The
methods optionally include adding up the number of nucleic acids of
interest, or the plurality of additional nucleic acids, or both, in
the reaction mixture or the sample, or both. A ratio of the nucleic
acid of interest or the plurality of additional nucleic acids in
the reaction mixture to the sum of the nucleic acid of interest
and/or the plurality of additional nucleic acids in the reaction
mixture or sample can be determined. From this, a concentration or
proportion of the nucleic acid of interest in the reaction mixture
or sample can be determined. Similarly, the sum of the number of
nucleic acids of interest and the plurality of additional nucleic
acids can provide an indication of the total number of nucleic
acids in the reaction mixture.
[0023] For any or all of the methods herein, aliquotting the sample
or reaction mixture can comprise diluting the sample into a
plurality of reaction containers (e.g., wells in a microtiter
plate), and/or flowing the sample into a microfluidic dilution
channel or chamber. In microfluidic embodiments, the sample is
optionally diluted in the microfluidic dilution channel or chamber
(a form of dilution module in the systems of the invention),
whereby the sample is aliquotted into multiple diluted aliquots in
the microfluidic dilution channel or chamber. Optionally, the part
or all of the aliquotting/dilution process can be multiplexed for
high throughput, e.g., by flowing a plurality of samples into the
device or reaction containers simultaneously. Samples, aliquots,
reaction mixtures, etc., can be flowed under pressure (e.g., into
the microfluidic device) or via electroosmosis, or by any other
available method. For convenience in microfluidic embodiments, the
sample can be diluted from a common reaction component reservoir,
e.g., comprising some or all of the reaction and/or buffer
components for the amplification reactions (e.g., polymerase,
primers, locus specific reagents, labels, salts, magnesium, water
and/or the like). Alternately, one or more component can be located
in one or more additional reservoirs and the components can be
mixed prior to amplification. Desirably, any or all of these steps
can be practiced in a continuous flow format, or utilizing the
stopped flow/simultaneous image analysis methods noted herein.
[0024] The concentration of the nucleic acids of interest and/or
any additional nucleic acid is optionally low in the methods of the
invention, e.g., about 1 molecule per aliquot. For example, the
sample can be diluted to a concentration of about 1 molecule of
interest per nanoliter or less. Optionally, diluted aliquots are
each diluted to the same degree; however, diluted aliquots can also
be differentially diluted (e.g., to form a dilution series). The
volume of the aliquots can be quite low to keep reagent costs low,
e.g., in microfluidic applications. For example, the aliquots can
be less than about 100 nl in volume, e.g., less than about 10 nl in
volume, or, e.g., about 1 nl in volume or less.
[0025] In a number of embodiments, at least one of the reaction
mixtures is in an aqueous solution (the enzymes used in typical
amplification reactions typically function well in an aqueous
environment) dispersed as an emulsion. This can take the form of
individually resolved reaction mixture droplets in a microfluidic
device, fluid in reservoirs of a microtiter plate, or other forms
such as where at least one of the reaction mixtures is formulated
in an aqueous phase of an emulsion comprising aqueous droplets
suspended in an immiscible liquid (in this embodiment,
amplification can be performed on the reaction mixture when it is
formulated in the emulsion). In the emulsion embodiment, the
nucleic acid of interest is optionally present as a single copy in
at least one aqueous droplet of the aqueous phase prior to
performing the amplification reaction. The nucleic acid of interest
is detected in the emulsion after the amplification reaction is
performed. Optionally, a plurality of additional nucleic acids are
also formulated in the aqueous phase of the emulsion and the method
comprises detecting the plurality of additional nucleic acids. As
with other embodiments herein, statistical analysis can be
performed on, e.g., the ratio of the nucleic acid sizes nucleic
acids in the emulsion, e.g., to determine the concentration and/or
proportions of the nucleic acids of interest having a given length
in the emulsion.
[0026] In any of the methods herein, at least 10 of the reaction
mixtures are optionally low copy reaction mixtures (e.g.,
comprising 100 or fewer, usually 50 or fewer, typically 10 or
fewer, generally 2 or fewer and often 1 or fewer copies of the
nucleic acid of interest and/or of the additional nucleic acid).
Optionally, at least 25, at least 50, at least 100, at least 150,
at least 500 or more of the reaction mixtures are low copy reaction
mixtures. The low copy reaction mixtures can comprise at least 10,
at least 25, at least 50, at least 100, at least 150 at least 500
or more single or zero copy reaction mixtures comprising 1 or fewer
copies of the nucleic acid of interest. The reaction mixtures can,
and often do, comprise no copies of the nucleic acid of interest.
Thus, a plurality of the reaction mixtures can comprise a plurality
of zero copy reaction mixtures that comprise no copies of the
nucleic acid of interest. That is, at least about 10, 25, 50, 100,
150, 500, 1,000 or even 10,000 or more of the reaction mixtures can
be zero copy reaction mixtures that have no copies of the nucleic
acid of interest. In one aspect, the invention provides the ability
to rapidly search through many such zero copy reaction mixtures to
individually identify a full length or fragmented nucleic acid of
interest.
[0027] In several embodiments of the invention, the sample
comprises at least one additional nucleic acid that is different
than the nucleic acid of interest. The additional nucleic acid can,
and often does, exist at a higher copy number in the sample than
the nucleic acid of interest. The additional nucleic acid can be a
known nucleic acid (e.g., a control or hybridization blocking
nucleic acid) or can itself be unknown with respect to part or all
of the composition (a common occurrence where the nucleic acid of
interest is to be detected in a biological sample, e.g., a cell or
tissue sample from a patient). For example, the additional nucleic
acid can be present at a concentration at least about 100.times.,
at least about 1,000.times., at least about 10,000.times., at least
about 100,000.times., at least about 1,000,000.times. or greater as
high as the nucleic acid of interest in the sample (that is, can
have at least about 100.times., at least about 1,000.times., at
least about 10,000.times., at least about 100,000.times., at least
about 1,000,000.times. or greater as many copies as the nucleic
acid of interest in the sample). By screening sufficient numbers of
sample aliquots, the nucleic acid of interest can be detected
regardless of its relative concentration.
[0028] Optionally, the additional nucleic acid can be detected
independent of the nucleic acid of interest. A ratio of the nucleic
acid of interest to the additional nucleic acid can be determined,
e.g., for statistical analysis of the nucleic acid of interest
and/or the additional nucleic acid. The number of nucleic acids in
the reaction mixture (whether the nucleic acid(s) of interest, the
additional nucleic acids, or other nucleic acids) can be added up
and the concentration of the nucleic acids (or the relative
concentrations) can be determined in the sample, or in any of the
various aliquots and reaction mixtures herein. In some embodiments
the ratio(s) and/or quantities of fragmented nucleic acid of
interest, nucleic acid of a given length, and/or an additional
nucleic acid can be determined using methods of the invention.
[0029] The nucleic acid of interest can be essentially any
detectable nucleic acid. Examples include SNPs, low copy nucleic
acids, cancer associated nucleic acids, infective or pathogen
associated nucleic acids, forensic nucleic acids, and the like.
Because of the ability of the methods of the invention to identify
extremely low copy number nucleic acids, and/or distinguish nucleic
acids by size, the invention is suitably applied to early stage
disease diagnosis where cancer cells or pathogens are present at
low concentrations. For example, colon cancer cells can be present
in stool samples, but, at least in the early stages of colon
cancer, the concentration of cancer cell DNA is small compared to
the overall DNA in such a sample (typically much less than 1% of
the cells from which the DNA sample was derived). Typically,
nucleic acids from the cancer cells is relatively more full length
than nucleic acids from other (apoptotic) cells in the sample. The
present invention can be used to identify, proportion, and quantify
cancer DNA in such a sample, providing a new method for disease
diagnosis and prognostication. Similar approaches can be used to
identify cancerous DNAs or pathogen nucleic acids from any fluid or
tissue from which such samples are normally taken or derived, e.g.,
blood, urine, serum, plasma, saliva, tears, sputum, stool,
ejaculatory fluid, cervical swabs, vaginal secretions, or the like.
From these samples, infective/pathogenic agents such as viruses
(e.g., HIV, herpes virus, pox virus, etc.), parasites (e.g.,
malarial parasites (Plasmodium), nematodes, etc.), bacteria (e.g.,
pathogenic E. coli, salmonella, etc.) can be identified. Where the
pathogen is present at a relatively low concentration relative to
related non-pathogenic organisms (e.g., pathogenic E. coli are
present at an initially low concentration in the gut, as compared
to non-pathogenic E. coli), the methods are particularly suitable.
Methods can distinguish nucleic acids from living bacteria from
those of lysed bacteria, e.g., in a clinical sample.
[0030] Most typically, the methods of the invention utilize
thermocyclic amplification reactions, although non-thermocyclic
reactions (e.g., using denaturants in place of heat, a procedure
that is relatively practical in microscale applications) can also
be used. In one typical class of embodiments, the reaction mixtures
are subjected to one or more amplification reaction(s) by
thermocycling the reaction mixtures in one or more microscale
amplification chamber or channel. A variety of thermocycling
methods can be used in a microscale device (or in reaction
containers), e.g., heating by applying electrical current to fluid
of the reaction mixture (e.g., in the microscale amplification
chambers or channels), resistively heating a heating element that
contacts or is in proximity to the reaction mixture (e.g., in the
microscale amplification chambers or channels), heating with a
Joule-Thompson or Peltier device, or any other available heating or
heating and cooling method(s).
[0031] Optionally, the components of the system can be treated with
one or more reagents between operational runs to reduce cross
contamination between operations. For example, the amplification
channel can have acid or base flowed into the channel between
amplification reactions to reduce unwanted contamination from one
or more previous amplification products.
[0032] In a convenient class of embodiments, detecting can include
real time homogenous PCR detection, e.g., via use of TaqMan.TM.
probes (operating by detecting a double-labeled probe before,
during, or after polymerase-mediated digestion of the double
labeled probe), use of molecular beacons, or the like. Real time
detection can be omitted, e.g., simply by detecting amplicons via
labeled probes, e.g., after separation of the amplicon from
unlabeled probe.
[0033] Optionally, the detecting step(s) can include quantifying
the nucleic acids of interest in the reaction mixtures, or the
sample, or both. Alternately, the nucleic acids can be quantified
separate from the detection step. In either case, quantifying the
nucleic acid of interest optionally comprises detecting the nucleic
acid in a plurality of single-copy reaction mixtures and performing
statistical or probabilistic analysis to determine a percentage or
distribution of reaction mixtures comprising a single copy of the
nucleic acid of interest. The statistical or probabilistic analysis
can comprise any available technique or combination thereof, e.g.,
Poisson analysis, Monte Carlo analysis, application of a genetic
algorithm, neural network training, Markov modeling, hidden Markov
modeling, multidimensional scaling, partial least squares (PLS)
analysis, or principle component analysis (PCA).
[0034] In many of the methods, the initial starting concentration
of a nucleic acid of interest (e.g., full and/or fragmented forms)
can be determined, e.g., by detecting a reproducible shape, length,
width, height, volume or area of associated signal(s) for the
nucleic acid of interest in a given reaction mixture. For example,
the signal can be detected from a label bound to the nucleic acid
of interest. The shape, length, width, height, volume or area is
optionally correlated to a number of nucleic acids interest present
in one of the reaction mixtures, and/or present in the sample based
upon a Taylor-Aris dispersion calculation, or a thermal diffusivity
calculation, or both, or by comparison to an empirically observed
set of reaction mixtures having a known number of starting nucleic
acids for amplification. Thus, in one aspect, the invention
comprises calculating diffusion, or dispersion, or both, of one or
more amplified nucleic acids in the given reaction mixture, and
correlating the diffusion, or the dispersion, or both, to a number
of copies of the nucleic acid of interest in one of the given
reaction mixtures prior to amplification.
[0035] Systems and/or kits adapted for practicing the methods
herein are a feature of the invention. The systems and/or kits can
include system instructions (e.g., embodied in a computer or in a
computer readable medium, e.g., as system software) for practicing
any of the method steps herein. Fluid handling elements for
storing, transferring, aliquotting, or diluting samples, e.g.,
microfluidic handling elements, and detector elements can also be
components of the systems and kits herein. In addition, packaging
materials, integration elements (e.g., instrument cases, power
supplies, etc.), instructions for using the systems and kits and
the like can be features of the invention.
[0036] In one embodiment, the invention provides a system for
detecting low copy nucleic acids of interest in a sample. The
system includes a dilution module that dilutes the sample into
multiple aliquots and a microfluidic device comprising an
amplification channel or chamber configured to thermocycle one or
more of the multiple aliquots. A detector integral with or proximal
to the microfluidic device is also included, where the detector is
configured to detect one or more amplified copies of the nucleic
acid of interest in or on the microfluidic device. System
instructions that direct the dilution module to aliquot the sample
into a plurality of aliquots, including a plurality of zero copy
aliquots comprising no copies of the nucleic acids of interest and
one or more single copy aliquot comprising a single copy of the
nucleic acid of interest are also included. Typically, the system
also includes system software that correlates a reproducible signal
shape, length, width, volume or area occupied by amplified copies
of the nucleic acid of interest, as detected by the detector, to
the number of copies of the nucleic acid of interest present in one
of the aliquots, or to the number of copies of the nucleic acid of
interest present in the sample, or both. The system can typically
evaluate the absolute or relative number of nucleic acids of
interest having different lengths to provide concentrations and/or
proportions of the nucleic acids. Any or all of the system
components can be selected to operate such that a sample of
interest is continuously flowed during operation of the system.
Alternately, the stopped flow/simultaneous image analysis methods
noted herein can be applied.
[0037] In a related embodiment, systems for quantifying one or more
low copy nucleic acid of interest in a sample are provided. In the
systems, a dilution module dilutes the sample into multiple
aliquots. A microfluidic device comprising an amplification channel
or chamber is configured to thermocycle one or more of the multiple
aliquots. A detector integral with or proximal to the microfluidic
device is configured to detect a reproducible shape, length, width,
volume or area occupied by signals from amplified copies of the
nucleic acid of interest (often hybridized to a detectable probe or
represented by released but previously hybridized probe) present in
one of the aliquots following thermocycling of the reaction mixture
aliquots. The system can also include system software that
correlates the shape, length, width, volume or area occupied by
amplified copies of the nucleic acid of interest to the number of
copies of the nucleic acid of interest present in one of the
aliquots, or to the number of copies of the nucleic acid of
interest present in the sample, proportion of nucleic acids with
different lengths, etc. Optionally, the system includes system
instructions that direct the dilution module to aliquot the sample
into a plurality of aliquots, including a plurality of zero copy
aliquots comprising no copies of the nucleic acids of interest and
one or more single copy aliquot comprising a single copy of the
nucleic acid of interest.
[0038] For many of the above system embodiments, the dilution
module can optionally be integral with the microfluidic device,
e.g., as a dilution channel. The microfluidic device can also
include one or more electrodes positioned to flow electrical
current into the microchamber or channel. Flow of current into the
microchamber or channel can be used to heat fluid in the
microchamber or channel. The microfluidic device optionally
includes or is coupled to one or more heating element (e.g., a
resistive heating element, a Peltier device or a Joule Thompson
device) positioned within or proximal to the microchamber or
channel, which heats fluid in the microchamber or channel.
[0039] The detector is typically configured to detect one or more
electromagnetic energy signal in or on the microfluidic device,
although other in device sensors (e.g., pH, conductivity, etc.) can
also be used. For example, the detector can detect fluorescence,
luminescence, and/or fluorescence polarization of the sample.
Optionally, in some embodiments, the detector can be an off-device
instrument, such as, e.g., size selective chromatography
instrumentation or a mass spectrometer.
[0040] The system optionally comprises software with instructions
for performing any of the method steps herein. For example, the
system can include statistical or probabilistic system software
that performs one or more statistical or probabilistic analysis of
signals received from one or more of the aliquots subjected to
thermocycling. For example, the statistical or probabilistic
analysis can include Poisson analysis, Monte Carlo analysis,
application of a genetic algorithm, neural network training, Markov
modeling, hidden Markov modeling, multidimensional scaling, PLS
analysis, and/or PCA analysis. The statistical or probabilistic
analysis optionally comprises quantitatively determining a
concentration, proportion, or number of the nucleic acids of
interest in the sample.
[0041] The systems above also optionally include fluid handling or
storage features such as sample storage modules that store the
samples until they are to be diluted by the dilution module, a
sample retrieval module that retrieves the sample from the sample
storage module and delivers it to the dilution module, or the like.
These features are optionally designed to provide for continuous
flow of fluid (e.g., comprising the sample) through the system
(thereby providing for higher sample throughput). Alternately, or
in combination, stopped flow/simultaneous image analysis can be
used in the systems herein.
[0042] Important aspects of the present invention are methods and
systems to determine whether a nucleic acid of interest is at least
a given length based on the presence, or absence of signals from
low or single copy reactions mixtures. The reaction mixtures for
such determinations typically contain two or more probes
complimentary to sequences at positions spaced along one or more
strands of the nucleic acid of interest. Coincident detection of
two or more probes in the reaction mixture can indicate that
individual nucleic acid molecules are not fragmented between probe
hybridization sites. Systems useful in determining length by the
two probe single copy reaction mixture techniques can include
dilution modules and microfluidic devices to prepare and detect the
reaction mixtures, and computers to interpret and correlate signal
data acquired from detectors.
[0043] Methods of determining whether a nucleic acid of interest in
a sample comprises at least a given length can include contacting
the nucleic acid of interest in a reaction mixture with two or more
different probes having detectable markers, and flowing the nucleic
acid into a detection region to detect one or more signals from the
probes. Coincident detection of two or more signals from different
probes can indicate the nucleic acid of interest is not fragmented
between the probes. Detection of a single signal can indicate the
nucleic acid is fragmented. Such determinations can be considered
assays of integrity for a nucleic acid of interest in a sample.
Samples for nucleic acid length determinations and differentiations
include, e.g., whole blood, serum, plasma, stool, urine, vaginal
secretions, ejaculatory fluid, synovial fluid, a biopsy,
cerebrospinal fluid, amniotic fluid, sputum, saliva, lymph, tears,
sweat, and urine.
[0044] In a preferred embodiment of differentiating lengths of
nucleic acids of interest in a sample, an amplification reaction
can be used to enhance the sensitivity of the assay. The nucleic
acid of interest is contacted with a first primer pair and a second
primer pair having at least one primer that is outside of the
sequence defined by the first primer pair, the nucleic acid of
interest is amplified in a reaction mixture in a microchannel or
microchamber with polymerase extensions from the primers to produce
first amplicons defined by the first primer pair or second
amplicons defined by the second primer pair. First and second
probes complimentary to the first and second amplicon and having
detectable markers are introduced into the reaction mixture to
hybridize with available complimentary sequences, and one or more
signals are detected from the probes. Detection of a signal from
only one of the probes indicates a fragmented nucleic acid of
interest and detecting signals from both probes indicates a nucleic
acid that is not fragmented. In preferred embodiments of this
sensitive assay, the reaction mixture detected contains only a
single copy of the nucleic acid of interest. Reaction mixtures
detected in the methods can be homogenous mixtures, e.g., not
requiring separation of labeled constituents before detection of
signals.
[0045] In many embodiments, the concentration of the nucleic acid
in samples is adjusted so that desired numbers of low, single, and
zero copy reactions can be independently detected. The adjustment
can be a concentration, e.g., by immunoprecipitation, capture to a
solid support, or ultrafiltration. The adjustment can be a
dilution, e.g., by serial dilution or fluidic mixing. In one
embodiment, the nucleic acid of interest is diluted or concentrated
to provide a concentration of about 1 molecule per nanoliter or
less in a reaction mixture.
[0046] In many embodiments, detection results from multiple low
copy, single copy, and zero copy reactions are compiled to obtain
confirmatory data and to allow statistical inferences with a
suitable level of confidence. For example, it is an aspect of the
invention that for quantitative results it is preferred to aliquot
a sample into at least 25 reaction mixtures comprising 2 or fewer
copies of the nucleic acid of interest each for hybridization with
probes and counting the number of aliquots resulting in detection
of a signal from one probe and/or signals from two or more probes.
It is preferred that the aliquotting (concentration, dilution,
and/or segregation into a small volume) result in one or more
reaction mixtures having single copies or zero copies of the
nucleic acid of interest; particularly for quantitation or
proportion analyses. From the compiled data, the number of one
marker signals and two marker signals can be evaluated to determine
a proportion of nucleic acids of interest having different lengths.
Thresholds can be established for confident correlation of some
such proportions with certain disease states.
[0047] In many embodiments, it is desirable to amplify relatively
short sections of the nucleic acid of interest, e.g., so that
random breakage is less likely to fragment the nucleic acid between
the primers or so the amplicons can act as consistent control
materials. In one aspect, one primer pair in an amplification acts
as a control for amplification and/or hybridization efficiency. It
is preferred that these primer pairs define amplicons of about 100
base pairs in length. Primer pairs for probe target amplicons are
preferably about the same length for each probe and can range from
more than about 1000 base pairs to about 20 base pairs, or from
about 200 base pairs to about 50 base pairs, or about 100 base
pairs. In certain embodiments, described herein, e.g., where the
amplicons include shorter amplicons and larger amplicons that
overlap the shorter amplicons, the larger amplicons generally range
in length from about 5000 base pairs to about 200 base pairs, or
about 1000 base pairs. In most examples of amplified
determinations, at least one of the probes is complimentary to the
amplicon sequence defined by one primer pair but not complimentary
to the amplicon sequence defined by another primer pair.
[0048] Amplifications in the methods of determining length are
generally provided by constituting amplification reactions
containing a polymerizing enzyme to increase the amount of target
(e.g., nucleic acid of interest) sequence, increase the amount of
hybridized probe, or increase the signal from such probes.
Amplifying a nucleic acid in the methods typically involves
incorporation of a polymerase into the amplifying reaction, such as
a heat stable DNA polymerase for a polymerase chain reaction (PCR),
a reverse-transcriptase for RT-PCR, ligase for a ligase chain
reaction (LCR), a Q-.beta. replicase, or enzymes for
RNA/transcription mediated techniques.
[0049] Probes used in the length determinations can have different
specificity for sequences along the length of the nucleic acid of
interest. The detectable markers on probes with different
specificity can have the same signal or, preferably, probes
hybridizing to different compliments on the nucleic acid or
associated amplicons have detectably different signals. The probes
can have any suitable detectable markers, but preferred markers are
based on fluorescent dyes. In particular, probes are favored that
include a fluorescent resonant energy transfer (FRET) detectable
marker or a molecular beacon (MB) marker.
[0050] Methods of determining given lengths for nucleic acids of
interest can quantify the nucleic acid and its fragmentation state.
Such quantitation can simply involve counting the number of signals
from probes in low or single copy reactions and calculating an
amount of the nucleic acid based on known dilution factors,
efficiency factors, standard curves, and the like. The quantifying
can separately determine the amount of various fragmentation forms
of the nucleic acid of interest based on the number of signals from
two or more different probes, e.g., having different detectable
marker signals. Signal parameters, such as shape, volume, width,
height, length, area, or ratio, of the one or more signal (e.g.,
chart peaks) can be interpreted to confirm an actual signal (i.e.,
that the signal is not an artifact) and/or to indicate a certain
quantity of a nucleic acid in a sample. Quantification can be based
on comparison of signal peak parameters to an internal standard
signal. Optionally, quantitation can be based on comparison of
signals from two or more reaction mixtures comprising different
degrees of amplification to standard reaction mixtures with similar
degrees of amplification. In this embodiment, different degrees of
amplification can be obtained by flowing reaction mixtures through
a thermocycler at different flow rates, flowing reaction mixtures
different distances into a thermocycler, retaining reaction
mixtures in a thermocycler for different amounts of time, or
exposing reaction mixtures to different numbers of amplification
cycles.
[0051] Samples containing unknown amounts of a nucleic acid of
interest can be quantified by comparing to their signal peaks to
sets of standard signal peaks. For example, a nucleic acid of
interest in a sample can be quantified by amplifying a dilution
series of standard materials containing known amounts of the
nucleic acid of interest through a certain number of amplification
cycles, detecting signals associated with standard amplicons
produced from the standard materials, amplifying the sample nucleic
acid of interest the number of amplification cycles, detecting a
signal associated with sample amplicons produced from the sample
nucleic acid of interest, and comparing one or more standard
amplicon signals to the sample amplicon signal to determine a
concentration value for the nucleic acid of interest in the sample.
Sample and standard signal parameters for comparison can include,
e.g., the shape of their signal peaks, points of inflection on the
signal peaks, slopes of the signal peaks, signal peak amplitudes,
signal peak areas, signal peak widths at half height, and/or the
like. The reliability of results can be enhanced through various
schemes of repeated testing. For example, the amplifying,
detecting, and comparing steps can be repeated one or more times,
with different numbers of amplification cycles, to determine
additional concentration values for the sample nucleic acid of
interest for statistical evaluation providing more precise or more
accurate concentration value results for the nucleic acid of
interest in the sample.
[0052] Improved assay results can be obtained by gathering signal
data after amplifications through two or more different numbers of
cycles. A major benefit of running the quantitative assay at
different amplifications is to broaden the usable range of the
assay. Typically, statistical evaluation of the additional data
provided by analysis at multiple amplification levels can enhance
other assay parameters, such as precision, accuracy, and
sensitivity. Quantifying a nucleic acid of interest in a sample
based on detection of multiple amplifications can include:
amplifying the nucleic acid of interest through more than one
number of amplification cycles, detecting signals associated with
amplicons produced from two or more of the amplification cycle
numbers, preparing a sample curve of a signal parameter versus
number of amplification cycles, and comparing one or more
identifiable points from the sample curve to a standard curve of
the identifiable points versus concentration to quantify the
nucleic acid of interest. Exemplary identifiable points from signal
curves include points of inflection, points having a certain slope,
points having a certain signal amplitude, points having a certain
fraction of a maximum signal amplitude, and/or the like.
[0053] Such quantitative assays, relying on identifiable points
from signal versus cycle curves, can be used to quantitate or
proportion fragmented and unfragmented nucleic acid of interest in
evaluations of integrity. Proportions of fragmented and given
length nucleic acid of interest can be determined in a sample by:
amplifying the nucleic acid of interest through a plurality of
amplification cycles in a reaction mixture defining two or more
different amplicons of the nucleic acid of interest; detecting,
from homogenous reaction mixtures, different signals associated
with each of the different amplicons after at least two different
numbers of amplification cycles; preparing sample curves for each
of the different signals versus numbers of amplification cycles;
and, comparing one or more identifiable points from the sample
curves to one or more standard curves describing nucleic acid of
interest concentrations versus identifiable points. Each of the
amplicons can be relatively or absolutely quantitated to determine
the amount of nucleic acid of interest sequences in the sample. In
preferred embodiments, the amplification reaction mixtures detected
are low copy or single copy reaction mixtures, thus allowing
unambiguous determinations of fragmented and given length nucleic
acid. That is, coincident detection of two or more or the different
signals from low or single copy mixtures can indicate a nucleic
acid of a given length, or the detection of a one of the different
signals can indicate a fragmented nucleic acid. As discussed
elsewhere herein, the number of amplification cycles experienced by
samples and standards can be controlled, e.g., by flowing the
amplification reactions through a thermocycler at different flow
rates, flowing the amplification reactions different distances into
a thermocycler, retaining the amplification reactions in a
thermocycler for different amounts of time, or exposing the
amplification reactions to different numbers of amplification
cycles.
[0054] Systems for differentiating the lengths of nucleic acids of
interest in a sample can be used to practice many of the methods
described herein. The systems can basically include a microfluidic
device with an amplification microchannel or microchamber
containing one or more reaction mixtures under conditions that
provide one or more amplicons of the nucleic acid of interest, a
detector integral with or proximal to the microfluidic device and
configured to detect the amplicons as one or more signals from a
homogenous mixture, and a software system that interprets one or
more coincidently detected signals to lengths of one or more
individual nucleic acid molecules of interest to differentiate
lengths of the nucleic acids of interest. High throughput aspects
of the system can be advanced by provision of multiple
amplification channels in the microfluidic device. The system can
include affinity molecules, such as oligonucleotides, on a solid
support to capture nucleotides of interest before or during
preparation of the reaction mixture, or to capture amplicons for
detection. Other system elements, e.g., to enhance high throughput
aspects of the invention include sample storage modules, sample
retrieval modules, and computers.
[0055] The systems of the invention can incorporate a dilution
module to adjust the concentration of reaction mixture
constituents. The dilution module can be configured to dilute the
sample to a concentration providing one or more single copy
reaction mixtures for nucleic acids of interest in the
amplification microchannel or microchamber. Such a dilution module
can be equipment to prepare serial multiwell plate dilutions, or a
dilution channel in the microfluidic device. The system can include
instructions that direct the dilution module to aliquot the sample
or reaction mixture into a plurality of aliquots, including a
plurality of zero copy aliquots comprising no copies of the nucleic
acid of interest and one or more single copy aliquots comprising a
single copy of the nucleic acid of interest. Such dilutions (or
concentrations) can provide substantial numbers of non-overlapping
reaction mixture aliquots for discrete counting of signals.
[0056] Reaction mixtures of the systems can include constituents
associated with amplification of the nucleic acid, hybridization
reactions of primers or probes, and/or detection of detectable
marker signals. A typical reaction mixture for detection of length
can include the nucleic acid of interest, a first primer pair, a
second primer pair with at least one primer complimentary to a
sequence of the nucleic acid of interest outside a sequence defined
by the first primer pair, and a polymerase that can synthesize
amplicons defined by the primer pairs. In some embodiments, a
control primer pair defines amplicons of 100 base pairs or less in
length while a test primer pair defines longer amplicons ranging in
length from about 100 base pairs to about 3000 base pairs. In this
embodiment, the control amplicons usually overlap sequences of the
longer amplicons with probe signals for longer and control probes
indicating the proportion of the nucleic acid that is fragmented.
In other embodiments, the two sets of primer pairs define amplicons
of about the same length but the amplicons do not overlap. In this
embodiment, coincident signals from a low or single copy reaction
can indicate the nucleic acid of interest is of a given length (the
length defined by the probes and the distance between them).
[0057] Amplification reaction mixtures and/or hybridization
mixtures can include one or more probes to determine the length of
a nucleic acid of interest. The probes can have one or more
detectable markers and a sequence complimentary to one or more of
the amplicons so that the detectable markers provide a signal
detectable by the detector. The probes can be complimentary to an
amplicon sequence defined by one primer pair but not complimentary
to an amplicon sequence defined by another primer pair, while, in
some methods described above, the probes can be complimentary to a
sequence common to both a first amplicon and a second amplicon. The
presence of two different probes with the same marker in a single
copy reaction can be inferred, e.g., from the amplitude of a signal
received, However, in many embodiments, two or more different
probes each comprise different signals for easy independent
monitoring of coincident signals. For example detectable markers on
different probes can have different fluorescent emission
wavelengths. The probes can be, e.g., fluorescent resonant energy
transfer (FRET) detectable marker or a molecular beacon (MB)
marker. In a particularly preferred embodiment, the probe has a
detectable marker includes a quencher removable from the FRET probe
by nuclease activity, so that one or more positive signals can be
detected from a homogenous mixture against low levels of background
noise.
[0058] Systems to determine length can include amplification
channels or chambers that provide conditions for amplification of a
reaction constituent. In preferred systems, the chambers are
thermocyclers and the nucleic acid of interest is amplified by a
polymerase reaction. The amplification microchannel or microchamber
can include, e.g., electrodes to apply a heating current to the
microchannel, a resistive heating element, a Joule-Thompson device,
a Peltier device, and/or the like. The amplification microchannel
or microchamber can be configured to thermocycle the reaction
mixture producing amplicons of the nucleic acid of interest in a
volume sufficiently small to substantially separate amplification
products of a single nucleic acid of interest molecule from other
nucleic acid of interest molecules in the sample or from additional
nucleic acids in the sample. In the systems of the invention the
amplicons can be detected without resolution of different amplicons
or different probes, e.g., in a size selective media or affinity
media.
[0059] Software systems can work in computers to enhance the high
throughput aspects of the methods of determining length and
automate interpretation of detected signals. For example, the
system software can interpret signal volumes, widths, heights,
lengths, areas, and/or ratios, from the detector to indicate a
number of copies of the nucleic acid of interest in the sample, a
number of the nucleic acids of interest having a given length, or a
proportion of nucleic acids of interest having different
lengths.
[0060] Detectors in the systems can detect signals from any
suitable detectable marker. Detectors can include technologies,
such as, e.g., fluorometers, charge coupled device, lasers, enzymes
or chromogenic enzyme substrates, photo multiplier tubes,
spectrophotometers, scanning detectors, microscopes,
galvo-scanners, and/or the like. In preferred embodiments the
detector can independently detect signals from two or more
detectable markers with different signals; e.g., a fluorometer
detector that can simultaneously detect emissions at two or more
frequencies.
[0061] Many of the above methods or systems can be used in
combination. Additional features of the invention will become
apparent upon review of the following.
BRIEF DESCRIPTION OF THE FIGURES
[0062] FIG. 1 schematically illustrates a chip design for an
8-channel PCR sipper chip used in many of the Examples herein.
[0063] FIG. 2 is a graph of percent amplification versus input copy
number for 2 experimental runs, with a comparison to a predicted
(Poisson) value.
[0064] FIG. 3, Panels A and B provide peak area and peak width bar
graphs.
[0065] FIG. 4, Panels A-D are graphs illustrating peak width for
amplification reactions.
[0066] FIG. 5 is a graphical analysis of single molecule
amplification peak widths.
[0067] FIG. 6 is a schematic representation of a system of the
invention.
[0068] FIG. 7 is a schematic representation of a system of the
invention.
[0069] FIG. 8 is a schematic representation of a stopped flow
system that uses simultaneous image processing of a network of
channels to scan for nucleic acids of interest.
[0070] FIG. 9 is a schematic of a fluidic network after
thermocycling. Spots represent the fluorescence "clouds" from
single copy amplification reactions. The spots are counted for
quantitative PCR analysis.
[0071] FIG. 10 is a data graph showing single molecule DNA
amplification.
[0072] FIG. 11 is a data graph showing single molecule DNA
amplification (6 passes).
[0073] FIG. 12 is a data graph showing single molecule DNA
amplification (3 panels).
[0074] FIG. 13 provides a graph of detection of 2 mutation sites
relevant to cancer detection developed on-chip using TaqMan
probes.
[0075] FIG. 14 shows a schematic diagram of low to single copy
detection of nucleic acids of interest amplified using two amplicon
sequences that do not overlap.
[0076] FIG. 15 shows a schematic diagram of low to single copy
detection of nucleic acid of interest amplified using two amplicon
sequences that overlap at a sequence complimentary to at least one
probe.
[0077] FIGS. 16A to 16B show schematic diagrams of amplification
curves generated by flowing amplification reactions different
distances within an actively cycling amplification region.
[0078] FIG. 17 shows detected signal peaks for a series of nucleic
acid standard materials with amplification cycles numbering 25
cycles or 40 cycles.
[0079] FIGS. 18A to 18C show schematic charts demonstrating the
quantitation of a nucleic acid of interest based on the number of
amplification cycles required to reach an identifiable point of
maximum slope.
[0080] FIG. 19 shows a schematic diagram of a system of the
invention for differentiating the length of nucleic acids of
interest.
DETAILED DESCRIPTION
[0081] The present invention derives, in part, from a surprising
conceptual shift in considering how rare nucleic acids can be
amplified and detected in or from a sample. In the past, detection
of rare nucleic acids was performed by trying to find ways of
improving the specificity and sensitivity of amplification and
detection reactions. This is because the better the reaction can
specifically amplify and identify a nucleic acid of interest, the
better the reliability and throughput of the system. Considering a
simple analogy, when trying to find a needle in a haystack, prior
art thinking focuses on more efficient ways of extracting the
needle from the haystack.
[0082] The present invention takes an entirely different approach
to identifying nucleic acids of interest. Instead of trying to fish
the nucleic acid of interest out of a complex sample directly, the
entire sample is simply deconstructed into low copy number aliquots
and the low copy number aliquots are subjected to amplification
reactions and individual detection until the nucleic acid of
interest is found. Continuing with the simple analogy, the entire
haystack is broken apart into individual pieces of hay and each is
examined to see if it is hay or needle. This low or single copy
amplification concept can provide analyses with high sensitivity
against a very low background.
[0083] Such low or single copy amplifications can be especially
useful in the present invention for evaluation of a nucleic acid
length. For example, hybridization of a dot blotted sample with a
pair probes specific to opposite ends of a target nucleic acid can
yield ambiguous results on the integrity of the target. Whether or
not the target nucleic acid is fragmented, signals from both probes
will be detected on the same blot. However, should the target be
subjected to single copy amplification and hybridization with the
probes in an isolated reaction mixture, detection of signals from
both probes would indicate target not fragmented between sequences
complimentary to the probes. On the other hand, amplification and
hybridization of a single copy target nucleic acid fragment would
result in detection of a signal from only one of the probes.
Therefore, coincident signals from two probes can indicate a
full-length target and detection of a signal from only one of the
probes can indicate the presence of a fragmented target in a single
copy reaction.
[0084] Modem high-throughput systems make this new conceptual
approach possible, i.e., the ability to run massively high numbers
of amplification reactions at low cost, e.g., using microfluidic
amplification technologies, makes it possible to much more
exhaustively sample for any particular nucleic acid of interest in
a sample. The continuous flow or high throughput stopped flow
nature of these systems further facilitates the approach.
Furthermore, examination of a sample by such exhaustive sampling
methods provides a great deal of quantitative information (and the
concomitant possibility of statistical analysis) with respect to
the composition of the sample and the proportions of fragmented or
unfragmented nucleic acid of interest. This, in turn, provides
diagnostic and prognostic information associated with to the
abundance (or relative abundance) of the nucleic acids of
interest.
[0085] Definitions
[0086] It is to be understood that this invention is not limited to
particular devices or biological systems, or amplification methods,
which can, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting. As used in
this specification and the appended claims, the singular forms "a",
"an" and "the" optionally include plural referents unless the
content clearly dictates otherwise. Thus, for example, reference to
"a microfluidic device" optionally includes a combination of one,
two or more devices.
[0087] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0088] An "aliquot" is a portion of a component of interest (e.g.,
a sample or reaction mixture). The aliquot can be diluted,
concentrated or undiluted as compared to the component of
interest.
[0089] A "nucleic acid of interest" is any nucleic acid to be
amplified, detected and/or quantified in a sample. A nucleic acid
of interest can be detected and identified in fragmented form
and/or in unfragmented form using methods and systems of the
invention.
[0090] An "amplification reaction" is a reaction that 1) results in
amplification of a template, or 2) would result in amplification of
a template if the template were present. Thus, an "amplification
reaction" can be performed on a sample aliquot that comprises a
nucleic acid to be amplified, or on a sample aliquot that does not
comprise the nucleic acid. Actual amplification of a template is
not a requirement for performing an amplification reaction.
[0091] As used herein, a "reaction mixture" refers to a mixture of
constituents of an amplification reaction and/or a hybridization
reaction. An aliquot of a reaction mixture containing a nucleic
acid of interest, or not, can still be considered a reaction
mixture. A single copy reaction mixture includes constituents of a
reaction mixture in a volume where a nucleic acid of interest, and
any associated amplicons, do not overlap with another nucleic acid
of interest or its associated amplicons.
[0092] A "zero copy" reaction mixture or aliquot is a reaction
mixture or aliquot that has no copies of the relevant nucleic acid
(e.g., a nucleic acid of interest, or an additional nucleic acid).
It can comprise nucleic acids from a sample other than the relevant
nucleic acid(s), or it can be completely devoid of any template
nucleic acids from the sample.
[0093] A "single copy" reaction mixture has 1 copy of the relevant
nucleic acid. The reaction mixture can be an amplification reaction
mixture or hybridization mixture containing, e.g., a single copy of
a nucleic acid of a given length, or fragment thereof.
[0094] A "low copy" reaction mixture or aliquot is a reaction
mixture or aliquot that has only a few copies of the relevant
nucleic acid(s). Typically, such a reaction will have 50 or fewer,
generally 25 or fewer, usually 10 or fewer and often 5 or fewer, 2
or fewer or 1 or fewer copies of the relevant nucleic acid(s).
[0095] A "high copy" nucleic acid reaction mixture or aliquot has
at least 1 order of magnitude more copies than the low copy number
reaction mixture or aliquot, and generally 2, 3, 4, or even 5 or
more orders of magnitude more than the low copy number reaction
mixture.
[0096] A nucleic acid is "quantified" or "quantitated" in a sample
when an absolute or relative amount of the nucleic acid in a sample
is determined. This can be expressed as a number of copies, a
concentration of the nucleic acid, a ratio or proportion of the
nucleic acid to some other constituent of the sample (e.g., another
nucleic acid), or any other appropriate expression.
[0097] A "given length" of a nucleic acid of interest, as used
herein, refers to a distance between two probes hybridized to the
nucleic acid plus the sequences complimentary to the probes. The
given length can be a known distance, measured, e.g., in units of
base pairs, or an unknown distance determined to exist as an
unfragmented sequence, e.g., by detection of coincident signals
from a low or singly copy reaction mixture.
[0098] As used herein, the term "different probes" refers to probes
complimentary to or specifically hybridizing to different target
sequences under stringent hybridization conditions.
[0099] As used herein, the term "different detectable markers"
refers to detectable markers that provide signals distinguishable
by a detector in the invention.
[0100] Nucleic Acids and Samples of Interest
[0101] The nucleic acid of interest to be detected in the methods
of the invention can be essentially any nucleic acid. The sequences
for many nucleic acids and amino acids (from which nucleic acid
sequences can be derived via reverse translation) are available. No
attempt is made to identify the hundreds of thousands of known
nucleic acids, any of which can be detected in the methods of the
invention. Common sequence repositories for known nucleic acids
include GenBank EMBL, DDBJ and the NCBI. Other repositories can
easily be identified by searching the internet. The nucleic acid
can be an RNA (e.g., where amplification includes RT-PCR or LCR) or
DNA (e.g., where amplification includes PCR or LCR), or an analogue
thereof (e.g., for detection of synthetic nucleic acids or
analogues thereof). Any variation in a nucleic acid can be
detected, e.g., a mutation, a single nucleotide polymorphism (SNP),
an allele, an isotype, a fragment, a full-length nucleic acid, an
amplicon, etc. Further, because the present invention is
quantitative, one can detect variations in expression levels,
fragmentation, or gene copy numbers by the methods.
[0102] In general, the methods of the invention are particularly
useful in screening samples derived from patients for the nucleic
acids of interest, e.g., from bodily fluids and/or waste from the
patient. This is because samples derived from relatively large
volumes of such materials can be screened in the methods of the
invention (removal of such materials is also relatively
non-invasive). The nucleic acids of interest (e.g., present in
cancer cells) can easily comprise 1% or less of the related nucleic
acid population of the sample (e.g., about 1%, 0.1%, 0.001%,
0.0001% or less of the alleles for a gene of interest). Thus, whole
blood, serum, plasma, stool, urine, vaginal secretions, ejaculatory
fluid, synovial fluid, a biopsy, cerebrospinal fluid, and amniotic
fluid, sputum, saliva, lymph, tears, sweat, or urine, or the like,
can easily be screened for rare nucleic acids or fragmentation by
the methods of the invention, as can essentially any tissue of
interest. These samples are typically taken, following informed
consent, from a patient by standard medical laboratory methods.
[0103] Prior to aliquotting and amplification, nucleic acids are
optionally purified from the samples by any available method, e.g.,
those taught in Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in Enzymology volume 152 Academic Press, Inc.,
San Diego, Calif. ("Berger"); Sambrook et al., Molecular Cloning--A
Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook"); and/or
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 2002) ("Ausubel"). A plethora of kits are also commercially
available for the purification of nucleic acids from cells or other
samples (see, e.g., EasyPrep.TM., FlexiPrep.TM., both from
Pharmacia Biotech; StrataClean.TM., from Stratagene; and,
QIAprep.TM. from Qiagen). Alternately, samples can simply be
directly subjected to amplification, e.g., following aliquotting
and dilution. One advantage of single molecule detection is that
the low concentration of sample components in the reaction can
reduce the need for nucleic acid purification. That is, dilution of
the sample reduces the abundance of unwanted components at the same
time it distributes the nucleic acid of interest into reaction
mixtures.
[0104] One preferred class of nucleic acids of interest to be
detected in the methods herein are those involved in cancer. Any
nucleic acid that is associated with cancer can be detected in the
methods of the invention, e.g., those that encode over expressed or
mutated polypeptide growth factors (e.g., sis), over expressed or
mutated growth factor receptors (e.g., erb-B1), over expressed or
mutated signal transduction proteins such as G-proteins (e.g.,
Ras), or non-receptor tyrosine kinases (e.g., abl), or over
expressed or mutated regulatory proteins (e.g., myc, myb, jun, fos,
etc.) and/or the like. In a preferred embodiment, specific or
arbitrary nucleic acids of interest are screened for the amount of
fragmentation, with high fragmentation generally associated with
apoptosis of normal cells and less fragmentation associated, e.g.,
with sloughing of cancer cells. In general, cancer can often be
linked to signal transduction molecules and corresponding oncogene
products, e.g., nucleic acids encoding Mos, Ras, Raf, and Met; and
transcriptional activators and suppressors, e.g., p53, Tat, Fos,
Myc, Jun, Myb, Rel, and/or nuclear receptors. p53, colloquially
referred to as the "molecular policeman" of the cell, is of
particular relevance, as about 50% of all known cancers can be
traced to one or more genetic lesion in p53.
[0105] Taking one class of genes that are relevant to cancer as an
example for discussion, many nuclear hormone receptors have been
described in detail and the mechanisms by which these receptors can
be modified to confer oncogenic activity have been worked out. For
example, the physiological and molecular basis of thyroid hormone
action is reviewed in Yen (2001) "Physiological and Molecular Basis
of Thyroid Hormone Action" Physiological Reviews 81(3): 1097-1142,
and the references cited therein. Known and well characterized
nuclear receptors include those for glucocorticoids (GRs),
androgens (ARs), mineralocorticoids (MRs), progestins (PRs),
estrogens (ERs), thyroid hormones (TRs), vitamin D (VDRs),
retinoids (RARs and RXRs), and the peroxisome proliferator
activated receptors (PPARs) that bind eicosanoids. The so called
"orphan nuclear receptors" are also part of the nuclear receptor
superfamily, and are structurally homologous to classic nuclear
receptors, such as steroid and thyroid receptors. Nucleic acids
that encode any of these receptors, or oncogenic forms thereof, can
be detected in the methods of the invention. About 40% of all
pharmaceutical treatments currently available are agonists or
antagonists of nuclear receptors and/or oncogenic forms thereof,
underscoring the relative importance of these receptors (and their
coding nucleic acids) as targets for analysis by the methods of the
invention.
[0106] As already mentioned, one preferred class of nucleic acids
of interest are those that are diagnostic of colon cancer, e.g., in
samples derived from stool. Colon cancer is a common disease that
can be sporadic or inherited. The molecular basis of various
patterns of colon cancer is known in some detail. In general,
germline mutations are the basis of inherited colon cancer
syndromes, while an accumulation of somatic mutations is the basis
of sporadic colon cancer. In Ashkenazi Jews, a mutation that was
previously thought to be a polymorphism may cause familial colon
cancer. Mutations of at least three different classes of genes have
been described in colon cancer etiology: oncogenes, suppressor
genes, and mismatch repair genes. One example nucleic acid encodes
DCC (deleted in colon cancer), a cell adhesion molecule with
homology to fibronectin. An additional form of colon cancer is an
autosomal dominant gene, hMSH2, that comprises a lesion. Familial
adenomatous polyposis is another form of colon cancer with a lesion
in the MCC locus on chromosome #5. For additional details on Colon
Cancer, see, Calvert et al. (2002) "The Genetics of Colorectal
Cancer" Annals of Internal Medicine 137 (7): 603-612 and the
references cited therein. For a variety of colon cancers and colon
cancer markers that can be detected in stool, see, e.g., Boland
(2002) "Advances in Colorectal Cancer Screening: Molecular Basis
for Stool-Based DNA Tests for Colorectal Cancer: A Primer for
Clinicians" Reviews In Gastroenterological Disorders Volume 2,
Supp. 1 and the references cited therein. As with other cancers,
mutations in a variety of other genes that correlate with cancer,
such as Ras and p53, are useful diagnostic indicators for cancer.
In another aspect, detection of fragmentation levels using methods
of the present invention can be particularly useful in detection of
colon cancer. For example, as the amount of total patient DNA
available in a stool specimen is low, the amplification aspect of
the present invention can be beneficial to examination of the DNA.
Whereas the DNA from cells sloughed from the normal colon lining is
generally degraded into fragments, e.g., of about 100 base pairs in
length, DNA entering the colon lumen from a colon tumor cells can
remain generally unfragmented. Detecting the presence of a
proportion of unfragmented nucleic acids over a certain threshold
in a stool specimen can correlate to presence of a colon
cancer.
[0107] Cervical cancer is another preferred target for detection,
e.g., in samples obtained from vaginal secretions. Cervical cancer
can be caused by the papova virus and has two oncogenes, E6 and E7.
E6 binds to and removes p53 and E7 binds to and removes PRB. The
loss of p53 and uncontrolled action of E2F/DP growth factors
without the regulation of pRB is one mechanism that leads to
cervical cancer. Furthermore, as with colon cancer, detecting the
presence of a proportion of unfragmented nucleic acids over a
certain threshold in a vaginal swab can correlate to the presence
of a cervical cancer.
[0108] Another preferred target for detection by the methods of the
invention is retinoblastoma, e.g., in samples derived from tears.
Retinoblastoma is a tumor of the eyes which results from
inactivation of the pRB gene. It has been found to transmit
heritably when a parent has a mutated pRB gene (and, of course,
somatic mutation can cause non-heritable forms of the cancer).
[0109] Neurofibromatosis Type 1 can be detected in the methods of
the invention. The NF1 gene is inactivated, which activates the
GTPase activity of the ras oncogene. If NF1 is missing, ras is
overactive and causes neural tumors. The methods of the invention
can be used to detect Neurofibromatosis Type 1 in CSF or via tissue
sampling.
[0110] Many other forms of cancer are known and can be found by
detecting, e.g., associated genetic lesions, fragmentation
proportions, or absolute concentrations of full-length nucleic
acids of interest using the methods of the invention. Cancers that
can be detected by detecting appropriate lesions or fragmentation
values include cancers of the lymph, blood, stomach, gut, colon,
testicles, pancreas, bladder, cervix, uterus, skin, and essentially
all others for which an associated genetic lesion or fragmentation
threshold exists. For a review of the topic, see, The Molecular
Basis of Human Cancer Coleman and Tsongalis (Eds) Humana Press;
ISBN: 0896036340; 1st edition (August 2001).
[0111] Similarly, nucleic acids from pathogenic or infectious
organisms can be detected by the methods of the invention, e.g.,
for infectious fungi, e.g., Aspergillus, or Candida species;
bacteria, particularly E. coli, which serves a model for pathogenic
bacteria (and, of course certain strains of which are pathogenic),
as well as medically important bacteria such as Staphylococci
(e.g., aureus), or Streptococci (e.g., pneumoniae); protozoa such
as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and
flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.);
viruses such as (+) RNA viruses (examples include Poxviruses e.g.,
vaccinia; Picornaviruses, e.g. polio; Togaviruses, e.g., rubella;
Flaviviruses, e.g., HCV; and Coronaviruses), (-) RNA viruses (e.g.,
Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV;
Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses),
dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e.,
Retroviruses, e.g., H[V and HTLV, and certain DNA to RNA viruses
such as Hepatitis B. Single and low copy amplification methods of
the invention can be useful in many cases, e.g., in exudates from
bacterial infections to identify living (having full length nucleic
acids) versus dead and lysed pathogens (having fragmented nucleic
acids).
[0112] A variety of nucleic acid encoding enzymes (e.g., industrial
enzymes) can also be detected according to the methods herein, such
as amidases, amino acid racemases, acylases, dehalogenases,
dioxygenases, diarylpropane peroxidases, epimerases, epoxide
hydrolases, esterases, isomerases, kinases, glucose isomerases,
glycosidases, glycosyl transferases, haloperoxidases,
monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile
hydratases, nitrilases, proteases, phosphatases, subtilisins,
transaminase, and nucleases. Similarly, agriculturally related
proteins such as insect resistance proteins (e.g., the Cry
proteins), starch and lipid production enzymes, plant and insect
toxins, toxin-resistance proteins, Mycotoxin detoxification
proteins, plant growth enzymes (e.g., Ribulose 1,5-Bisphosphate
Carboxylase/Oxygenase, "RUBISCO"), lipoxygenase (LOX), and
Phosphoenolpyruvate (PEP) carboxylase can also be detected.
[0113] Aliquotting the Sample
[0114] The sample can be aliquotted and/or diluted using standard
or microfluidic fluid handling approaches (or combinations
thereof). Standard fluid handling approaches for
dilution/aliquotting include, e.g., pipetting appropriate volumes
of the sample into microtiter trays and adding an appropriate
diluent. These operations can be performed manually or using
available high throughput fluid handlers, such as, e.g., those
designed to use serially dilute solutions in microtiter trays. High
throughput equipment (e.g., incorporating automated pipettors and
robotic microtiter tray handling) is preferred, as the present
invention contemplates making and using high numbers of aliquots of
a sample of interest.
[0115] Many automated systems for fluid handling are commercially
available and can be used for aliquotting and/or diluting a sample
in the context of the present invention. For example, a variety of
automated systems are available from the Zymark Corporation (Zymark
Center, Hopkinton, Mass.), which utilize various Zymate systems
(see also, http://www.zymark.com/), which typically include, e.g.,
robotics and fluid handling modules. Similarly, the common
ORCA.RTM. robot, which is used in a variety of laboratory systems,
e.g., for microtiter tray manipulation, is also commercially
available, e.g., from Beckman Coulter, Inc. (Fullerton, Calif.). In
any case, a conventional high throughput systems can be used in
place of, or in conjunction with microfluidic systems (for example,
conventional systems can be used to aliquot samples into microtiter
trays, from which microfluidic systems can draw materials) in
practicing the methods of the invention.
[0116] In one aspect, emulsions are created, where sample aliquots
comprise or consist of droplets within the emulsions. The emulsions
can be amplified by standard thermocyclic reactions and amplified
nucleic acids detected within droplets of the emulsions using
standard equipment (e.g., flow cytometers, microscope stations, or
CCD arrays).
[0117] Microfluidic systems provide a preferred fluid handling and
amplification technology that can conveniently be applied to the
present invention. In typical embodiments, samples are drawn into
microfluidic devices that comprise networks of microscale cavities
(channels, chambers, etc., having at least one dimension less than
about 500 .mu.M in size and often less than about 100 .mu.M) and
the samples are mixed, diluted, aliquotted or otherwise manipulated
in the network of cavities (e.g., channels and/or chambers). For
example, the microscale device can comprise one or more capillary,
in fluid communication with the network, extending outward from a
body structure of the microscale device. Negative pressure (vacuum)
is applied to the capillary and fluids are drawn into the network
from a container (e.g., a well on a microtiter tray). This process
can be multiplexed by using a device that comprises multiple
capillary channels, permitting many samples to be drawn into the
network and processed simultaneously. Alternately, multiple samples
can be sequentially drawn into the microfluidic device and routed
internally to multiple channels for simultaneous processing and
analysis. Sample interfaces with dried samples can also be
performed using this basic system, e.g., by partly or completely
expelling fluid from the capillary to hydrate samples prior to
drawing them into the microfluidic device (the fluid is typically
contacted to the samples as a hanging drop on the tip of the
capillary and then drawn back into the capillary). For either
approach, see also, U.S. Pat. No. 6,482,364 to Parce, et al. (Nov.
19, 2002) MICROFLUIDIC SYSTEMS INCLUDING PIPETTOR ELEMENTS; U.S.
Pat. No. 6,042,709 to Parce, et al. (Mar. 28, 2000) MICROFLUIDIC
SAMPLING SYSTEM AND METHODS; U.S. Pat. No. 6,287,520 to Parce, et
al. (Sep. 11, 2001) ELECTROPIPETTOR AND COMPENSATION MEANS FOR
ELECTROPHORETIC BIAS and U.S. Pat. No. 6,235,471 to Knapp, et al.
(May 22, 2001) CLOSED-LOOP BIOCHEMICAL ANALYZERS. Essentially any
fluid manipulation (aliquotting, diluting, heating and cooling) can
be performed in the network using available methods. Details
regarding dilution and aliquotting operations in microscale devices
can be found in the patent literature, e.g., U.S. Pat. No.
6,149,870 to Parce, et al. (Nov. 21, 2000) APPARATUS FOR IN SITU
CONCENTRATION AND/OR DILUTION OF MATERIALS IN MICROFLUIDIC SYSTEMS;
U.S. Pat. No. 5,869,004 to Parce, et al. (Feb. 9, 1999) METHODS AND
APPARATUS FOR IN SITU CONCENTRATION AND/OR DILUTION OF MATERIALS IN
MICROFLUIDIC SYSTEMS; and U.S. Pat. No. 6,440,722 to Knapp, et al.
(Aug. 27, 2002) MICROFLUIDIC DEVICES AND METHODS FOR OPTIMIZING
REACTIONS. Samples and components to be mixed/diluted or aliquotted
can be brought into the microscale device through pipettor elements
or from reaction component reservoirs on the device itself, or,
commonly, both. For example, the sample can be brought into the
microfluidic device through a pipettor channel and diluted and
supplied with common reagents from an on device dilution and/or
reagent reservoir(s). Locus specific reagents (e.g., amplification
primer pairs) can be on the device in wells, or stored off the
device, e.g., in microtiter plates (in which case they can be
accessed by the pipettor channel). Any or all of these operations
can be performed in a continuous or stopped flow format.
[0118] The functions the chip performs typically include reaction
assembly (assembly of reaction mixtures), thermocycling, and acting
as a "cuvette" for an optical system during an imaging (detection)
step. In the reaction assembly, the reaction mixture components
(particularly magnesium and the enzyme) which get combined at the
last second before heating begins are assembled. This is called a
"hot start" and provides advantages of specificity. During
thermocycling, the system optionally provides both constant fluid
movement and a continuous sequence of temperature changes. During
imaging, a high data rate CCD is useful in providing an adequate
dynamic range using the dispersion/diffusion methods of
quantification.
[0119] Commercial systems that perform all aspects of fluid
handling and analysis that can be used in the practice of the
present invention are available. Examples include the 250 HTS
system and AMS 90 SE from Caliper Technologies (Mountain View,
Calif.). These systems performs experiments in serial, continuous
flow fashion and employ a "chip-to-world" interface, or sample
access system, called a sipper through which materials in microwell
plates are sipped into a capillary or capillaries attached to the
chip and drawn into the channels of the chip. There they are mixed
with components of interest and a processing and result detection
steps are performed.
[0120] Whether conventional fluid handling or microfluidic
approaches (or both) are used, the aliquotting and/or dilution
events can be performed to achieve particular results. For example,
a sample can be diluted equally in each aliquot, or, alternately,
the aliquots can be differentially diluted (e.g., a dilution series
can be made). The aliquots themselves can be of a volume that is
appropriate to the fluid handling approach being used by the
system, e.g., on the order of a few microliters for microtiter
plates to 100 nL, 10 nL or even 1 nL or less for microfluidic
approaches.
[0121] The aliquots can be selected to have high or low copy
numbers of any relevant nucleic acid (e.g., for low copy number
aliquots, 50 or fewer, generally 25 or fewer, usually 10 or fewer
and often 5 or fewer, 2 or fewer or 1 or fewer copies of the
relevant nucleic acid(s)). The number of aliquots generated will
depend on the size of the sample and the amount of quantitative
information desired by the practitioner. For example, where simple
detection of a rare nucleic acid is desired, enough low and/or
single copy number aliquots are made of the sample to detect the
nucleic acid in one of the aliquots. Where more quantitative
information is needed, enough copies are made to provide reliable
statistical information, e.g., to a given confidence value. In
either case, this can include anywhere from 1 aliquot to 10.sup.9
or more aliquots, e.g., 10, 100, 1,000, 10,000, 100,000, 1,000,000,
1,000,000,000 or more aliquots. There is no theoretical limit on
the number of aliquots that can be made and assessed for a nucleic
acid of interest according to the present invention, though there
are practical considerations with respect to the throughput of the
system and the size of the sample (the lower the throughput, the
fewer aliquots can be analyzed in a given time; the larger the
sample size the more aliquots can be made of the sample). Using
microfluidic approaches, reagent usage (and concomitant reagent
costs) can be minimized. By formatting the system to provide for
continuous flow of sample and reagents, including, optionally,
during amplification, the systems of the invention can greatly
speed the process of searching many different samples for a nucleic
acid of interest. Similarly, if stopped flow approaches are used,
simultaneous processing of signals from PCR reactions can be used
to speed the process of searching samples for a nucleic acid of
interest. In the examples below, about 150 aliquots for each
dilution range was sufficient to provide reasonable quantitative
information for Poisson statistics for model samples. Obviously,
more or fewer aliquots can be used in the methods as well.
[0122] In many of the embodiments herein, it is worth noting that
many of the aliquots will have zero copies of the nucleic acid of
interest, due to the rarity of the relevant nucleic acid in the
sample (and the dilution that is chosen). This does not present a
detection problem in a continuous flow analysis system--the flow
rate can be used to calculate how many aliquots have passed
(undetected) by a detector prior to detection of the nucleic acid
of interest. In non-continuous flow systems (e.g., microwell plate
based systems), one can simply count blank reactions (wells lacking
amplification product) to determine the frequency of amplification
of the nucleic acid of interest. In any event, anywhere from 1 to
10.sup.6 or more zero copy reactions can be made and assessed by
the system, e.g., about 10, 25, 50, 100, 500, 1,000, 10,000,
100,000, or 1,000,000 or more zero copy reactions can be detected
in the process of detecting a nucleic acid of interest. Similarly,
additional nucleic acids other than the nucleic acid of interest
(e.g., controls, or alternate alleles of a nucleic acid of interest
that are also amplified by the relevant locus specific reagent) can
be detected (or not detected) by the system. The proportion of such
alternate nucleic acids in the system to the nucleic acid of
interest can range from less than 1 to 10.sup.9 or more, e.g.,
1.times., 10.times., 100.times., 1,000.times., 10,000.times.,
100,000.times., 1,000,000.times., 1,000,000,000.times. or more.
[0123] Furthermore, as demonstrated in the examples and figures
herein, the continuous flow format is a surprisingly efficient
system, meaning that a high proportion of single molecules that get
into the system are amplified. This efficiency is useful in
ensuring that very rare molecules are detected, if present, for
example in a biowarfare or infectious disease detection
applications. Evidence for high efficiency is in the examples,
tables and figures herein. Typically, the systems of the invention
can be used to amplify at least 90%, generally 95%, often 99% or
more of the rare molecules that are present in sample of interest,
or that are present in a collection of aliquots that are subjected
to amplification. Efficiency factors can be determined, e.g.,
empirically, for adjustment of mathematical formulas for more
accurate quantitative interpretations of signal data.
[0124] Amplifying the Aliquots
[0125] The methods of the invention include amplifying one or more
sequences of a nucleic acid of interest from a sample or aliquot
and, optionally, one or more additional nucleic acids. Typically
two or more sequences of a nucleic acid of interest are amplified
at separated positions to allow interpretation of the nucleic acid
length. Any available amplification method can be used, including
PCR, RT-PCR, LCR, and/or any of the various RNA mediated
amplification methods. PCR, RT-PCR and LCR are preferred
amplification methods for amplifying a nucleic acid of interest in
the methods of the invention. Real time PCR and/or RT-PCR (e.g.,
mediated via TaqMan.TM. probes or molecular beacon-based probes)
can also be used to facilitate detection of amplified nucleic
acids.
[0126] It is expected that one of skill is generally familiar with
the details of these amplification methods. Details regarding these
amplification methods can be found, e.g., in Sambrook (2000);
Ausubel (2002) and Innis (1990), all above. Additional details can
be found in PCR: A Practical Approach (The Practical Approach
Series) by Quirke et al. (eds.). (1992) by Oxford University
Press.
[0127] Additional details can also be found in the literature for a
variety of applications of PCR. For example, details regarding
amplification of nucleic acids in plants can be found, e.g., in
Plant Molecular Biology (1993) Croy (ed.) BIOS Scientific
Publishers, Inc. Similarly, additional details regarding PCR for
cancer detection can be found in any of a variety of sources, e.g.,
Bernard and Wittwer (2002) "Real Time PCR Technology for Cancer
Diagnostics Clinical Chemistry 48(8): 1178-1185; Perou et al.
(2000) "Molecular portraits of human breast tumors" Nature
406:747-52; van't Veer et al. (2002) "Gene expression profiling
predicts clinical outcome of breast cancer" Nature 415:530-6;
Rosenwald et al. (2001) "Relation of gene expression phenotype to
immunoglobulin mutation genotype in B cell chronic lymphocytic
leukemia" J Exp Med 194: 1639-47; Alizadeh et al. (2000) "Distinct
types of diffuse large B-cell lymphoma identified by gene
expression profiling" Nature 403:503-11; Garber et al. (2001)
"Diversity of gene expression in adenocarcinoma of the lung" Proc
Natl Acad Sci U S A 98: 13784-9; Tirkkonen et al. (1998) "Molecular
cytogenetics of primary breast cancer by CGH" Genes Chromosomes
Cancer 21:177-84; Watanabe et al. (2001) "A novel amplification at
17q21-23 in ovarian cancer cell lines detected by comparative
genomic hybridization" Gynecol Oncol 81:172-7, and many others.
[0128] Molecular Beacons
[0129] In one aspect, real time PCR is performed on the various
aliquots or reaction mixtures described herein, e.g., using
molecular beacons or TaqMan.TM. probes. A molecular beacon (MB) is
an oligonucleotide or PNA which, under appropriate hybridization
conditions, self-hybridizes to form a stem and loop structure. The
MB has a label and a quencher at the termini of the oligonucleotide
or PNA; thus, under conditions that permit intra-molecular
hybridization, the label is typically quenched (or at least altered
in its fluorescence) by the quencher. Under conditions where the MB
does not display intra-molecular hybridization (e.g., when bound to
a target nucleic acid, e.g., to a region of an amplicon during
amplification), the MB label is unquenched.
[0130] Details regarding standard methods of making and using MBs
are well established in the literature and MBs are available from a
number of commercial reagent sources. See also, e.g., Leone et al.
(1995) "Molecular beacon probes combined with amplification by
NASBA enable homogenous real-time detection of RNA." Nucleic Acids
Res. 26:2150-2155; Tyagi and Kramer (1996) "Molecular beacons:
probes that fluoresce upon hybridization" Nature Biotechnology
14:303-308; Blok and Kramer (1997) "Amplifiable hybridization
probes containing a molecular switch" Mol Cell Probes 11:187-194;
Hsuih et al. (1997) "Novel, ligation-dependent PCR assay for
detection of hepatitis C in serum" J Clin Microbiol 34:501-507;
Kostrikis et al. (1998) "Molecular beacons: spectral genotyping of
human alleles" Science 279:1228-1229; Sokol et al. (1998) "Real
time detection of DNA:RNA hybridization in living cells" Proc.
Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al. (1998)
"Multicolor molecular beacons for allele discrimination" Nature
Biotechnology 16:49-53; Bonnet et al. (1999) "Thermodynamic basis
of the chemical specificity of structured DNA probes" Proc. Natl.
Acad. Sci. U.S.A. 96:6171-6176; Fang et al. (1999) "Designing a
novel molecular beacon for surface-immobilized DNA hybridization
studies" J. Am. Chem. Soc. 121:2921-2922; Marras et al. (1999)
"Multiplex detection of single-nucleotide variation using molecular
beacons" Genet. Anal. Biomol. Eng. 14:151-156; and Vet et al.
(1999) "Multiplex detection of four pathogenic retroviruses using
molecular beacons" Proc. Natl. Acad. Sci. U.S.A. 96:6394-6399.
Additional details regarding MB construction and use is found in
the patent literature, e.g., U.S. Pat. No. 5,925,517 (Jul. 20,
1999) to Tyagi et al. entitled "Detectably labeled dual
conformation oligonucleotide probes, assays and kits;" U.S. Pat.
No. 6,150,097 to Tyagi et al (Nov. 21, 2000) entitled "Nucleic acid
detection probes having non-FRET fluorescence quenching and kits
and assays including such probes" and U.S. Pat. No. 6,037,130 to
Tyagi et al (Mar. 14, 2000), entitled "Wavelength-shifting probes
and primers and their use in assays and kits."
[0131] MBs are robust reagents for detecting and quantifying
nucleic acids, including in real time, e.g., during PCR, LCR or
other nucleic acid amplification reactions (e.g., MBs can be used
to detect targets as they are formed). A variety of commercial
suppliers produce standard and custom molecular beacons, including
Cruachem (cruachem.com), Oswel Research Products Ltd. (UK;
oswel.com), Research Genetics (a division of Invitrogen, Huntsville
Ala. (resgen.com)), the Midland Certified Reagent Company (Midland,
Tex. mcrc.com) and Gorilla Genomics, LLC (Alameda, Calif.). A
variety of kits which utilize molecular beacons are also
commercially available, such as the Sentinel.TM. Molecular Beacon
Allelic Discrimination Kits from Stratagene (La Jolla, Calif.) and
various kits from Eurogentec SA (Belgium, eurogentec.com) and
Isogen Bioscience BV (The Netherlands, isogen.com).
[0132] MB components (e.g., oligos, including those labeled with
fluorophores or quenchers) can be synthesized using conventional
methods. For example, oligos or peptide nucleic acids (PNAs) can be
synthesized on commercially available automated oligonucleotide/PNA
synthesis machines using standard methods. Labels can be attached
to the oligos or PNAs either during automated synthesis or by
post-synthetic reactions which have been described before see,
e.g., Tyagi and Kramer (1996) "Molecular beacons: probes that
fluoresce upon hybridization" Nature Biotechnology 14:303-308 and
U.S. Pat. No. 6,037,130 to Tyagi et al (Mar. 14, 2000), entitled
"Wavelength-shifting probes and primers and their use in assays and
kits." and U.S. Pat. No. 5,925,517 (Jul. 20, 1999) to Tyagi et al.
entitled "Detectably labeled dual conformation oligonucleotide
probes, assays and kits." Additional details on synthesis of
functionalized oligos can be found in Nelson, et al. (1989)
"Bifunctional Oligonucleotide Probes Synthesized Using A Novel CPG
Support Are Able To Detect Single Base Pair Mutations" Nucleic
Acids Research 17:7187-7194. Labels/quenchers can be introduced to
the oligonucleotides or PNAs, e.g., by using a controlled-pore
glass column to introduce, e.g., the quencher (e.g., a
4-dimethylaminoazobenzene-4'-sulfonyl moiety (DABSYL). For example,
the quencher can be added at the 3' end of oligonucleotides during
automated synthesis; a succinimidyl ester of
4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) can be used when
the site of attachment is a primary amino group; and
4-dimethylaminophenylazo- phenyl-4'-maleimide (DABMI) can be used
when the site of attachment is a sulphydryl group. Similarly,
fluorescein can be introduced in the oligos, either using a
fluorescein phosphoramadite that replaces a nucleoside with
fluorescein, or by using a fluorescein dT phosphoramadite that
introduces a fluorescein moiety at a thymidine ring via a spacer.
To link a fluorescein moiety to a terminal location,
iodoacetoamidofluorescein can be coupled to a sulphydryl group.
Tetrachlorofluorescein (TET) can be introduced during automated
synthesis using a 5'-tetrachloro-fluorescein phosphoramadite. Other
reactive fluorophore derivatives and their respective sites of
attachment include the succinimidyl ester of 5-carboxyrhodamine-6G
(RHD) coupled to an amino group; an iodoacetamide of
tetramethylrhodamine coupled to a sulphydryl group; an
isothiocyanate of tetramethylrhodamine coupled to an amino group;
or a sulfonylchloride of Texas red coupled to a sulphydryl group.
During the synthesis of these labeled components, conjugated
oligonucleotides or PNAs can be purified, if desired, e.g., by high
pressure liquid chromatography or other methods.
[0133] TaqMan.TM. Probes
[0134] PCR quantification using dual-labeled fluorogenic
oligonucleotide probes, commonly referred to as "TaqMan.TM."
probes, can be performed according to the present invention. These
probes are composed of short (e.g., 20-25 base)
oligodeoxynucleotides that are labeled with two different
fluorescent dyes. On the 5' terminus of each probe is a reporter
dye, and on the 3' terminus of each probe a quenching dye is found.
The oligonucleotide probe sequence can be complementary to an
internal target sequence present in a PCR amplicon. When the probe
is intact, energy transfer occurs between the two fluorophores and
emission from the reporter is quenched by the quencher (fluorescent
resonant energy transfer or FRET). During the extension phase of
PCR, the probe is cleaved by 5' nuclease activity of the polymerase
used in the reaction, thereby releasing the reporter from the
oligonucleotide-quencher and producing an increase in reporter
emission intensity.
[0135] Accordingly, TaqMan.TM. probes are oligonucleotides that
have a label and a quencher, where the label is released after
hybridization and during amplification by the exonuclease action of
the polymerase used in amplification. This provides a real time
measure of amplification during synthesis. A variety of TaqMan.TM.
reagents are commercially available, e.g., from Applied Biosystems
(Division Headquarters in Foster City, Calif.) as well as from a
variety of specialty vendors such as Biosearch Technologies (e.g.,
black hole quencher probes).
[0136] General Probe Synthesis Methods
[0137] In general, synthetic methods for making oligonucleotides,
including probes, molecular beacons, PNAs, LNAs (locked nucleic
acids), etc., are well known. For example, oligonucleotides can be
synthesized chemically according to the solid phase phosphoramidite
triester method described by Beaucage and Caruthers (1981),
Tetrahedron Letts., 22(20):1859-1862, e.g., using a commercially
available automated synthesizer, e.g., as described in
Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168.
Oligonucleotides, including modified oligonucleotides can also be
ordered from a variety of commercial sources known to persons of
skill. There are many commercial providers of oligo synthesis
services, and thus this is a broadly accessible technology. Any
nucleic acid can be custom ordered from any of a variety of
commercial sources, such as The Midland Certified Reagent Company
(mcrc@oligos.com), The Great American Gene Company (www.genco.com),
ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc.
(Alameda, Calif.) and many others. Similarly, PNAs can be custom
ordered from any of a variety of sources, such as PeptidoGenic
(pkim@ccnet.com), HTI Bio-products, inc. (www.htibio.com), BMA
Biomedicals Ltd (U.K.), Bio-Synthesis, Inc., and many others.
[0138] Amplification in Microfluidic Systems
[0139] A number of high throughput approaches to performing PCR and
other amplification reactions have been developed, e.g., involving
amplification reactions in microfluidic devices, as well as methods
for detecting and analyzing amplified nucleic acids in or on the
devices. Details regarding such technology is found, e.g., in the
technical and patent literature, e.g., Kopp et al. (1998) "Chemical
Amplification: Continuous Flow PCR on a Chip" Science, 280
(5366):1046; U.S. Pat. No. 6,444,461 to Knapp, et al. (Sep. 3,
2002) MICROFLUIDIC DEVICES AND METHODS FOR SEPARATION; U.S. Pat.
No. 6,406,893 to Knapp, et al. (Jun. 18, 2002) MICROFLUIDIC METHODS
FOR NON-THERMAL NUCLEIC ACID MANIPULATIONS; U.S. Pat. No. 6,391,622
to Knapp, et al. (May 21, 2002) CLOSED-LOOP BIOCHEMICAL ANALYZERS;
U.S. Pat. No. 6,303,343 to Kopf-Sill (Oct. 16, 2001) INEFFICIENT
FAST PCR; U.S. Pat. No. 6,171,850 to Nagle, et al. (Jan. 9, 2001)
INTEGRATED DEVICES AND SYSTEMS FOR PERFORMING TEMPERATURE
CONTROLLED REACTIONS AND ANALYSES; U.S. Pat. No. 5,939,291 to
Loewy, et al. (Aug. 17, 1999) MICROFLUIDIC METHOD FOR NUCLEIC ACID
AMPLIFICATION; U.S. Pat. No. 5,955,029 to Wilding, et al. (Sep. 21,
1999) MESOSCALE POLYNUCLEOTIDE AMPLIFICATION DEVICE AND METHOD;
U.S. Pat. No. 5,965,410 to Chow, et al. (Oct. 12, 1999) ELECTRICAL
CURRENT FOR CONTROLLING FLUID PARAMETERS IN MICROCHANNELS; Service
(1998) "Microchips Arrays Put DNA on the Spot" Science
282:396-399), Zhang et al. (1999) "Automated and Integrated System
for High-Throughput DNA Genotyping Directly from Blood" Anal. Chem.
71:1138-1145 and many others.
[0140] For example, U.S. Pat. No. 6,391,622 to Knapp, et al. (May
21, 2002) CLOSED-LOOP BIOCHEMICAL ANALYZERS and the references
cited therein describes systems comprising microfluidic elements
that can access reagent storage systems and that can perform PCR or
other amplification reactions by any of a variety of methods in the
microfluidic system. For example, the microfluidic system can have
one or more capillaries extending outwards from the body structure
of the microfluidic system for drawing materials into the body
structure. Within the body structure are microfluidic cavities
(channels, chambers, or the like having at least one dimension
smaller than about 500 microns, and, typically smaller than about
100 microns) in which the amplification reactions are performed.
The capillaries that extend out from the body structure can access
standard reagent storage elements (microtiter plates, or the like)
by drawing fluid into the capillary, e.g., due to application of a
vacuum or electroosmotic force. Similarly, the capillaries can
access dried reagent libraries on substrates (e.g., the
LibraryCard.TM. reagent array made by Caliper Technologies) by
partly or completely expelling fluid to rehydrate library members
and then by drawing the rehydration fluid back into the capillary.
For example, the capillary can partly expel fluid to form a hanging
drop on the capillary, which is then contacted to the material to
be hydrated. The material in the hanging drop is then drawn back
into the capillary. In any case, molecular beacons or TaqMan.TM.
probes can be incorporated into the relevant amplification reaction
and detected in the microfluidic device to provide for real time
PCR detection. Alternately, PCR amplicons can be detected by
conventional methods, such as hybridization to a labeled probe,
e.g., prior to or following a separation operation that separates
unhybridized probe from hybridized probe. For example, an
electrophoretic separation can be performed in a channel of the
microscale device.
[0141] Conventional High Throughout Systems
[0142] In an alternative embodiment, standard fluid handling
approaches are used in place of, or in conjunction with,
microfluidic approaches. PCR can be performed in standard reaction
vessels (e.g., microtiter plates), as can dilutions or other
operations relevant to the present invention. Various
high-throughput systems are available for non-microfluidic
approaches to fluid handling (typically involving plates comprising
several reaction chambers, e.g., 96 well, 384 well or 1536 well
microtiter plates). These approaches can utilize conventional
robotics to perform fluid handling operations and can use
conventional commercially available thermocyclers to perform
amplification reactions. See above, for a discussion of automated
fluid handling systems.
[0143] Detecting the Amplified Nucleic Acids
[0144] Any available method for detecting amplified nucleic acids
can be used in the present invention. Common approaches include
real time amplification detection with molecular beacons or
TaqMan.TM. probes, detection of intercalating dyes (ethidium
bromide or sybergreen), detection of labels incorporated into the
amplification probes or the amplified nucleic acids themselves,
e.g., following electrophoretic separation of the amplification
products from unincorporated label), and/or detection of secondary
reagents that bind to the nucleic acids. Details on these general
approaches is found in the references cited herein, e.g., Sambrook
(2000), Ausubel (2002), and the references in the sections herein
related to real time PCR detection. Additional labeling strategies
for labeling nucleic acids and corresponding detection strategies
can be found, e.g., in Haugland (1996) Handbook of Fluorescent
Probes and Research Chemicals Sixth Edition by Molecular Probes,
Inc. (Eugene Oreg.); or Haugland (2001) Handbook of Fluorescent
Probes and Research Chemicals Eighth Edition by Molecular Probes,
Inc. (Eugene Oreg.) (Available on CD ROM).
[0145] Amplified nucleic acids (amplicons) can be detected in
homogenous (substantially unseparated) reaction mixtures or
solutions (e.g., using molecular beacons or TaqMan.TM. probes) or
during or after separation (e.g., by electrophoresis). Details on
these strategies can be found in the preceding references.
[0146] Amplification and detection are commonly integrated in a
system comprising a microfluidic device in the present invention.
Available microfluidic systems that include detection features for
detecting nucleic acids include the 250 HTS system and AMS 90 SE
from Caliper Technologies (Mountain View, Calif.), as well as the
Agilent 2100 bioanalyzer (Agilent, Palo Alto, Calif.). Additional
details regarding systems that comprise detection (and
separation/detection) capabilities are well described in the patent
literature, e.g., the references already noted herein and in Parce
et al. "High Throughput Screening Assay Systems in Microscale
Fluidic Devices" WO 98/00231.
[0147] In general, the devices herein optionally include signal
detectors, e.g., which detect fluorescence, phosphorescence,
radioactivity, pH, charge, absorbance, luminescence, temperature,
magnetism or the like. Fluorescent detection is especially
preferred and generally used for detection of amplified nucleic
acids (however, upstream and/or downstream operations can be
performed on amplicons, which can involve other detection methods,
such as mass spectroscopy or size exclusion).
[0148] The detector(s) optionally monitor one or a plurality of
signals from an amplification reaction and/or hybridization
reaction. For example, the detector can monitor optical signals
which correspond to "real time" amplification assay results. The
detector can monitor a single type of signal, or, e.g.,
simultaneously monitor multiple different signals.
[0149] Example detectors include photo multiplier tubes,
spectrophotometers, CCD arrays, scanning detectors, microscopes,
galvo-scanns and/or the like. Amplicons or other components which
emit a detectable signal can be flowed past the detector, or,
alternatively, the detector can move relative to the site of the
amplification reaction (or, the detector can simultaneously monitor
a number of spatial positions corresponding to channel regions, or
microtiter wells e.g., as in a CCD array). Detectors in the present
invention can detect signals from probes associated with nucleic
acids of the invention that flow into one or more detection
regions, e.g., of a microfluidic device.
[0150] The detector can include or be operably linked to a computer
(or other logic device), e.g., which has software for converting
detector signal information into assay result information (e.g.,
presence of a nucleic acid of interest, the length of a nucleic
acid of interest, proportions of nucleic acid of interest lengths,
and/or correlations with disease states), or the like.
[0151] Signals are optionally calibrated, e.g., by calibrating the
microfluidic system by monitoring a signal from a known source. For
example, signals can be calibrated against a reference light
source, internal reference signals, or normalized for detection of
positive signals over background.
[0152] A microfluidic system can also employ multiple different
detection systems for monitoring signals in the system. Detection
systems of the present invention are used to detect and monitor the
materials in a particular channel region (or other reaction
detection region). Once detected, the flow rate and velocity of any
cells or droplets in the channels can be optionally measured by
sensors and controlled as described above.
[0153] Examples of detection systems useful in methods and systems
of the invention can include optical sensors, temperature sensors,
pressure sensors, pH sensors, conductivity sensors, and the like.
Each of these types of sensors is readily incorporated into the
microfluidic systems described herein. In these systems, such
detectors can be placed either within or adjacent to the
microfluidic device or one or more channels, chambers or conduits
of the device, such that the detector is within sensory
communication with the device, channel, or chamber. The phrase
"within sensory communication" of a particular region or element,
as used herein, generally refers to the placement of the detector
in a position such that the detector is capable of detecting the
property of the microfluidic device, a portion of the microfluidic
device, or the contents of a portion of the microfluidic device,
for which that detector was intended. For example, a pH sensor
placed in sensory communication with a microscale channel is
capable of determining the pH of a fluid disposed in that channel.
Similarly, a temperature sensor placed in sensory communication
with the body of a microfluidic device is capable of determining
the temperature of the device itself.
[0154] Particularly preferred detection systems include optical
detection systems for detecting an optical property of a material
within the channels and/or chambers of the microfluidic devices
that are incorporated into the microfluidic systems described
herein. Such optical detection systems are typically placed
adjacent to a microscale channel of a microfluidic device, and are
in sensory communication with the channel via an optical detection
window that is disposed across the channel or chamber of the
device. Optical detection systems include systems that are capable
of measuring the light emitted from material within the channel,
the transmissivity or absorbance of the material, as well as the
materials spectral characteristics. In preferred aspects, the
detector measures an amount of light emitted from the material,
such as a fluorescent or chemiluminescent material. As such, the
detection system will typically include collection optics for
gathering a light based signal transmitted through the detection
window, and transmitting that signal to an appropriate light
detector. Microscope objectives of varying power, field diameter,
and focal length are readily utilized as at least a portion of this
optical train. The light detectors are optionally
spectrophotometers, photodiodes, avalanche photodiodes,
photomultiplier tubes, diode arrays, or in some cases, imaging
systems, such as charged coupled devices (CCDs) and the like. The
detection system is typically coupled to a computer, via an analog
to digital or digital to analog converter, for transmitting
detected light data to the computer for analysis, storage and data
manipulation.
[0155] In the case of fluorescent materials such as labeled
amplicons, the detector typically includes a light source that
produces light at an appropriate wavelength for activating the
fluorescent material, as well as optics for directing the light
source through the detection window to the material contained in
the channel or chamber. The light source can be any number of light
sources that provides an appropriate wavelength, including lasers,
laser diodes, and LEDs. Other light sources are used in other
detection systems. For example, broad band light sources are
typically used in light scattering/transmissivity detection
schemes, and the like. Typically, light selection parameters are
well known to those of skill in the art.
[0156] The detector can exist as a separate unit, but can also be
integrated with the system or microfluidic device, into a single
instrument. Integration of these functions into a single unit
facilitates connection of these instruments with the computer, by
permitting the use of few or a single communication port(s) for
transmitting information between the controller, the detector and
the computer.
[0157] Counting and Statistically Analyzing Nucleic Acids of
Interest
[0158] One feature of the present invention is that it provides for
robust quantitation of rare (and other) nucleic acids in a sample.
This robust quantitation provides the ability to perform
statistical or probabilistic analysis of the sample. For example,
Poisson analysis, Monte Carlo analysis, application of genetic
algorithms, neural network training, Markov modeling, hidden Markov
modeling, multidimensional scaling, partial least squares (PLS)
analysis, or principle component analysis (PCA) can all be applied
to data generated by the present invention. These statistical
evaluations can be used to determine, e.g., the concentration,
abundance, or length proportions of a given nucleic acid in a
sample and to correlate abundance or proportions to diagnosis or
prognosis associated with the diagnosis or prognosis.
[0159] General references that are useful in understanding how to
generate and analyze data, as well as other relevant concepts
include: Neil Weiss (1999) Introductory Statistics & Elementary
Statistics Edition: 5.sup.th ISBN:0201434490; Berinstein (1998)
Finding Statistics Online: How to Locate the Elusive Numbers You
Need. Medford, N.J.: Information Today; Everitt, (1998) The
Cambridge Dictionary of Statistics New York: Cambridge University
Press; Kotz (1988). Encyclopedia of Statistical Sciences, vol. 1-9
plus supplements New York: Wiley; Dillon and Goldstein (1984).
Multivariate Analysis: Methods and Applications New York: Wiley;
Tabachnick and Fidell (1996) Using Multivariate Statistics New
York: HarperCollins College Publishers; Box et al. (1978)
Statistics for Experimenters New York: Wiley; Cornell (1990)
Experiments with Mixtures New York: Wiley; John, P. W. M. (1998)
Statistical Design and Analysis of Experiments Philadelphia: SIAM;
Gibas and Jambeck (2001) Bioinformatics Computer Skills O'Reilly,
Sebastipol, Calif.; Pevzner (2000) Computational Molecular Biology
and Algorithmic Approach, The MIT Press, Cambridge Mass.; Durbin et
al. (1998) Biological Sequence Analysis: Probabilistic Models of
Proteins and Nucleic Acids, Cambridge University Press, Cambridge,
UK; and Rashidi and Buehler (2000) Bioinformatic Basics:
Applications in Biological Science and Medicine CRC Press LLC, Boca
Raton, Fla.
[0160] Calculating Diffusion and Dispersion
[0161] One feature of the invention is the discovery that the
highly reproducible peak parameters, e.g., amplitude, width area,
and/or shape features of a signal from an amplification reaction
can be correlated to the starting copy number for the reaction
and/or used to discriminate signals of interest from background
fluctuations. This correlation can be performed at the theoretical
level, taking thermal diffusivity and Taylor Aris diffusion into
account, or it can be performed by comparison to standards (e.g.,
comparisons to peak shapes, e.g., heights, widths, or general shape
profiles for amplification reactions that have known copy numbers
for starting materials). The same or different peak parameters can
be evaluated in interpretation of detector signals for two on more
probes in determination of nucleic acid length.
[0162] For theoretical calculation approaches, a label is typically
initially confined in a region -h<x<h, as a function of time
(t) and spatial position (x) with respect to the peak center (x=0)
and the concentration (C) of the label, or of a component
corresponding to the label (e.g., the nucleic acid of interest), is
equal to 1/2 C.sub.o {erf[(h-x)/(2Dt).sup.1/2)]}, where C.sub.o is
the initial concentration at time t=0, erf is an error function,
and D is a coefficient of overall dispersion. D is equal to the sum
of thermal diffusion and Taylor dispersion (D.sub.T) in the system.
In turn, the Taylor dispersion (D.sub.T) is dependent on the
dimensions and shape of the microfluidic cavity through which the
label is flowed, the flow velocity (u) and the thermal diffusivity
(D). Typically, D=K(d.sup.2u.sup.2)/D, where K is a proportionality
factor which is a function of the microfluidic cavity through which
the label is flowed and d is a characteristic microfluidic cavity
length. For example, where the microfluidic cavity is a circular
channel and K=1/192, d is the diameter of the circular channel and
D=D+D.sub.T. Further details on thermal diffusivity and Taylor Aris
dispersion can be found in MICROFLUIDIC SYSTEMS AND METHODS FOR
DETERMINING MODULATOR KINETICS, U.S. Ser. No. 09/609,030 By Andrea
Chow, Filed Jun. 30, 2000.
[0163] Additional System Details
[0164] The systems of the invention can include microfluidic
devices, reaction mixtures, detectors, sample storage elements
(microtiter plates, dried arrays of components, etc.), flow
controllers, amplification devices or microfluidic modules,
computers and/or the like. These systems can be used for
aliquoting, amplifying and analyzing the nucleic acids of interest.
The microfluidic devices, amplification components, detectors and
storage elements of the systems have already been described in some
detail above. The following discussion describes appropriate
controllers and computers, though many configurations are available
and one of skill would be expected to be familiar in their use and
would understand how they can be applied to the present
invention.
[0165] Flow Controllers
[0166] A variety of controlling instrumentation is optionally
utilized in conjunction with the microfluidic devices described
herein, for controlling the transport and direction of fluids
and/or materials within the devices of the present invention, e.g.,
by pressure-based or electrokinetic control.
[0167] For example, in many cases, fluid transport and direction
are controlled in whole or in part, using pressure based flow
systems that incorporate external or internal pressure sources to
drive fluid flow. Internal sources include microfabricated pumps,
e.g., diaphragm pumps, thermal pumps, Lamb wave pumps and the like
that have been described in the art. See, e.g., U.S. Pat. Nos.
5,271,724, 5,277,556, and 5,375,979 and Published PCT Application
Nos. WO 94/05414 and WO 97/02357. The systems described herein can
also utilize electrokinetic material direction and transport
systems.
[0168] Preferably, external pressure sources are used, and applied
to ports at channel termini. These applied pressures, or vacuums,
generate pressure differentials across the lengths of channels to
drive fluid flow through them. In the interconnected channel
networks described herein, differential flow rates on volumes are
optionally accomplished by applying different pressures or vacuums
at multiple ports, or preferably, by applying a single vacuum at a
common waste port and configuring the various channels with
appropriate resistance to yield desired flow rates. Example systems
are described in U.S. Ser. No. 09/238,467 filed Jan. 28, 1999.
[0169] Typically, the controller systems are appropriately
configured to receive or interface with a microfluidic device or
system element as described herein. For example, the controller
and/or detector, optionally includes a stage upon which a
microfluidic device is mounted to facilitate appropriate
interfacing between the controller and/or detector and the device.
Typically, the stage includes an appropriate mounting/alignment
structural element, such as a nesting well, alignment pins and/or
holes, asymmetric edge structures (to facilitate proper device
alignment), and the like. Many such configurations are described in
the references cited herein.
[0170] The controlling instrumentation discussed above is also
optionally used to provide for electrokinetic injection or
withdrawal of material downstream of the region of interest to
control an upstream flow rate. The same instrumentation and
techniques described above are also utilized to inject a fluid into
a downstream port to function as a flow control element.
[0171] Computer
[0172] As noted above, either or both of the controller system
and/or the detection system can be coupled to an appropriately
programmed processor or computer (logic device) which functions to
instruct the operation of these instruments in accordance with
preprogrammed or user input instructions, receive data and
information from these instruments, and interpret, manipulate and
report this information to the user. As such, the computer is
typically appropriately coupled to one or both of these instruments
(e.g., including an analog to digital or digital to analog
converter as needed).
[0173] The computer typically includes appropriate software for
receiving user instructions, either in the form of user input into
a set parameter fields, e.g., in a GUI, or in the form of
preprogrammed instructions, e.g., preprogrammed for a variety of
different specific operations. The software then converts these
instructions to appropriate language for instructing the operation
of the fluid direction and transport controller to carry out the
desired operation. The computer then receives the data from the one
or more sensors/detectors included within the system, and
interprets the data, either provides it in a user understood
format, or uses that data to initiate further controller
instructions, in accordance with the programming, e.g., such as in
monitoring and control of flow rates (including for continuous
flow), temperatures, applied voltages, and the like.
[0174] The systems and/or kits can include system instructions
(e.g., embodied in a computer or in a computer readable medium,
e.g., as system software) for practicing any of the method steps
herein. For example, the system optionally includes system software
that correlates a shape, length, width, volume and/or area occupied
by amplified copies of the nucleic acid of interest, as detected by
the detector, to the number of copies of the nucleic acid of
interest present in one of the aliquots, or to the number of copies
of the nucleic acid of interest present in the sample, or both.
Similarly, the system optionally includes system instructions that
direct the dilution module to aliquot the sample into a plurality
of aliquots, including a plurality of zero copy aliquots comprising
no copies of the nucleic acids of interest and one or more single
copy aliquot comprising a single copy of the nucleic acid of
interest.
[0175] The statistical functions noted above can also be
incorporated into system software, e.g., embodied in the computer,
in computer memory or on computer readable media. For example, the
computer can include statistical or probabilistic system software
that performs one or more statistical or probabilistic analysis of
signals received from one or more of the aliquots subjected to
amplification (e.g., via thermocycling). For example, the
statistical or probabilistic analysis can include Poisson analysis,
Monte Carlo analysis, application of a genetic algorithm, neural
network training, Markov modeling, hidden Markov modeling,
multidimensional scaling, PLS analysis, and/or PCA analysis. The
statistical or probabilistic analysis software optionally
quantitatively determines a concentration, proportion, or number of
the nucleic acids of interest in the sample.
[0176] Computers and software of the systems receive and evaluate
signal data from one or more analyses to provide quantitation
and/or proportionality determinations for nucleic acids of
interest. In a basic form, e.g., the amplitude or integrated area
of a signal can be adjusted with a conversion factor for an output
in desired units, such as, e.g., copies per nL, ng/.mu.L, and the
like. Alternately, one or more standard materials of known
concentration can be analyzed to provide data for regression
analyses wherein changes in detectable signals with changes in
concentration are expressed as an equation (standard curve) from
which unknown concentrations can be determined by insertion of one
or more signal parameters into the equation. In a particular
embodiment, quantitation of a nucleic acid of interest can be based
on the number of amplification cycles required to obtain a signal
of a certain intensity.
[0177] In the present invention, the computer typically includes
software for the monitoring of materials in the channels.
Additionally, the software is optionally used to control
electrokinetic or pressure modulated injection or withdrawal of
material. The injection or withdrawal is used to modulate the flow
rate as described above, to mix components, and the like.
[0178] Example System
[0179] FIGS. 6 and 7 provide a schematic illustration of a model
system of the invention. As shown in FIG. 6, system 600 includes
microfluidic device 601. Device 601 includes main channel 604
fabricated therein. Amplification components are flowed, e.g., from
reservoir 606, e.g., by applying a vacuum at vacuum source 608
(and/or at any of the reservoirs or wells noted below) through main
channel 604. Amplification components can also be flowed from wells
610 or 612 and into main channel 604, for example to form a
reaction mixture. Materials can be also flowed from wells 606 or
608, or materials can be flowed into these wells, e.g., when they
are used as waste wells, or when they are coupled to a vacuum
source. Flow from wells 614, 612, 610, 606, or 608 can be performed
by modulating fluid pressure, or by electrokinetic approaches.
Instead of the arrangement of channels depicted in FIGS. 6 and 7,
an arrangement such as the device of FIG. 1 can be substituted. A
variety of other appropriate microfluidic configurations are set
forth in the references noted herein.
[0180] Materials relevant to performing the amplification reactions
can be flowed from the enumerated wells, or can be flowed from a
source external to Device 601. As depicted, the integrated system
can include pipettor channel 620 (sipper), e.g., protruding from
device 601, for accessing an outside source of reagents. For
example, as depicted, pipettor channel 620 can access microwell
plate 622, which includes samples or sample aliquots, or locus
specific reagents, or other reagents useful in the practice of the
invention in the wells of the plate. Aliquots or reagents relevant
to amplification can be flowed into channel 604 through pipettor
channel 620. Detector 624 is in sensory communication with channel
604, detecting signals resulting, e.g., from the interaction of a
label with an amplicon as described above. Detector 624 is operably
linked to Computer 626, which digitizes, stores and manipulates
signal information detected by detector 624.
[0181] Voltage/pressure controller 628 controls voltage, pressure,
or both, e.g., at the wells of the system, or at vacuum couplings
fluidly coupled to channel 604 (or the other channels, wells, or
chambers noted above). Optionally, as depicted, computer 626
controls voltage/pressure controller 628. In one set of
embodiments, computer 626 uses signal information to select further
reaction parameters. For example, upon detecting amplification of a
nucleic acid of interest in a well from plate 622, the computer
optionally directs withdrawal of additional aliquots from the well
for analysis through pipettor channel 620, e.g., to deliver
different concentrations of the aliquot to the amplification
reaction. Similarly, upon determining that no nucleic acid is
present (a zero copy reaction) computer 626 can direct controller
628 to process another aliquot. If statistical information is
desired, computer 626 directs controller 628 to perform appropriate
fluid manipulations to generate enough data for the statistical
analysis. Computer 626 is optionally coupled to or comprises a user
viewable display, permitting control of the computer by the user
and providing a readout for the user to view results detected by
the system.
[0182] FIG. 7 depicts an alternate embodiment, in which a solid
phase array of reagents or samples is accessed by a microfluidic
system. As shown in FIG. 7, system 700 includes microfluidic device
701. Device 701 includes pipettor channel 720 and a microfluidic
network fabricated within the device. Amplification components,
such as primer pairs, polymerases, buffers, probes, etc., are
flowed through device 701, typically by applying pressure (positive
or negative) and/or electrokinetic pressure in the microfluidic
network.
[0183] As depicted, the integrated system can include pipettor
channel 720, e.g., protruding from device 701, for accessing an
outside source of reagents. For example, as depicted, pipettor
channel 720 can access solid phase array 725, which includes
samples or sample aliquots, or locus specific reagents, or other
reagents useful in the practice of the invention. Fluids are partly
or completely expelled from channel 720 to rehydrate materials on
array 725. For example, channel 720 can comprise a hanging drop
that is used to rehydrate materials, with the drop being withdrawn
into channel 720 for distribution into microfluidic device 701.
Detector 724 is in sensory communication with device 701 and
computer/controller 726. Computer/controller 726 can be operated in
a manner similar to computer 626 of FIG. 6. In either case,
computer 626 or computer controller 726 optionally control movement
of tray 622 or array 725, and/or microfluidic device 601 or 701 to
permit the relevant pipettor channel to process samples or other
materials on the array or in the wells of the tray.
[0184] Many variations of the above system are also appropriate.
For example, many types of heating systems can be used in the
present invention. For example, winding the channel around fixed
heating areas can be performed. Robotics or fluid system elements
can be used to heat fluids in multiple different temperature water
baths (e.g., 3 baths for a typical amplification reaction at
typical annealing, reaction and dissociation conditions).
[0185] Additional Kits Details
[0186] The present invention also provides kits for carrying out
the methods described herein. In particular, these kits typically
include system components described herein, as well as additional
components to facilitate the performance of the methods by an
investigator.
[0187] The kit also typically includes a receptacle in which the
system component is packaged. The elements of the kits of the
present invention are typically packaged together in a single
package or set of related packages. The package optionally includes
reagents used in the assays herein, e.g., buffers, amplification
reagents, sizing probe pairs, standard reagents, and the like, as
well as written instructions for carrying out the assay in
accordance with the methods described herein. In the case of
prepackaged reagents, the kits optionally include pre-measured or
pre-dosed reagents that are ready to incorporate into the methods
without measurement, e.g., pre-measured fluid aliquots, or
pre-weighed or pre-measured solid reagents that may be easily
reconstituted by the end-user of the kit.
[0188] Generally, the microfluidic devices described herein are
optionally packaged to include reagents for performing the device's
preferred function. For example, the kits can include any of
microfluidic devices described along with assay components,
reagents, sample materials, control materials, or the like. Such
kits also typically include appropriate instructions for using the
devices and reagents, and in cases where reagents are not
predisposed in the devices themselves, with appropriate
instructions for introducing the reagents into the channels and/or
chambers of the device. In this latter case, these kits optionally
include special ancillary devices for introducing materials into
the microfluidic systems, e.g., appropriately configured
syringes/pumps, or the like (in one preferred embodiment, the
device itself comprises a pipettor element, such as an
electropipettor for introducing material into channels and chambers
within the device). In the former case, such kits typically include
a microfluidic device with necessary reagents predisposed in the
channels/chambers of the device. Generally, such reagents are
provided in a stabilized form, so as to prevent degradation or
other loss during prolonged storage, e.g., from leakage. A number
of stabilizing processes are widely used for reagents that are to
be stored, such as the inclusion of chemical stabilizers (i.e.,
enzymatic inhibitors, microcides/bacteriostats, anticoagulants),
the physical stabilization of the material, e.g., through
immobilization on a solid support, entrapment in a matrix (i.e., a
gel), lyophilization, or the like.
EXAMPLES
[0189] The following examples are offered to illustrate, but not to
limit the claimed invention. It is understood that the examples and
embodiments described herein are for illustrative purposes only and
that various modifications or changes in light thereof will be
suggested to persons skilled in the art and are to be included
within the spirit and purview of this application and scope of the
appended claims.
[0190] Single Molecule Amplification and Detection of DNA in a
Microfluidic Format
[0191] Introduction
[0192] The amplification of a desired region of DNA by polymerase
chain reaction (PCR) has revolutionized the field of molecular
biology. In conventional formats of PCR, which use many microliters
of fluids during amplification, the starting DNA copy number is
typically at least hundreds to tens of thousands of molecules.
Recent advances in microfluidics have demonstrated that it is
feasible to miniaturize PCR down by a thousand fold to a
nanoliter-reaction volume range. When the sample concentration
remains constant, the starting number of DNA template in such a
small volume can drop below a cutoff copy number that could be
considered statistically unacceptable in some applications. For
instance, in single nucleotide polymorphism (SNP) analysis, if the
starting copy number is too low (below about a few tens of copies),
the amplification of the two different alleles from a heterozygous
sample may not amplify equally in quantity due to statistical
fluctuations, possibly causing uncertainty in a correct SNP
identification for that sample.
[0193] In the theoretical limit, only one DNA copy is necessary as
a starting template for a PCR reaction. From such a reaction, the
amplified product is a pure "clone" of a single parent DNA
template, instead of a mixture of many DNA parent templates. Single
molecule amplification and detection results in some interesting
applications that are not achievable otherwise. One such
application is the detection of cancer genes. This example
describes (1) a method to perform single molecule PCR using
microfluidic technology, (2) analysis and detection of single
molecule amplification, and (3) example applications using single
molecule PCR detection for cancer detection.
[0194] We have experimentally demonstrated that single molecule PCR
is possible in a microfluidic channel. In experiments in the
absence of flow, there is evidence in support of single molecule
PCR in that localized "clouds" of fluorescent probes (corresponding
to amplification products) were observed along the heated
amplification microchannel (thus also representing a "detection
region"). The evidence for single molecule PCR is more definitive
in a sipper chip continuous flow format, in which a very large
number of experiments can easily be conducted to obtain adequate
statistics to support experimental observations.
[0195] Continuous Flow Protocol
[0196] Using a microfluidic sipper chip as shown in the chip design
schematic of FIG. 1, a DNA sample (e.g., a genomic DNA) was brought
onto chip 100 through a sipper using a pressure gradient into
distribution channel 105. Under continuous flow, in an
assembly-line fashion, the sample was first mixed with a common
reagent from an on-chip reagent reservoir through common reagent
channel 106, then split into 8 equal aliquots into 8 independent
analysis channels 110-118. Each aliquot was mixed with
locus-specific reagents supplied from a channel-specific chip
reservoir to form a reaction mixture, then flowed through heated
region 130 comprising metal traces proximal to amplification
microchannel 110-118 to provide controlled heated regions of chip
100. Reagent addition for channel specific reagents into channels
110-118 provides an elegant microfluidic method of providing for an
on-chip "hot start," in which all of the reagents are added to
analysis channels just before amplification. The temperature of the
region was cycled appropriately (temperature set points and
respective dwell times are controlled) for PCR conditions in the
channels in heated region 130. Heated channel lengths and fluid
velocity are chosen such that the total PCR cycles meet a desired
number, usually between 25 to 40 cycles (though inefficient PCR
approaches that have short cycle times and high cycle numbers can
also be used; See also, U.S. Pat. No. 6,303,343 to Kopf-Sill (Oct.
16, 2001) entitled INEFFICIENT FAST PCR). 8 channel detection
region 135 comprises an appropriate detector for detecting PCR
amplicons in channels 110-118.
[0197] Amplification and Detection of Rare Molecules
[0198] We used the PCR sipper chip illustrated in FIG. 1 to
demonstrate single molecule PCR amplification, experimentally, in a
continuous flow format. DNA samples with increasing dilution, in
concentrations down to less than 1 molecule per nL, were prepared
in a microtiter plate which supplies the samples to the sipper (on
chip dilution could be performed in alternate embodiments). Due to
statistical fluctuations in sampling very low concentration DNA
down to below one molecule per channel, it is expected that some
channels will show amplification signals and some will not. The
fraction of tests at which amplification is observed is best
described by Poisson statistics.
[0199] Table 1 summarizes results of a set of PCR experiments when
the average copy number of DNA in each of the 8 channels varied
from 0.02 to 48. For each DNA concentration, 8 PCR experiments were
done simultaneously. The number of occurrences of a measurable PCR
fluorescent signal for each sample was recorded in Table 1, with
the maximum occurrence being 8 and the minimum being 0. The percent
of occurrence of PCR was calculated and compared with a Poisson
statistics prediction. A very good agreement between the measured
and predicted percent occurrence of PCR was found. Table 2
summarizes a replication of similar sets of experiments on another
day. FIG. 2 is a graphical comparison of predicted (Poisson) and
measured statistics (Run 1 and 2) for both sets of experiments.
Predicted and actual measurements show close agreement.
1TABLE 1 AVERAGE NUMBER OF COPIES IN THE CHANNEL PLATE PASS 0.02
0.05 0.1 0.2 0.4 0.8 1.5 3 6 12 24 48 1 0 0 0 2 2 3 5 6 6 8 8 8 2 0
0 3 0 0 5 7 8 8 8 8 8 3 0 0 1 1 2 2 5 7 8 8 8 8 4 2 0 0 1 3 2 5 7 8
8 8 8 5 0 0 2 0 1 4 3 7 8 8 8 8 6 1 0 2 2 3 5 5 7 7 8 8 8 7 2 1 0 0
3 3 5 7 8 8 8 8 8 0 3 1 4 1 2 6 7 8 8 7 8 9 1 2 0 3 1 5 8 7 7 8 8 8
10 0 1 0 1 2 5 7 8 8 8 8 8 11 0 0 0 1 4 4 5 7 8 8 8 8 12 0 0 0 0 3
8 7 8 8 8 8 8 13 0 0 0 1 3 3 5 7 8 8 8 8 14 0 0 1 1 2 3 6 6 8 8 8 8
15 0 0 0 0 2 6 6 8 8 8 8 8 16 0 0 0 0 1 5 3 8 8 8 8 8 17 0 0 0 0 0
2 5 6 8 8 8 8 18 0 1 0 2 2 4 4 7 8 8 8 8 19 0 0 0 2 1 2 5 8 8 8 8 8
TOTAL 6 8 10 21 36 73 102 136 148 152 151 152 POSSIBLE 152 152 152
152 152 152 152 152 152 152 152 152 (actual/possible) % 4% 5% 7%
14% 24% 48% 67% 89% 97% 100% 99% 100% POISSON 2% 5% 10% 18% 33% 55%
78% 95% 100% 100% 100% 100% PREDICT
[0200]
2TABLE 2 AVERAGE NUMBER OF COPIES IN THE CHANNEL PLATE PASS 0.02
0.05 0.1 0.2 0.4 0.8 1.5 3 6 12 24 48 1 0 0 1 1 5 4 5 7 8 8 8 8 2 1
1 0 2 2 3 5 6 7 8 8 8 3 0 0 2 2 2 3 2 8 8 8 8 8 4 1 0 0 0 3 3 5 5 8
8 8 8 5 0 0 1 1 1 2 5 8 8 8 8 8 6 0 0 0 2 2 3 4 8 7 8 8 8 7 0 0 0 2
1 4 5 7 8 8 8 8 8 0 1 2 0 0 3 5 6 8 8 8 8 9 0 0 0 2 1 2 3 6 8 8 8 8
10 1 0 0 0 3 1 4 6 7 8 8 8 11 0 0 1 0 4 5 5 6 8 8 8 8 12 0 0 0 2 3
2 3 7 7 7 8 8 13 3 1 4 0 1 1 4 6 7 8 8 8 14 0 1 2 1 1 4 7 6 7 8 8 8
15 0 0 2 1 0 5 4 8 8 8 8 8 16 0 0 0 1 1 4 3 7 8 8 8 8 17 0 0 0 2 3
4 4 4 8 8 8 8 18 0 0 1 0 4 0 6 7 8 8 8 8 19 0 1 0 1 1 3 2 8 8 8 8 8
20 1 0 1 3 2 2 4 7 8 8 8 8 TOTAL 7 5 17 23 40 58 85 133 154 159 160
160 POSSIBLE 160 160 160 160 160 160 160 160 160 160 160 160
(actual/ 4% 3% 11% 14% 25% 36% 53% 83% 96% 99% 100% 100% possible)
% POISSON 2% 5% 10% 18% 33% 55% 78% 95% 100% 100% 100% 100%
PREDICT
[0201] In a continuous flow mode, sipped samples broaden in plug
length due to molecular diffusion and flow-induced dispersion. For
a sipped sample containing tens or hundreds of copies of starting
DNA templates, the effect of diffusion and dispersion on the width
of the fluorescence PCR probe region can be predicted by
considering Taylor-Aris dispersion. For single molecule PCR,
similar calculations can be performed, and the peak shape of the
fluorescent product is less broad than a large sample plug
counterpart. The narrower peak is mainly due to the starting region
from which DNA is amplified being narrower in the single molecule
case (a few nm instead of hundreds of .mu.m). FIGS. 3A and 3B
summarize analysis of peak area and peak (slug) width as a function
of starting copy number of DNA in channels. The lower copy number
amplifications in fact showed narrow peaks as expected (and
vice-versa).
[0202] Evidence for the system amplifying and typing single
molecules also includes the fact that when the sample is a
heterozygote, all peaks are positive for one or the other TaqMan
probe, but not both.
[0203] There are other uses for single molecule typing that can be
performed according to the present invention as well. For example,
two TaqMan.TM. or molecular beacon assays can be developed for
sequences that are located close together in the genome. Those
assays can be used to determine whether the proximal sequences are
present on the same amplified molecule. This is an indirect way of
doing a sizing assay: one can ask whether individual molecules have
both TaqMan.TM./beacon sites, providing an indication of how often
molecules are of a size that encompasses both sites. One can also
type the two sites, providing a haplotyping method.
[0204] Experiment to Monitor PCR Amplification On-Chip by Measuring
Fluorescence Generated by TagMan Probe Cleavage.
[0205] This example provides an experiment to monitor PCR
amplification on-chip by measuring fluorescence generated by TaqMan
probe cleavage. FIG. 5 shows the peak width at 1/2 max vs.
calculated input copy number per channel (on-chip).
[0206] For this experiment, all necessary PCR reagents were loaded
on-chip. One DNA sample was diluted in assay buffer in a 384-well
plate (0.72 ng/.mu.L to 11.5 ng/.mu.L). The amplification cycle
time was 17 seconds (5 seconds at 90.degree. C., 7 seconds at
58.degree. C. and 5 seconds at 72.degree. C.). All injected samples
were subjected to a total of 35 amplification cycles. Samples were
injected for a total of 200 seconds, with a buffer wash (between
samples) of 350 seconds. Width of PCR signal (peak) was measured at
1/2 the peak maximum for each microfluidic channel on-chip (8
total). Data shows that amplification of a single molecule in any
channel produces the same width, in time (approximately 40-50
seconds). As more molecules (copies) are injected onto the chip,
they begin to overlap, causing the width of the peak to increase in
time. However, with long injection times, some single molecules
show up on the edges of the injected slug of DNA.
[0207] Application of Methods to Allele Typing
[0208] In cancer research, detection of cancer genes is very
difficult because the mutated gene usually occurs at a much lower
concentration then the wild type in a sample. The ability to detect
amplification from a single molecule would solve the problem of
detecting a low concentration of a mutated gene with a high
concentration of wild type in the background since one can now
study a single clone at a time. The use of the microfluidic sipper
chip format with parallelized PCR on the chip speeds up the rate at
which a single clone is examined at a time, to the point where it
is practical to do a massive number of PCRs to find the few mutated
genes responsible for cancer that exist in a given sample. FIG. 4
illustrates raw fluorescence intensity measurements for SNP
analysis at very low starting copy number to below 1 copy per
channel on average. These data show the possibility of detecting
SNP at single molecule PCR conditions.
[0209] FIGS. 10-12 show additional data from additional
experimental runs, demonstrating single molecule amplification. As
shown in FIGS. 10-11, a first set of experimental data with 100% of
a first SNP allele is shown on the left, while a second set of
experimental data with 1% of a second SNP allele (and 99% of the
first allele) is shown on the right. The top signal line on the
figure is data using a first dye detection system (which provides a
longer wavelength "red" signal) for detecting amplification of the
first allele, while the bottom line is data from a second dye
detection system (a shorter wavelength "blue" signal) for detecting
amplification of the second allele. The data represents about 700
total detected DNA molecules in one sample slug. As shown, only the
right side shows signal peaks corresponding to amplification of the
second SNP. The data prove that a system of the invention can
accurately amplify and detect rare molecules within a large
population. That is, as a model, two DNA samples were mixed, each
homozygous for the two alleles of a SNP. In this experiment, single
DNA molecules for one allele that were present in a large
population of DNA molecules of the other allele were detected (5-7
low copy alleles in about 700 for this case). FIG. 11 provides
results for 6 separate experimental runs, demonstrating that
characteristic peak shapes from molecule to molecule is constant,
providing experimental evidence that both PCR and dispersion of the
resulting amplicons are very reproducible. In fact, a
LabChip.RTM.-based system, as in the present invention, allows
unlimited sensitivity to rare molecules in that: 1) it is
practical, in a microfluidic system, to spread the sample out
through the channel such that rare molecules are present amongst
smaller numbers of wild-type molecules (reducing the problems
created by proportional amplification of starting materials in each
aliquot); and 2) reproducible fluidic handling and analysis gives a
predictable single molecule peak shape that can be used to
discriminate between molecular signals and random signal
fluctuations.
[0210] FIG. 12 provides a titration of a first SNP against a second
SNP, showing that the signal from the amplicons corresponding to
the first SNP ("FAM DNA," in the upper trace) and the second SNP
("VIC DNA," lower trace) show an appropriate signal correlation.
The given percentages correspond to the percentage of DNA from a
first homozygous sample (both alleles in the first homozygous
sample are "FAM", that is, the material from the homozygous sample
is "FF" homozygous) and a second homozygous sample (both alleles in
the are VIC DNA sample, "VV"). In this context, "FAM DNA" stands
for a DNA sequence that is probed for by a specific oligo sequence
with a FAM dye label, while "VIC DNA" stands for a DNA sequence
that is probed for by a specific oligo sequence with a VIC dye
label. "FF" stands for a homozygous DNA sample for the "FAM"
(oligo) sequence and "VV" stands for a homozygous DNA sample for
the "VIC" (oligo) sequence.
[0211] Demonstration of Detection of Cancer Markers
[0212] FIG. 13 provides an example of detection of 2 mutation sites
relevant to cancer detection developed on-chip, using TaqMan
probes. To demonstrate the relevance of the system of the invention
to cancer diagnostics, it was used to test a number of cancer
(e.g., colorectal cancer) markers using TaqMan probes. Two of those
assays are shown in FIG. 13: one for the K-RAS gene and one for the
p53 gene, both diagnostic markers for a variety of cancers, such as
colon cancer. The data trace shows fluorescence at two wavelengths
vs. time for one microfluidic channel. Two TaqMan probes, one
specific for the normal allele, and one specific for the mutant
allele, were designed and tested in this on-chip assay format. The
presence of normal DNA is detected with the wild-type probe (a
"red" signal, designated in the black and white reproduction on the
top data trace) and mutant DNA molecules are detected with the
mutant probe (a "blue" signal, represented in the black and white
reproduction as the bottom data trace). Most of the DNA molecules
(approximately 500) in the sample slug are normal, shown by the
high "red" top fluorescent signal and low "blue" bottom fluorescent
signal. This signal is produced by the allele-specific (red, top)
and background (blue, bottom) TaqMan probe cleavage surrounding the
amplification products of normal genomic DNA molecules. When a
mutant molecule (synthetic DNA template with the appropriate point
mutation) traverses the system, it is amplified and recognized as a
large blue (bottom) peak (with red (top) background peaks).
[0213] A Device and Method of Single Molecule Amplification by
Microfluidics that Permits Accurate Analysis of Heterogeneous
Nucleic Acid Mixtures.
[0214] Continuous flow PCR systems allow for spatial separation of
individual low, single copy, and zero copy amplification reaction
mixtures in a microfluidic processing environment. Normally,
spatial separation is used to separate different reactions, where
the starting template concentration is high enough to ensure
accurate representation of alleles coming from both parents (e.g.,
about 50 genome equivalents are often used). In the present
invention, the same task is accomplished by diluting the DNA enough
such that individual template molecules are separated such that the
amplification and detection products for each one are fluidically
separated. If the detection product is allele specific, a signal
for only one of the two alleles is detected. One can the count the
results for each allele, giving the genotype quite accurately. The
disadvantage for genotyping by this method is that the throughput
decreases: one needs many reactions to get a genotype, instead of
just one. Genotyping is typically performed with one reaction
because the starting concentrations in a two allele system is
usually about 50/50 (or at least on the same order of magnitude)
and the signal-to-noise ratio of the genotyping biochemistry is
good.
[0215] If, however, the representation of different alleles in the
starting sample differs enormously, the genotyping biochemistry is
not good enough to give an accurate quantitation of the
under-represented allele. In fact, as a practical matter, it is
very difficult to use many typical detection biochemistries for
detection of alleles that are present in as few as 1 in 5 copies.
In cancer, the mutant/normal ratio can be quite low (1 in
thousands) and therefore undetectable by conventional biochemical
methods. On the other hand, if one amplifies single molecules, the
reactions can be repeated and flowed in a continuous system for as
long as desired--and there is no theoretical limit of detection
Oust a practical one: if the mutant genotype is very rare, many
reactions will have to be detected, e.g., in the continuous flow
high throughput format).
[0216] This also provides a strategy for quantifying infectious
agents by PCR. Today, that is done by PCR or RT-PCR which depends
on a cycle-by-cycle quantification and comparison to a standard
curve of template molecules amplified under similar conditions. In
the present invention, we flowed the sample at a known flow rate
and measured the amplifications per unit volume as a more precise
and quantitative determination of the template concentration. One
can accomplish the same thing by amplifying dilute concentrations
of the sample in wells. When the total number of positive wells
equaled e.sup.0=0.37, there was a high statistical probability that
each well had only a single template molecule in it. One could also
have more than one molecule present in the flow stream at any given
time if an independent and reliable way of measuring the copy
number is used.
[0217] Single Molecule PCR in a Microfluidic Device Under Stop-Flow
Conditions for Virus Detection & Analysis.
[0218] The desired sensitivity for virus detection (e.g., about
50-100 copies/ml) make it a challenging application for detection
using a microfluidic platform, due to the mismatch between
processing volume on the chip and the initial sample volume.
However, one of the features of PCR in a microfluidic device
demonstrated in this application is the ability to quantitate
single copies of nucleic acids. This allows one to count the number
of infected cells, or virus particles in a sample of interest, at
biologically relevant concentrations of cells or virus particles.
In this example, we describe quantitative single molecule PCR from
a starting volume on the order of 10 microliters (an initial
pre-concentration step taking the sample from .about.1 ml to about
10 ul can be performed by standard techniques, e.g.
immunoprecipitation or hybridization capture into magnetic
beads).
[0219] The .about.10 ul of concentrated solution containing e.g.,
>50 virus particles can be completely processed (or a
substantial fraction of the volume processed) on a microfluidic
chip in the following manner. The sample is mixed on-chip with the
reagents necessary for PCR (at, e.g., a 1:1 ratio), e.g. primers,
probes, dNTPs, etc. The mixture is pressure loaded into a
microfluidic network that has a holdup volume on the order of 10 ul
(see, FIG. 8), and the flow is stopped. As shown, the schematic
device of FIG. 8 comprises PCR reagent well 801, sample well 802
vacuum/waste well 803, imaging area 804 (a detection region) and
microfluidic network 805. The contents of the network are then
thermocycled by applying heat externally to the chip, or,
optionally, via resistive or Joule heating. Upon completion of
thermocycling, the chip is imaged to locate all of the "clouds" of
fluorescence (see, FIG. 9), each corresponding, typically, to a
single copy of DNA from a virus particle.
[0220] FIG. 9 is a schematic of the fluidic network of FIG. 8 after
thermocycling. Spots 806 represent the fluorescence "clouds" from
single amplicons (e.g., in one example, virus particle amplicons).
Spots 806 can be counted for quantitative PCR. For this particular
application, it is likely that it is most efficient to image the
entire fluidic network of the chip simultaneously (e.g., CCD
imaging), rather than in a continuous flow mode with the detector
(e.g., a photodiode) at a fixed point. However, continuous flow
can, alternatively, be used. The active area of the chip for
imaging is typically on the order of 20.times.30 mm (but can,
optionally, be smaller or larger). This area is compatible with
high resolution imaging (.about.1-2 um resolution) via techniques
commonly used for DNA array applications. These can include CCD
imaging, confocal laser scanning, and/or the like.
[0221] The dynamic range for quantification is typically at least
2-3 orders of magnitude, depending, in part, on the size of the
chip. For a typical size of 20.times.30 mm, the dynamic range is
about 2 orders of magnitude. One way to estimate the dynamic range
is to examine the average separation between copies, and then
compute the expected diffusion distances over the course of an
experiment. A rough calculation to demonstrate that these types of
volumes can be processed on a microfluidic chip is presented
below.
[0222] To determine whether further concentration of a 10 uL sample
down to the nL range was necessary, the following calculation was
performed. The conclusion reached was that further concentration
was not necessary.
[0223] If one loads a series of parallel channels (e.g., 64) that
are 30 um deep, 120 um wide, and 30 mm long, the total volume in
these channels is 6.2 .mu.L. If it is further assumed that in the
6.2 .mu.L, half of the volume comes from PCR reagents and the other
half comes from the original 10 .mu.L sample, then roughly 3 .mu.L
out of 10 .mu.L are sampled per run, which is a reasonable volume
from a statistical sampling or a practical ease of use standpoint.
Furthermore, if the 10 .mu.L concentrated sample contains 100
particles from an initial starting 1 mL volume of plasma, then one
can detect about 30 PCR clouds per run, if the PCR efficiency is
close to 100%. These clouds would be 62 mm apart, on average, from
each other along the channel, or about 1 cloud in every 2
channels.
[0224] The next issue addressed is chip size and detection
practicality. If the 64 (2{circumflex over ( )}n binary split)
parallel channels are packed together with 200 um landing area
between, they will occupy 21 mm. So an area of 30 mm.times.21 mm
can be imaged (or scanned) to find the 30 PCR clouds (in stop flow
mode) that should appear in the channels. This is similar to the
size of a typical DNA assay chip, meaning that available chip
scanners can be used for the detection.
[0225] In summary, if 1 .mu.L is concentrated to 10 .mu.L and
placed into a chip well, further concentration is unnecessary for
detection. If anything is done to increase the volume (such as the
addition of neutralization chemical(s) to an elution buffer,
addition of lysing agents, etc.), a further concentration step can
be desirable. To avoid adding lysing agents, it can be desirable to
do an ultrasonic lysing of particles in the 10 .mu.L solution in
the well before aliquoting.
[0226] The following is one example protocol for quantitative
analysis by the above methods: 1) Off chip concentration, e.g., by
affinity capture (a standard technique) and elution to reduce the
sample from 1 mL to 10 .mu.L; 2) Place the 10 .mu.L concentrate in
chip well, apply ultrasonic power to lyse particles; 3) Load the
DNA sample into parallel channels with on chip addition of PCR
reagents by pressure, then stop flow; 4) Activate external heater
to perform PCR in stop flow mode for all channels; and, 5) Image or
scan the channel to detect signs of single molecule PCR.
[0227] One aspect of the invention provides methods to ensure
stopped flow conditions on a chip. There are a number of methods
that can be employed. For example, one can use temperature
sensitive materials (e.g. polymers), to create the stop-flow
condition. A simple method to achieve stopped flow is to use
standard chip-capillary or chip-tubing connections combined with a
valve.
[0228] Methods and Devices for Determining Whether Nucleic Acids of
Interest are of a Given Length
[0229] Single molecule amplification techniques of the invention
can be used to unambiguously determine whether a nucleotide of
interest in a reaction mixture is, e.g., fragmented or has a given
length between probes. For example, simultaneous signals from two
or more different probes hybridized to opposite ends of a nucleic
acid of interest in a single copy reaction mixture can provide a
high level of confidence that the nucleic acid is not fragmented.
This contrasts with conventional methods, such as dot blot
hybridizations to multiple target nucleic acid copies, wherein
coincidental detection of probes to each end of the nucleic acid of
interest can indicate the presence of full length nucleic acid of
interest and/or the presence of a one or more pair of unassociated
fragmented nucleic acids of interest.
[0230] The methods and systems for determining length can detect
the presence of individual full length or fragmented nucleic acids
of interest, as well as provide counts indicating proportions or
concentrations of fragmented nucleic acids of interest, even in
complex mixtures containing large amounts of additional nucleic
acids. Such counts can be subjected to statistical analyses for
reporting of validated assay results and correlation to associated
disease states.
[0231] Methods of Determining the Fragmentation Status of Nucleic
Acids
[0232] Samples containing nucleic acids of interest can be diluted
and/or contained in volumes small enough to provide reaction
volumes that include on the average about 1 individual nucleic acid
of interest (single copy) in a reaction mixture. If the single copy
nucleic acid of interest is hybridized with two probes
complimentary to separated sequences on the nucleic acid of
interest single copy, detection of the two hybridized probes at the
same time in the reaction mixture indicates the nucleic acid is not
fragmented between the separate probe target sequences. On the
other hand, if the nucleic acid is fragmented between the target
sequences, the single copy reaction mixture will contain only one
of the fragments and only one of the hybridized probes would be
detected. By diluting and/or subdividing the sample into such
single copy reaction mixtures, one can confidently determine
whether one or more nucleic acids of interest include at least a
length (a given length) including the sequences complimentary to
particular probes and the nucleic acid between the probes.
[0233] Methods of determining whether a nucleic acid is of a given
length can generally include, e.g., adjusting the concentration of
a sample containing the nucleic acid; contacting the nucleic acid
with probes, primer pairs, and a polymerase in a reaction mixture;
amplifying the nucleic acid to produce specifically detectable
amplicons; hybridizing probes to the nucleic acid and/or amplicons
in the reaction mixture; flowing reaction mixture constituents into
a detection region; detecting signals from hybridized probes; and,
interpreting the signals to provide fragment and given length
nucleic acid quantities or proportions. Such quantities or
proportions can be correlated to disease states that may be
associated with the sample source.
[0234] The concentration of nucleic acids of interest can be
adjusted to provide useful numbers of low copy, single copy, and/or
zero copy reaction mixtures in the methods of determining whether
the nucleic acids are of a given length. Samples can be
concentrated, e.g., by ultrafiltration, affinity capture, or
immunoprecipitation so that a suitable copy number is obtained in
reaction mixtures and detectors of the invention. Samples can be
diluted, e.g., by serial dilution in microwell plates or admixing
with buffers or reagents in a fluidic system dilution channel, to
obtain a desirable concentration of nucleic acids. In many
microfluidic embodiments, a concentration of about one nucleic acid
of interest molecule per nanoliter is desirable for reaction or
detection. In methods of the invention, the nucleic acid of
interest can be adjusted to a range from about 100 molecules per nL
to about 0.01 molecules per nL in the reaction mixtures, or from
about 10 molecules per nL to about 0.1 molecules per nL, or from
about 3 molecules per nL to about 1 molecule per nL.
[0235] It is generally useful in the methods of determining a given
length to amplify the nucleic acid of interest to a larger number
of probe target molecules for enhanced sensitivity. One can
envision detecting unamplified individual nucleic acids of interest
by using probes with highly specific or highly intense detectable
marker signals (e.g., amplified or sandwich probes). However, in a
preferred embodiment, the nucleic acids of interest are amplified
in a reaction mixture containing a polymerizing enzyme that can
make copies (amplicons) of sequences (and/or compliments) from
nucleic acids of interest. In a more preferred embodiment, the
nucleic acid(s) of interest are amplified by contacting with two or
more primer pairs and a polymerase enzyme in a polymerase chain
reaction (PCR).
[0236] Typically, the PCR amplification reaction takes place in an
amplification microchannel or microchamber of a microfluidic
device. This can provide high throughput, low sample use, and
isolation of single copy reaction mixtures. For example, reaction
mixture constituents including a temperature stable DNA polymerase
and one or more primer pairs can flow into a temperature controlled
amplification microchannel with the nucleic acid of interest.
Amplicons can be extended by polymerization from primer pairs
hybridized to the nucleic acid of interest at specific locations,
thus the primers define the amplicons. As is well known in the art
of PCR, and previously discussed above, repeated cycles of nucleic
acid melting, annealing to primers, and primer extension by the
polymerase can increase by many orders of magnitude the amount of
nucleic acid having the sequence (and second strand compliment)
defined by the primer pairs. In the methods of determining a given
nucleic acid length, two or more primer pairs are typically
provided in the reaction mixture to amplify two or more regions of
the nucleic acid of interest associated with the intended probes.
Regions of the amplified nucleic acid can include sequences
complimentary to the probes and spaced a given (known or unknown)
sequence length apart. In some embodiments, the probes themselves
can be members of the primer pairs defining the amplicons.
[0237] The nucleic acid of interest (and/or any amplicons) can be
contacted with two or more different probes to determine whether
the nucleic acid of interest includes the given length between
sequences complimentary to the probes. Where the nucleic acids of
interest are single stranded, two probes can hybridize to
complimentary sequences spaced by the given length along the
strand. Where the nucleic acids of interest are double stranded,
and/or have had a complimentary strand that was polymerized, e.g.,
in an amplification reaction, the probes can be complimentary to
and hybridize to sequences on either strand or both strands
separated at a distance of the given sequence length.
[0238] In an embodiment of the methods, the amplicons defined by
two or more primer pairs do not overlap and are separated by
sequences of about the given length, as shown in FIG. 14. If the
nucleic acid of interest 140 is not fragmented between the first
amplicon sequences defined by first primer pair 141 and the second
amplicon sequences defined by second primer pair 142, low or single
copy amplification reactions will include both first 143 and second
144 amplicons. Hybridization of such a reaction mixture with a
first and second probe, specific to the first and second amplicon
sequences, respectively, will provide coincident probe signals 145.
If the nucleic acid of interest 140 is fragmented between the first
amplicon sequences defined by first primer pair 141 and the second
amplicon sequences defined by second primer pair 142, single copy
amplification reactions will include either first 143 or second 144
amplicons, but not both. Hybridization of these single copy
reaction mixtures with the first and second probes, specific to the
first and second amplicon sequences, respectively, either first
probe signals 146, or second probe signals 147 will be detected,
but not both. Primer pairs in such amplifications typically define
amplicon sequences of about 100 base pairs, with the amplicons
ranging from more than about 5000 base pairs to about 20 base
pairs, or from about 50 base pairs to about 1000 base pairs.
[0239] In another embodiment, as shown in FIG. 15, first strand
primer 150 is shared between second strand primers 151 and 152 for
an amplification that overlaps at the sequence of the shorter
amplicon. If the nucleic acid of interest 153 is not fragmented
between the shorter amplicon 154 sequence and the longer second
strand primer 152 sequence, the amplification reaction will provide
both shorter 154 and longer 155 amplicons. In such a case,
hybridization to a probe specific to the shorter amplicon and a
probe specific to the longer amplicon will yield short probe
signals 156 coincident with long probe signals 157. If the nucleic
acid of interest 153 is fragmented between the shorter amplicon 154
sequence and the longer second strand primer 152 sequence, the
amplification reaction will provide only shorter 154 amplicon and
detection of a single copy reaction mixture will yield only short
probe signals 156. In some embodiments, the short amplicon can act
as a control sequence confirming the effectiveness of the
amplification and hybridization reactions. Optionally, the short
amplicon can act as an internal reference to indicate the total
number of total whole and fragmented nucleic acid of interest
present in the sample of reaction mixture. In embodiments where
nucleic acids in the sample are subject to random breakage (such as
occurs in apoptotic cells) the region defined in the shorter
amplicon can be far less likely to be fragmented than the region
defined in the longer amplicon. In these embodiments, where the
primer pairs share a common first strand primer, determination of a
given length can be based on detection a probe hybridizing to a
sequence outside of the shorter amplicon region, i.e., the
determination does not require coincidence of signals in a single
copy reaction. However, these embodiments can benefit, e.g., from
the discrete counting provided by single copy detection methods to
enhance the precision of counts and the confidence of statistical
inferences from the data. Primer pairs in such amplifications
typically define shorter amplicon sequences of about 100 base
pairs, as above. Primer pairs defining the longer amplicon are
typically separated by sequence lengths ranging from more than
about 5000 base pairs to about 200 base pairs, or from about 500
base pairs to about 2000 base pairs, or about 3000 base pairs.
[0240] Amplification reactions can be carried out using any
appropriate technique known in the art and as described in the
Amplifying the Aliquots section above. For example, the
amplification method can be PCR, RT-PCR, LCR, and/or any of the
various RNA mediated amplification methods.
[0241] Although basic methods of determining whether a nucleic acid
of interest has a given length include the use of one or two primer
pairs or probes, additional information about the length and
fragmentation status of a nucleic acid of interest can be obtained
by using more than two primer pairs or probes. The additional
probes and/or primer pairs can enhance the resolution of length
determination between and/or outside of the first and second primer
pairs and probes. For example, with an additional primer pair
amplifying a sequence between the first and second primer pairs,
detection of probed amplicons can yield additional information from
low or single copy reactions. In such a case, coincident detection
of only the first and additional probes can indicate the nucleic
acid of interest has the given length between the probes but a
break between the additional probe compliment and the second probe
compliment. Coincident detection of only the additional and second
signals can indicate the nucleic acid of interest has the given
length between the additional and second probe complimentary
sequences but a break between the first probe compliment and the
additional probe compliment. A non-fragmented nucleic of interest
in this case would have coincident signals from each of the first,
additional, and second probes in detection of a single copy
reaction. In another example, with the additional primer pair
amplifying a sequence outside sequences bracketed by the first and
second primer pairs, detection of amplicons interrogated with the
three appropriate probes can yield useful information from low or
single copy reactions. In such a case, coincident detection of only
the first and additional probes can indicate the nucleic acid of
interest has the given length between the probes but a break exists
between the first probe compliment and the second probe compliment.
Coincident additional and second signals can indicate the nucleic
acid of interest has the given length between the additional and
second probe compliments but a break exists between the first probe
compliment and the second probe compliment. A non-fragmented
nucleic of interest in this case would have coincident signals from
each of the first, additional, and second probes in a single copy
reaction. Such a nucleic acid of interest would have at least the
given length between the additional probe and the nearest of the
first or second probe, plus the given length between the first and
second probe. Further additional probes and/or associated primer
pairs can yield additional nucleic acid length information, as can
be appreciated by those skilled in the art.
[0242] Complimentary probes can specifically hybridize to nucleic
acids of interest and/or associated amplicons to provide one or
more signals from low or single copy hybridizations thereby
yielding information useful in determining whether the nucleic acid
has at least a given length. The probes can be hybridized under
conditions of stringency (e.g., buffer ionic strength and
temperature) suitable to provide the required level of specificity.
In many embodiments, probes are hybridized to nucleic acids under
highly stringent conditions. In preferred embodiments of
determining length, the probes are molecular beacon (MB) probes,
fluorescent resonance energy transfer (FRET) probes, or TaqMan.RTM.
probes, as described in the Amplification of Aliquots section
above. In preferred embodiments detectable markers provide
qualitatively different signals unique to each of the different
probes. Optionally, two or more different probes can have the same
signal and the coincident presence of the two probes in a single
copy reaction mixture can be detected as a signal, e.g., of double
amplitude or area.
[0243] Target nucleic acids of interest and/or associated amplicons
hybridized to complimentary probes can be subjected to marker
signal detection procedures. Amplification and/or hybridization
reaction mixtures can flow into a detection region for detection of
any signals present in the mixtures. Depending on configuration of
hardware, the detection region can be, e.g., microchambers or
microchannels, a region where the amplification reaction mixture
was formed or amplified, a region where the hybridization reaction
took place, a cuvette region downstream from reaction regions,
detection regions integral with or proximal to a microfluidic
device, and/or the like. The detector can be any type appropriate
to the marker signal and compatible with other system hardware, as
described above. The probes, hybridized and/or released from the
nucleic acid or amplicon, can be detected by flowing into or
through the detection region, the detector can be scanned across
the probes, or the probes can be detected in a two or three
dimensional detection region, e.g., using imaging technologies
known in the art.
[0244] Detected signals can be interpreted to provide detection of
a nucleic acid of interest and a determination of whether the
nucleic acid has at least a given length. As was discussed above
from the perspective of primers and amplicons, coincident detection
of one of more probes in a low or single copy reaction mixture can
provide information about the length of a nucleic acid of interest.
Theoretically, primers and amplicons are not necessary to
determining whether a nucleic acid of interest has a given length
or not, but amplification schemes can be useful to enhancing the
sensitivity of such determinations. Detection of signals in a low
or single copy reaction mixture from two or more probes that have
been specifically hybridized to a nucleic acid (or associated
amplicons) at sequences spaced a given distance along the nucleic
acid can indicate the nucleic acid is not fragmented between the
probes. Detection of a signal from only one probe can indicate the
presence of a break in the nucleic acid. Accumulated data of
detections from multiple low, single, and zero copy reaction
mixtures can yield information useful in quantitation,
proportioning, and correlating the nucleic acids present in
samples.
[0245] Nucleic acids of interest of differing length can be
quantified essentially as described throughout this specification,
particularly in the Counting and Statistically Analyzing a Nucleic
Acid of Interest section above. Signals detected from low, single,
and/or zero copy reaction mixtures can be interpreted and counted
to accumulate data useful in calculation of quantities. For
example, inferences can be made about the total amount of full
length or fragmented nucleic acid of interest in a sample based on
counts of coincident signals, solitary signals, and no signal (zero
copy) reactions. Appropriate adjustments can be made according to
dilution factors, efficiency factors, internal reference values,
and the like. Proportions of full length to fragmented nucleic
acids of interest can be determined based on the proportions of
associated signal counts. It is appreciated in the art that
acquisition of larger amounts of signal data can improve the
precision or accuracy of such quantitative determinations. For
example, it is preferred in the methods to evaluate a sample by
aliquotting the sample into at least 25 reaction mixtures with two
or fewer copies (including single copy and zero copy reactions) in
order to compile a statistically valid data set, e.g., to interpret
the fragmentation status of the sample, calculate the proportions
of fragments to unfragmented nucleic acids, to quantitate the
nucleic acids of interest, to make valid correlations to disease
states, and the like.
[0246] In another aspect, the shape, volume, width, height, length,
area, or ratio, of the one or more signals can be evaluated to
provide quantitative information about nucleic acids of interest.
These peak parameters of acquired signals can be subjected to
regression analyses to identify standard curve equations that most
closely reflect the parameter change with changed concentrations of
the nucleic acids of interest in samples. Where an assay includes
detection of different signals from two or more probes with
different detectable markers, the same or different peak parameters
can be input for regression analysis of the different detected
signals.
[0247] In one embodiment, the quantity of a nucleic acid of
interest in a sample can be determined by measuring the change in a
signal with increasing numbers of amplification cycles. Plotting
the change in signal strength with increasing amplification cycles
often results in a sigmoid curve. Certain precise points along the
curve, such as points of inflection, points with certain slopes,
points having a certain absolute signal strength, points having a
certain fraction of maximum (plateau) signal strength, and/or the
like, can be identified with high precision. Useful standard curves
can be prepared based on regression analyses of any of these
identifiable points from assay of standard materials of known
nucleic acid concentration. For example, a standard curve can be
prepared representing known nucleic acid of interest concentrations
versus amplification cycles required to attain the chosen
identifiable point. Such standard curves can be, e.g., plotted
curves of standard data or mathematical representations of such
curves. Concentrations of nucleic acids for unknown samples can be
determined with reference to the standard curve. Different degrees
of amplification can be consistently obtained, e.g., by flowing
amplification reactions through an active amplification region at
different rates, for different times, and/or different distances to
provide a series of reaction mixtures experiencing different
numbers of amplification cycles.
[0248] In one embodiment, different degrees of, e.g., PCR
amplification are provided by flowing the amplification reaction
mixture different distances in an actively cycling heated region of
a thermocycler channel. For example, amplification reactions can
flow into an actively cycling amplification region for a certain
distance before thermocycling is stopped. In this case, the front
edge of the flowing slug of reaction mixtures experiences more
amplification cycles while late entering mixtures nearer the
trailing edge experience fewer amplification cycles. If such a slug
of reaction mixtures were to continue flowing past a detector, the
detector signal output can be a reverse sigmoid curve as shown in
FIG. 16A. Alternately, a slug of amplification reaction mixtures
can flow into an inactive amplification region before starting
thermocycling so that reaction mixtures near the front edge flow
out of the thermocycler sooner to experience fewer amplification
cycles than reaction mixtures nearer the trailing edge. Detection
of this slug of reaction mixtures flowing past a detector can
provide a sigmoid curve detector signal output, as shown in FIG.
16B, indicating weak amplification at the front edge of the slug
and higher amplification for the trailing edge.
[0249] Information about the fragmentation state or integrity of
nucleic acids of interest can be correlated to disease states,
e.g., of the sample source organism. Correlation analyses known in
the art can be carried out, e.g., comparing qualitative,
quantitative, and/or proportion data on the length of certain
nucleic acids obtained using methods of the invention. For example,
disease states can be correlated to a quantity of unfragmented
nucleic acid, or a proportion of fragmented nucleic to nucleic acid
having a given length. A proportional threshold or quantitative
threshold can be established using statistical analyses to provide
an acceptable degree of confidence in identification of samples
possibly positive for the correlated disease state without
unacceptable false positive results. For example, a certain
proportion of fragmented to unfragmented nucleic acid in a stool or
cervical swab sample can identify the sample as likely to originate
from a patient having, e.g., colon or cervical cancer,
respectively.
[0250] Methods for Quantifying Nucleic Acids of Interest
[0251] Methods and systems of the invention can be used in various
formats to quantify nucleic acids of interest. The quantitative
assays can be configured to provide a desired quality of output
results. For example, assay parameters, such as, e.g., sensitivity,
accuracy, precision, and rates of false positives or false
negatives can be influenced by the design of particular assays.
Repetition of assays can increase precision. The quantitative
assays can be improved by evaluating signal peak parameters best
suited to provide valid results, e.g., with the desired range,
sensitivity and/or accuracy.
[0252] Comparison of a sample signal output peak to a series of
standard signal output peaks can indicate the concentration of a
nucleic acid of interest in a sample. For example, a series of
standard materials containing known mounts of the nucleic acid of
interest can each be amplified through the same number of
amplification cycles to produce a series of detectable signals, as
shown in FIG. 17. For each concentration of nucleic acid standard,
a different detectable signal peak associated with the amplicons
can be detected. In one embodiment, a sample with an unknown
concentration of the nucleic acid of interest is amplified the same
number of cycles as the standard materials. The resultant amplicons
are detected, with the same probe and detectable marker system as
for the standards, to provide a signal with certain distinctive
peak parameters. The signal from the sample can be evaluated to
identify signal peak parameters (e.g., the shape of signal peaks,
points of inflection on signal peaks, slopes of signal peaks,
signal peak amplitudes, signal peak areas, signal peak widths at
half height, etc.) most suitable for the analytical goal. For
example, peak area might provide the most accurate quantitative
comparison, while peak height might provide a more precise
comparison, and peak shape might provide suitable quantitative
comparisons over a broader range of concentrations. Identifying an
appropriate signal peak parameter for comparison in a particular
instance can be determined, e.g., using methods of assay
development and validation procedures well known in the art. The
precision and/or accuracy of nucleic acid quantification can be
enhanced by interpolating the comparison to intermediate values
between standard values, by running replicate standards, by
statistical analyses of repeated sample assays, by running
comparisons at two or more amplification levels, and/or the
like.
[0253] One method of enhancing reliability of nucleic acid
quantification by using multiple assay results depends on
comparison of amplification signal response curves between samples
and standards. For example, the amount of amplicon associated
signal generated over a number of amplification cycles can be
compared to the signals for standards of various known
concentrations. As shown schematically in FIG. 18A, a signal from
an amplified sample can start low 180, increase logarithmically at
some point 181, and taper off to a maximum signal plateau 182 with
increasing numbers of amplification cycles, thus describing a
sigmoid curve. Standard materials with different known
concentrations of the nucleic acid of interest can be amplified, as
shown in FIG. 18B, to provide a series of standard sigmoid curves.
Points along the sample and standard curves can be identified,
e.g., with various levels consistency. Identifiable points can be,
e.g., points of inflection, points having a certain slope, points
having a certain signal amplitude, points having a certain fraction
of a maximum (e.g., plateau asymptotic) signal amplitude, and the
like. A standard curve of concentration versus cycles to an
identifiable point can be prepared, as shown in FIG. 18C, so that
the concentration of the nucleic acid in a sample can be determined
from the number of amplification cycles it takes for the sample to
reach the identifiable point. For example, a point of maximum slope
183 (maximum rate of signal increase) can be precisely identified
on each of the standard curves. The cycles to maximum slope can be
plotted versus concentration to prepare a standard curve. The
nucleic acid of interest concentration of an unknown sample can be
read as the concentration 184 providing the number of cycles 185 to
the maximum slope from the standard curve. Of course, such
determinations do not necessarily require manual plotting of
standard curves or sample curves. As used herein, preparing curves
includes all means of expressing the relationships between relevant
factors (e.g., concentration and slope, signal parameter and
amplification cycles, identifiable points and concentration, etc.),
such as, e.g., data plotting, regression analysis, curve fitting,
determination of an equation, and/or the like whether accomplished
manually or with the aid of an analog or digital computer and
software. In this embodiment of nucleic acid of interest
quantitation, if is preferred standard and sample reaction mixtures
be cycled through different numbers of amplification cycles, e.g.,
as described in the Methods of Determining the Fragmentation Status
of Nucleic Acids section above.
[0254] The quantities and proportions of nucleic acids of a given
length and/or fragmented nucleic acids of interest can be
determined with high precision, e.g., by reference to standard
curves of concentration versus amplification cycles to identifiable
points generated for two or more probe signals. In this concept,
detectable signals associated with fragmented or unfragmented
nucleic acid of interest can be quantitated separately, e.g.,
according to the methods described in the paragraph above, to
evaluate the integrity of the nucleic acid of interest in a sample.
In one aspect, standard curves of concentration versus
amplification cycles to an identifiable point can be separately
plotted for signals associated with two or more amplicons of the
nucleic acid of interest. (A signal is associated with an amplicon,
e.g., if it originates from a nucleic acid probe that has
hybridized to a sequence of the amplicon and has detectable marker
providing the signal.) Cycles to the identifiable point for the
sample can be compared to the standard curves for each amplicon to
separately determine fragmented and given length nucleic acid of
interest concentrations. In a preferred embodiment, the signals are
detected from homogenous reaction mixtures. In another preferred
embodiment, the signals are detected from low or single copy
reaction mixtures to provide highly resolved data on the integrity
of the nucleic acid of interest.
[0255] Systems for Determining the Fragmentation Status of Nucleic
Acids
[0256] Systems of the invention can provide efficient processing
and well adapted hardware for high sensitivity differentiation of
the lengths of nucleic acids of interest in samples. Systems for
differentiating lengths of nucleic acids can be essentially as
described herein for single molecule amplifications, but, e.g.,
incorporating additional elements for amplification, detection,
interpretation, and/or correlation of multiple probes. The core
system for differentiating nucleic acid length includes, e.g., a
microfluidic device capable of containing low and single copy
reaction mixtures in microchannels or microchambers, detectors
capable of distinguishing one or more signals from a homogenous
reaction mixture, and a software system configured to interpret
single or coincidently detected signals to lengths of individual
nucleic acids from a sample. Additional subsystems can include
sample storage modules, retrieval modules, dilution modules, and
computers, as discussed above for single molecule amplification
systems in general.
[0257] An exemplary system for differentiating lengths of nucleic
acids, as shown in FIG. 19, can function as follows. Samples 190 in
wells of microtiter plates 191 can be held in storage module 192
until retrieval by retrieval module 193 and delivery for sampling
by microfluidic device 194. Samples are aspirated up a capillary
sipper tube 195 to microchannels 196 or microchambers of the
microfluidic device where integral dilution module 197 (e.g., a
dilution channel) appropriately dilutes the sample with reagents
198 and buffers 199 to constitute an amplification reaction
mixture. The reaction mixtures can be separately aliquotted into
each channel in multiple amplification region 200 where low,
single, and/or zero copy reaction mixtures can be exposed to
amplification conditions (e.g., thermocycling). Amplified reaction
mixture aliquots can flow into detection region 201 where signals
from detectable markers on one or more different probes can be
detected by detector 202. The detector can communicate with
computer 203 to transmit detector signals for interpretation and/or
correlation by system software.
[0258] Samples for nucleic acid differentiation in the systems can
be any that contain natural or unnatural nucleic acids. For
example, the samples can include a nucleic acid with single
nucleotide polymorphism (SNP), a cancer associated nucleic acid, a
nucleic acid from an infective agent, whole blood, serum, plasma,
stool, urine, a vaginal secretion, ejaculatory fluid, synovial
fluid, a biopsy, cerebrospinal fluid, amniotic fluid, or a forensic
nucleic acid. Samples can be stored in a storage module, as
described above, under environmental conditions of temperature,
light, and humidity conducive to long storage life of the samples.
Libraries of samples can be stored, e.g., as matrices of various
liquid aliquots in multiwell plates or dry spots on slides. The
samples can be located at positions in the storage module trackable
with an inventory system and accessible by a sample retrieval
system.
[0259] Retrieval modules can be employed in the systems of
differentiating nucleic acid length, e.g., to enhance the high
throughput capabilities of the system. The retrieval modules can
remove designated samples from a storage module and deliver them to
dilution modules or microfluidic devices for analysis. The
retrieval modules can have, e.g., robotic arms with 6 degrees of
freedom of movement, x-y plotted tray graspers, belt driven
conveyors, and/or the like. Bar code readers or radio frequency
identification systems can be incorporated into the retrieval
modules to identify and track tagged samples.
[0260] Reaction mixtures for determination of nucleic acid length
can be prepared to include a nucleic acid of interest, two or more
polymerase primer pairs, nucleotide triphosphates, buffers, and/or
two or more detectable probes. The reaction mixture constituents
can be combined in any order at any suitable time or place. The
reaction mixtures can be constituted outside the microfluidic
device, e.g., in microwell plates using manual or automated
pipettor systems. The reaction mixture can be diluted outside of
the microfluidic device, e.g., by manual serial dilutions or by
using an automated dilution module. The dilutions can provide a
concentration of the nucleic acid of interest resulting in desired
amounts of low, single, and zero copy reaction mixtures in
amplification microchannels, hybridization reactions, and/or
detection regions of the system. The reaction mixture can include,
e.g., two or more primer pairs defining amplicons and bracketing
sequences complimentary to probes a given distance apart on the
nucleic acid of interest. Preferred primer pairs define amplicons
ranging in size from about 50 base pairs to about 3000 base pairs,
or about 100 base pairs. In preferred embodiments, the primer pairs
have roughly the same melting temperatures and define amplicons of
about the same length. In one embodiment, the amplicons include a
shorter amplicon that overlaps a longer amplicon in a region having
sequences complimentary to one or more probes.
[0261] In one aspect, the systems can include, e.g., solid supports
to provide affinity concentrations of sample constituents for
analysis, or concentration of hybridization reaction mixture
constituents. Solid supports can have affinity elements, such as
oligonucleotides complimentary to reaction constituents (e.g.,
nucleic acids of interest, primers, probes, etc.), capable of
specifically capturing the constituents. The solid supports can be
useful in adjusting the concentration of nucleic acids of interest
for input into the systems. The solid supports can immobilize
constituents during amplification or hybridization steps. Probes
that have been hybridized to the nucleic acids of interest and/or
their associated amplicons can be captured in a detection region
for detection. Although certain reaction mixture constituents can
be immobilized on the solid supports while certain other
constituents can flow past to be removed, the mixture can still be
considered homogenous if different probes are present in the same
mixture at the time of detection.
[0262] One or more probes can be present in the amplification
reaction mixture and/or in the hybridization mixture to hybridize
with the nucleic acid of interest or associated amplicons. In some
embodiments, e.g., wherein detection of the longer amplicons
indicate unfragmented nucleic acid, signals from a single probe can
confirm the presence of unfragmented nucleic acid. However, in
preferred embodiments, the system includes two or more probes with
specificity for nucleic acid sequences separated a given distance
along the nucleic acid of interest. In many cases the signal can be
enhanced by providing alternate probes specific for sequences in
the second strand of a double stranded nucleic acid at about each
end of the given length; typically, the alternate probes hybridize
near the first strand probes but are not complimentary to the first
strand probes in order to avoid hybridization of primers to each
other. Preferred probes have significant signal changes associated
with binding to target nucleic acid of interest. This can provide,
e.g., a positive signal against a low background from a homogenous
mixture (i.e., without the need to separate unhybridized probe from
the reaction mixture). Although probes can have any of the variety
of detectable markers described above, preferred probes for
differentiating the length of a nucleic acid are molecular beacon
(MB), TaqMan.RTM., and fluorescent resonance energy transfer (FRET)
probes.
[0263] In an aspect of the invention, multiple assays can be run in
a single reaction mixture (multiplexing). For example, a reaction
mixture can include independently detectable probe pairs for more
than one nucleic acid of interest. A reaction mixture can be
constituted to include, e.g., a pair of probes complimentary to
sequences at the ends of a first given length of a first nucleic
acid of interest, and another pair of probes complimentary to
sequences at the ends of a second given length of another nucleic
acid of interest. The four probes can each have different
detectable markers so they can be individually detected in reaction
mixtures. Should a reaction mixture in the detector region
coincidently emit three probe signals, system software could
unambiguously interpret this to indicate the presence of a single
copy of an identifiable first nucleic acid of interest having the
given length and a single fragmented copy of the other nucleic
acid. One skilled in the art can envision other unambiguous signal
combinations using such a multiplexing scheme.
[0264] Microfluidic devices in systems for differentiating nucleic
acid lengths can include chips with reagent wells, gas, liquid and
electrical contact ports, sample sippers, microchannels and
microchambers, amplification microchannels, and detection regions,
as described above in the Example Systems section. In preferred
embodiments, the microfluidic device includes such features as a
sipper, multiple amplification channels (such as shown in FIG. 1),
and detection region configured for laser excitation and
fluorescent detection at multiple frequencies.
[0265] Detectors in the systems can be any configured with a
capability to detect two or more signals from a homogenous mixture
of reaction mixture constituents. The detector can be appropriate
for the type of detectable marker signal provided by the hybridized
probes. The detector can include, e.g., a fluorometer, a charge
coupled device, a laser, an enzyme, or an enzyme substrate, a photo
multiplier tube, a spectrophotometer, scanning detector,
microscope, a galvo-scanner, etc. The detectors can monitor a
detection region into which amplification reaction mixtures,
aliquots, hybridization reaction mixtures, or reaction mixture
constituents have flowed. As hybridization actuated fluorescent
probes are preferred in the methods, preferred detectors in the
systems are fluorometers. Although the presence of two probes with
the same detectable marker signals can be distinguished, it is
preferred that the signals be different and the detector be capable
of distinguishing two or more different signals coincidently from a
homogenous mixture. In a more preferred embodiment, the detector
comprises a laser light excitation source directed to the detection
region through optic fibers, and a photodiode array capable of
simultaneously detecting two or more emission wavelengths.
[0266] A software system can be an element of the system to
interpret one or more signals and/or to correlate the signals to a
disease state. The software system can include, e.g., algorithms to
count signals, calculate concentrations, calculate proportions,
prepare standard curves, and/or evaluate correlations, e.g., to
disease states. The software can interpret, e.g., coincidence of
two or more signals to indicate the presence of a nucleic acid of a
given length in the detection region. The software can interpret,
e.g., a single signal to indicate the presence of a fragmented
nucleic acid in the detection region. The software can calculate a
quantity of a nucleic acid of interest by factoring in information,
such as, e.g., the number of signal counts, reaction volumes,
dilution or concentration factors, efficiency factors, known input
values and constants, signal peak shape, and/or the like. The
software can interpret peak parameters, such as a volume, width,
height, length, area, and/or a ratio, of the signals detected by
the detector to indicate a number of copies of the nucleic acid of
interest in the sample, a number of the nucleic acids of interest
having a given length, or a proportion of nucleic acids of interest
having different lengths. Software systems can correlate results of
one or more signal detection to disease states, e.g., by comparing
a validated quantity or proportion threshold to assay results for a
sample.
[0267] Computers can be an important element of systems for
differentiating lengths of nucleic acids. Computers can coordinate
control activities in the system, such as, e.g., sample
identification, sample retrieval, sample sipping, control of
microchip pressures and voltages, receipt of detector signals, and
software interpretation of signals. In preferred embodiments, the
computer is in communication with the signal detector to receive,
store, and evaluate signals from sample or standard assays.
Computers in the systems can be as described in the Computers
section above. For example, systems in the present invention can
include, e.g., a digital computer with data sets and instruction
sets entered into a software system to practice the methods of
determining lengths described herein. The computer can be, e.g., a
PC (Intel x86 or Pentium chip--compatible with DOS.RTM., OS2.RTM.,
WINDOWS.RTM. operating systems) a MACINTOSH.RTM., Power PC, or
SUN.RTM. work station (compatible with a LINUX or UNIX operating
system) or other commercially available computer which is known to
one of skill. The computer can be, e.g., a simple logic device,
such as an integrated circuit or processor with memory, integrated
into the system. Software for interpretation of detector signals is
available, or can easily be constructed by one of skill using a
standard programming language such as Visualbasic, Fortran, Basic,
Java, or the like.
[0268] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art, from a reading of this disclosure, that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, many of the
techniques and apparatus described above can be used in various
combinations.
[0269] All publications, patents, patent applications, and/or other
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, and/or other
document were individually indicated to be incorporated by
reference for all purposes.
* * * * *
References