U.S. patent application number 11/837581 was filed with the patent office on 2008-09-04 for method and device for detecting the presence of a single target nucleic acid in samples.
This patent application is currently assigned to CYTONIX. Invention is credited to James F. BROWN, Jonathan E. SILVER.
Application Number | 20080213766 11/837581 |
Document ID | / |
Family ID | 25276672 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080213766 |
Kind Code |
A1 |
BROWN; James F. ; et
al. |
September 4, 2008 |
METHOD AND DEVICE FOR DETECTING THE PRESENCE OF A SINGLE TARGET
NUCLEIC ACID IN SAMPLES
Abstract
A method comprising for each individual sample of a plurality of
samples, loading at least one sample portion of the individual
sample into at least one respective sample chamber of a plurality
of sample chambers, subjecting the sample portions to at least a
first amplification step; and then determining whether sample
portions contain at least one molecule of the target nucleic acid.
For each sample portion, if the sample portion contains at least a
single molecule of the target nucleic acid, the sample portion
would attain a detectable concentration of the target nucleic acid
after a single round of amplification.
Inventors: |
BROWN; James F.; (Clifton,
VA) ; SILVER; Jonathan E.; (Bethesda, MD) |
Correspondence
Address: |
BURR & BROWN
PO BOX 7068
SYRACUSE
NY
13261-7068
US
|
Assignee: |
CYTONIX
Beltsville
MD
Health and Human Services United States of America as
represented by the Secretary
|
Family ID: |
25276672 |
Appl. No.: |
11/837581 |
Filed: |
August 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10798857 |
Mar 11, 2004 |
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11837581 |
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10131854 |
Apr 25, 2002 |
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10798857 |
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09563714 |
May 2, 2000 |
6391559 |
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10131854 |
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08838262 |
Apr 17, 1997 |
6143496 |
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09563714 |
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10131854 |
Apr 25, 2002 |
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08838262 |
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09563714 |
May 2, 2000 |
6391559 |
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10131854 |
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08838262 |
Apr 17, 1997 |
6143496 |
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09563714 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
B01L 3/5027 20130101;
B01L 3/5088 20130101; B01J 2219/00644 20130101; B01J 2219/00621
20130101; B01L 2300/0819 20130101; B01J 2219/00637 20130101; C12Q
1/6806 20130101; B01J 2219/00722 20130101; B01L 2200/16 20130101;
C12Q 1/6844 20130101; B01J 2219/00317 20130101; B01J 2219/00677
20130101; B01L 3/50851 20130101; B01L 2400/0409 20130101; C40B
60/14 20130101; B01L 2200/0642 20130101; B01L 2200/06 20130101;
C12Q 1/686 20130101; Y10S 436/809 20130101; Y10T 436/2575 20150115;
B01L 2300/0636 20130101; Y10S 436/805 20130101; B01J 2219/00659
20130101; B01L 2200/10 20130101; B01L 3/5085 20130101; C07H 21/00
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for detecting, for each of a plurality of sample
portions, whether the sample portion includes at least one molecule
of a target nucleic acid, said method comprising: for each
individual sample of a plurality of samples, loading at least one
sample portion of said individual sample into at least one
respective sample chamber of a plurality of sample chambers, each
said sample portion comprising at least a part of said individual
sample, whereby for each individual sample portion, if said sample
portion contains at least a single molecule of said target nucleic
acid, said sample portion would attain a detectable concentration
of said target nucleic acid within a portion of said sample portion
after a single round of amplification; subjecting said sample
portions to at least a first amplification step; and then for each
of a plurality of said sample portions, determining whether said
sample portion contains at least one molecule of said target
nucleic acid.
2. A method as recited in claim 1, wherein said first amplification
step is a homogeneous amplification step.
3. A method as recited in claim 1, wherein said first amplification
step is a thermocycle step
4. A method as recited in claim 1, wherein if a first sample
portion contains at least a single molecule of said target nucleic
acid, said first sample portion would attain a detectable
concentration of said target nucleic acid within a portion of said
first sample chamber after a single amplification step.
5. A method as recited in claim 1, wherein said determining whether
said sample portion contains at least one molecule of said target
nucleic acid is performed by carrying out a procedure which
generates signals having magnitude which is higher where said
detectable concentration is present than where said detectable
concentration is not present.
6. A method as recited in claim 5, wherein said procedure comprises
detecting fluorescence from fluor-labeled materials.
7. A method as recited in claim 5, wherein said procedure comprises
detecting at least one chemical property which changes upon
hybridization.
8. A method as recited in claim 5, wherein said procedure comprises
evaluating at least one property selected from among the group
consisting of agglutination, turbidity, phosphorescence, light
scattering, light absorbance, fluorescence energy transfer,
fluorescence quenching, fluorescence dequenching, time-delayed
fluorescence, chemiluminescence and calorimetric evaluation.
9. A method as recited in claim 1, wherein said sample chamber
comprises at least a portion of an inside of a microcapillary
device, and wherein said peaks are generated by detecting regions
within said microcapillary device in which said detectable
concentration is present.
10. A method as recited in claim 1, wherein each of said sample
chambers has a volume of about 1 picoliter or less.
11. A method as recited in claim 1, wherein each of said sample
chambers has a volume in the range of from about 1 picoliter to
about 1 microliter.
12. A method as recited in claim 1, wherein each of said sample
chambers has a volume of about 10 picoliters or less.
13. A method as recited in claim 1, wherein each of said sample
chambers has a volume of about 100 picoliters.
14. A method as recited in claim 1, wherein each of said sample
chambers has a volume of about 1 nanoliter.
15. A method as recited in claim 1, wherein each of said sample
chambers has a volume of 10 nanoliters or less.
16. A method as recited in claim 1, wherein each of said sample
chambers has a volume of about 10 nanoliters.
17. A method as recited in claim 1, wherein each of said sample
chambers has at least one dimension of 2 mm or less.
18. A method as recited in claim 1, wherein each of said sample
chambers has at least one dimension of 1 mm or less.
19. A method as recited in claim 1, wherein each of said sample
chambers has at least one dimension of 100 microns or less.
20. A method as recited in claim 1, wherein each of said sample
chambers has at least one dimension of 20 microns or less.
21. A method as recited in claim 1, wherein each of said sample
chambers has at least one dimension of a few microns or less.
22. A method as recited in claim 1, wherein each of said sample
portions has a volume of about 1 picoliter or less.
23. A method as recited in claim 1, wherein each of said sample
portions has a volume in the range of from about 1 picoliter to
about 1 microliter.
24. A method as recited in claim 1, wherein each of said sample
portions has a volume of about 10 picoliters or less.
25. A method as recited in claim 1, wherein each of said sample
portions has a volume which is nanoliter-sized.
26. A method as recited in claim 1, wherein each of said sample
portions has a volume of about 1 nanoliter or less.
27. A method as recited in claim 1, wherein each of said sample
portions has a volume of about 10 nanoliters or less.
28. A method as recited in claim 1, wherein each of said sample
portions has a volume of about 100 nanoliters or less.
29. A method as recited in claim 1, wherein each of said sample
portions has a volume of about 1 microliter or less.
30. A method as recited in claim 1, wherein each of said sample
portions is confined in at least one dimension by opposing barriers
separated by about 2 mm or less.
31. A method as recited in claim 1, wherein each of said sample
portions is confined in at least one dimension by opposing barriers
separated by about 1 mm or less.
32. A method as recited in claim 1, wherein each of said sample
portions is confined in at least one dimension by opposing barriers
separated by about 500 microns or less.
33. A method as recited in claim 1, wherein each of said sample
portions is confined in at least one dimension by opposing barriers
separated by about 100 microns or less.
34. A method as recited in claim 1, wherein each of said sample
portions is confined in at least one dimension by opposing barriers
separated by about 20 microns or less.
35. A method as recited in claim 1, wherein each of said sample
portions is confined in at least one dimension by opposing barriers
separated by a few microns or less.
36. A method as recited in claim 1, wherein each said sample
chamber comprises at least a portion of an inside of a
microcapillary device.
37. A method as recited in claim 36, wherein said microcapillary
device has an internal volume of about 100 nanoliters or less.
38. A method as recited in claim 36, wherein said microcapillary
device has an internal volume of about 10 nanoliters or less.
39. A method as recited in claim 36, wherein said microcapillary
device has an internal volume of about 1 nanoliter or less.
40. A method as recited in claim 36, wherein each of said sample
portions has a volume of about 60 nanoliters or less.
41. A method as recited in claim 36, wherein each of said sample
portions has a volume of about 100 nanoliters or less.
42. A method as recited in claim 36, wherein said microcapillary
device has an inner diameter in the range of from 20 micrometers to
75 micrometers.
43. A method as recited in claim 36, wherein said microcapillary
device has an inner diameter of about 100 micrometers.
44. A method as recited in claim 36, wherein said microcapillary
device has an inner diameter of about 100 micrometers or less.
45. A method as recited in claim 36, wherein said microcapillary
device has an inner diameter of about 500 micrometers or less.
46. A method as recited in claim 36, wherein said microcapillary
device has a length in the range of from about 1 mm to about 100
mm.
47. A method as recited in claim 36, wherein said microcapillary
device has a length of about 4 cm.
48. A method as recited in claim 36, wherein said microcapillary
device comprises at least two surfaces spaced from each other by
about 20 micrometers or less.
49. A method as recited in claim 1, wherein each said sample
chamber comprises at least a part of a porous sample structure.
50. A method as recited in claim 49, wherein at least one of said
porous sample structures comprises a plurality of pores, each of
said pores having a first end and a second end, said first end of
each of said pores being open.
51. A method as recited in claim 49, wherein at least one of said
porous sample structures comprises a plurality of pores, each of
said pores having a first end and a second end, said first and
second ends of each of said pores being open.
52. A method as recited in claim 49, wherein at least one of said
porous sample structures comprises a plurality of pores, each of
said pores having a first end and a second end, said first end of
each of said pores being hydrophobic.
53. A method as recited in claim 49, wherein said first porous
sample structure comprises a plurality of pores, each of said pores
having a hydrophilic interior.
54. A method as recited in claim 49, wherein at least one of said
porous sample structures comprises a plurality of pores, an
interior of each of said pores being hydrophilic, and an exposed
surface of said porous sample structure being hydrophobic.
55. A method as recited in claim 49, wherein at least one of said
porous sample structures comprises at least one structure selected
from the group consisting of microchannel arrays, structures having
pores formed therein, metal screens, plastic screens, glass
screens, ceramic screens, cellulosic screens, polymeric screens,
metal sieves, plastic sieves, glass sieves, ceramic sieves,
cellulosic sieves and polymeric sieves.
56. A method as recited in claim 49, wherein said porous sample
structures are positioned in a microfluidic device which comprises
a plurality of porous sample structures.
57. A method as recited in claim 49, wherein each of said sample
portions has an internal volume of about 1 picoliter or less.
58. A method as recited in claim 49, wherein each of said sample
portions has an internal volume of about 10 picoliters.
59. A method as recited in claim 49, wherein each of said sample
portions has an internal volume of about 10 nanoliters or less.
60. A method as recited in claim 49, wherein each of said sample
portions has an internal volume of about 100 nanoliters or
less.
61. A method as recited in claim 49, wherein each of said sample
portions has an internal volume in the range of from about 100
nanoliters to about 1 microliter.
62. A method as recited in claim 49, wherein said porous sample
structure has an internal volume of about 1 picoliter or less.
63. A method as recited in claim 49, wherein said porous sample
structure has an internal volume in the range of from about 1
picoliter to about 1 microliter.
64. A method as recited in claim 49, wherein said porous sample
structure has an internal volume of about 1 nanoliter or less.
65. A method as recited in claim 49, wherein said porous sample
structure has an internal volume of about 10 nanoliters or
less.
66. A method as recited in claim 49, wherein said porous sample
structure has an internal volume of about 100 nanoliters or
less.
67. A method as recited in claim 49, wherein said porous sample
structure has an internal volume of about 1 microliter or less.
68. A method as recited in claim 49, wherein said porous sample
structure has an internal volume of about 1 micron.
69. A method as recited in claim 1, wherein each of said sample
chambers contains at least one amplification targeting reagent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 10/798,857 (the entirety of which is
incorporated herein by reference), filed on Mar. 11, 2004, which is
a divisional application of U.S. patent application Ser. No.
10/131,854, filed on Apr. 25, 2002, which is a divisional
application of U.S. patent application Ser. No. 09/563,714, filed
on May 2, 2000 (now U.S. Pat. No. 6,391,559), which is a divisional
application of U.S. patent application Ser. No. 08/838,262, filed
Apr. 17, 1997 (now U.S. Pat. No. 6,143,496).
[0002] This application is also a continuation application of U.S.
patent application Ser. No. 10/131,854 (the entirety of which is
incorporated herein by reference), filed on Apr. 25, 2002, which is
a divisional application of U.S. patent application Ser. No.
09/563,714, filed on May 2, 2000 (now U.S. Pat. No. 6,391,559),
which is a divisional application of U.S. patent application Ser.
No. 08/838,262, filed Apr. 17, 1997 (now U.S. Pat. No.
6,143,496).
FIELD OF THE INVENTION
[0003] The present invention relates to the in vitro amplification
of a segment of nucleic acid, methods to analyze concentrations of
specific nucleic acids in sample fluids, and methods for detecting
amplification of a target nucleic acid sequence. The present
invention also relates to miniaturized analytical assemblies and
methods of filling miniaturized analytical assemblies.
BACKGROUND OF THE INVENTION
[0004] Nucleic acid amplification techniques such as polymerase
chain reaction (PCR), ligase chain reaction (LCR), strand
displacement amplification (SDA), and self-sustained sequence
replication (3SR) have had a major impact on molecular biology
research. In particular, PCR, although a relatively new technology,
has found extensive application in various fields including the
diagnosis of genetic disorders, the detection of nucleic acid
sequences of pathogenic organisms in clinical samples, the genetic
identification of forensic samples, and the analysis of mutations
in activated oncogenes. In addition, PCR amplification is being
used to carry out a variety of tasks in molecular cloning and
analysis of DNA. These tasks include the generation of specific
sequences of DNA for cloning or use as probes, the detection of
segments of DNA for genetic mapping, the detection and analysis of
expressed sequences by amplification of particular segments of
cDNA, the generation of libraries of cDNA from small amounts of
mRNA, the generation of large amounts of DNA for sequencing, the
analysis of mutations, and for chromosome crawling. During the next
few years, PCR, other amplification methods, and related
technologies are likely to find increasing application in many
other aspects of molecular biology.
[0005] Unfortunately, problems exist in the application of PCR to
clinical diagnostics. Development has been slow due in part to:
labor intensive methods for detecting PCR product; susceptibility
of PCR to carryover contamination--false positives due to
contamination of a sample with molecules amplified in a previous
PCR; and difficulty using PCR to quantitate the number of target
nucleic acid molecules in a sample. A need exists for a simple
method of quantitative analysis of target nucleic acid molecules in
a sample.
[0006] Recently, significant progress has been made in overcoming
some of the problems of clinical diagnostic nucleic acid
amplification through the development of automatable assays for
amplified product that do not require that the reaction vessel be
opened, thereby minimizing the risk of carryover contamination.
Most of these assays rely on changes in fluorescent light emission
consequent to hybridization of a fluorescent probe or probes to
amplified nucleic acid. One such assay involves the hybridization
of two probes to adjacent positions on the target nucleic acid. The
probes are labeled with different fluors with the property that
energy transfer from one fluor stimulates emissions from the other
when they are brought together by hybridization to adjacent sites
on the target molecule.
[0007] Another assay, which is commercially available, is the
"TaqMan" fluorescence energy transfer assay and kit, available from
Perkin Elmer, Applied Biosystems Division, Foster City, Calif. This
type of assay is disclosed in the publication of Holland et al.,
Detection of specific polymerase chain reaction product by
utilizing the 5'.fwdarw.3' exonuclease activity of Thermus
aquaticus DNA polymerase, Proc. Natl. Acad. Sci. USA, Vol. 88, pp.
7276-7280, August 1991, and in the publication of Livak et al.,
Oligonucleotides with Fluorescent Dyes at Opposite Ends Provide a
Quenched Probe System Useful for Detecting PCR Product and Nucleic
Acid Hybridization, PCR Methods and Applic., 4, pp. 357-362 (1995).
The "TaqMan" or 5' exonuclease assay uses a single nucleic acid
probe complementary to the amplified DNA and labeled with two
fluors, one of which quenches the other. If PCR product is made,
the probe becomes susceptible to degradation via an exonuclease
activity of Taq polymerase that is specific for DNA hybridized to
template ("TaqMan" activity). Nucleolytic degradation allows the
two fluors to separate in solution which reduces quenching and
increases the intensity of emitted light of a certain wavelength.
Because these assays involve fluorescence measurements that can be
performed without opening the amplification vessel, the risk of
carryover contamination is greatly reduced. Furthermore, the assays
are not labor intensive and are easily automated.
[0008] The TaqMan and related assays have provided new ways of
quantitating target nucleic acids. Early methods for quantitation
relied on setting up amplification reactions with known numbers of
target nucleic acid molecules and comparing the amount of product
generated from these control reactions to that generated from an
unknown sample, as reviewed in the publication by Sninsky et al.
The application of quantitative polymerase chain reaction to
therapeutic monitoring, AIDS 7 (SUPPL. 2), PP. S29-S33 (1993).
Later versions of this method used an "internal control", i.e., a
target nucleic acid added to the amplification reaction that should
amplify at the same rate as the unknown but which could be
distinguished from it by virtue of a small sequence difference, for
example, a small insertion or deletion or a change that led to the
gain or loss of a restriction site or reactivity with a special
hybridization probe, as disclosed in the publication by
Becker-Andre, et al., Absolute mRNA Quantification using the
polymerase chain reaction (PCR). A novel approach by a PCR aided
transcript titration assay (PATTY), Nucleic Acids Res., Vol. 17,
No. 22, pp. 9437-9446 (1989), and in the publication of Gilliland
et al., Analysis of cytokine mRNA and DNA: Detection and
quantitation by competitive polymerase chain reaction, Proc. Natl.
Acad. Sci. USA, Vol. 87, pp. 2725-2729 (1990). These methods have
the disadvantage that slight differences in amplification
efficiency between the control and experimental nucleic acids can
lead to large differences in the amounts of their products after
the million-fold amplification characteristic of PCR and related
technologies, and it is difficult to determine relative
amplification rates accurately.
[0009] Newer quantitative PCR methods use the number of cycles
needed to reach a threshold amount of PCR product as a measure of
the initial concentration of target nucleic acid, with DNA dyes
such as ethidium bromide or SYBR.TM. Green I, or "TaqMan" or
related fluorescence assays used to follow the amount of PCR
product accumulated in real time. Measurements using ethidium
bromide are disclosed in the publication of Higuchi, et al.,
Simultaneous Amplification and Detection of Specific DNA Sequences,
BIO/TECHNOLOGY, Vol. 10, pp. 413-417 (1.992). "TaqMan" assays used
to follow the amount of PCR product accumulated in real time are
disclosed in the publication of Heid et al., Real Time Quantitative
PCR, Genome Research, Vol. 6, pp. 986-994 (1996), and in the
publication of Gibson et al., A Novel Method for Real Time
Quantitative RT-PCR, Genome Research, Vol. 6, pp. 995-1001 (1996).
However, these assays also require assumptions about relative
amplification efficiency in different samples during the
exponential phase of PCR.
[0010] An alternative method of quantitation is to determine the
smallest amount of sample that yields PCR product, relying on the
fact that PCR can detect a single template molecule. Knowing the
average volume of sample or sample dilution that contains a single
target molecule, one can calculate the concentration of such
molecules in the starting sample. However, to accumulate detectable
amounts of product from a single starting template molecule usually
requires that two or more sequential PCRs have to be performed,
often using nested sets of primers, and this accentuates problems
with carryover contamination.
[0011] Careful consideration of the factors affecting sensitivity
to detect single starting molecules suggests that decreasing the
volume of the amplification reaction might improve sensitivity. For
example, the "TaqMan" assay requires near saturating amounts of PCR
product to detect enhanced fluorescence. PCRs normally saturate at
about 10.sup.11 product molecules/microliter (molecules/.mu.l) due
in part to rapid reannealing of product strands. To reach this
concentration of product after 30 cycles in a 10 .mu.l PCR requires
at least 10.sup.3 starting template molecules
(10.sup.3.times.2.sup.30/10 .mu.l.apprxeq.10.sup.11/.mu.l).
Somewhat less than this number of starting molecules can be
detected by increasing the number of cycles, and in special
circumstances even single starting molecules may be detectable as
described in the publication of Gerard et al., A Rapid and
Quantitative Assay to Estimate Gene Transfer into Retrovirally
Transduced Hematopoietic Stem/Progenitor Cells Using a 96-Well
Format PCR and Fluorescent Detection System Universal for
MMLV-Based Proviruses, Human Gene Therapy, Vol. 7, pp. 343-354
(1996). However, this strategy usually fails before getting to the
limit of detecting single starting molecules due to the appearance
of artifactual, amplicons derived from the primers (so called
"primer-dimers") which interfere with amplification of the desired
product.
[0012] If the volume of the PCR were reduced 1000-fold to .about.10
nanoliters (nl), then a single round of 30 cycles of PCR might
suffice to generate the saturating concentration of product needed
for detection by the TaqMan assay, i.e. 1.times.2.sup.30 per 10
nanoliters.apprxeq.10.sup.11 per microliter. Attempts have been
made to miniaturize PCR assemblies but no one has developed a
cost-effective PCR assembly which can carry out PCR in a
nanoliter-sized sample. Part of the problem with miniaturization is
that evaporation occurs very rapidly with small sample volumes, and
this problem is made worse by the need to heat samples to
.about.90.degree. C. during thermocycling.
[0013] In addition to potential advantages stemming from ability to
detect single target nucleic acid molecules, miniaturization might
also facilitate the performance of multiple different amplification
reactions on the same sample. In many situations it would be
desirable to test for the presence of multiple target nucleic acid
sequences in a starting sample. For example, it may be desirable to
test for the presence of multiple different viruses such as HIV-1,
HIV-2, HTLV-1, HBV and HCV in a clinical specimen; or it may be
desirable to screen for the presence of any of several different
sequence variants in microbial nucleic acid associated with
resistance to various therapeutic drugs; or it may be desirable to
screen DNA or RNA from a single individual for sequence variants
associated with different mutations in the same or different genes,
or for sequence variants that serve as "markers" for the
inheritance of different chromosomal segments from a parent.
Amplification of different nucleic acid sequences and/or detection
of different sequence variants usually requires separate
amplification reactions with different sets of primers and/or
probes. If different primer/probe sets were positioned in an array
format so that each small region of a reaction substrate performed
a different amplification/detection reaction, it is possible that
multiple reactions could be carried out in parallel, economizing on
time, reagents, and volume of clinical specimen.
[0014] A need therefore exists for a device that can form and
retain a sample volume of about 1.0 nanoliters or less and enable
amplification to be performed without significant evaporation. A
need also exists for a reliable means of detecting a single
starting target nucleic acid molecule to facilitate quantification
of target nucleic acid molecules. A need also exists for performing
multiple different, amplification and detection reactions in
parallel on a single specimen and for economizing usage of reagents
in the process.
SUMMARY OF THE INVENTION
[0015] According to the present invention, methods and apparatus
for performing nucleic acid amplification on a miniaturized scale
are provided that have the sensitivity to determine the existence
of a single target nucleic acid molecule. The invention also
provides analytical assemblies having sample retaining means which
form, isolate and retain fluid samples having volumes of from about
one microliter to about one picoliter or less. The invention also
provides a method of forming fluid samples having sample volumes of
from about one microliter to about one picoliter or less, and
retaining the samples under conditions for thermocycling. The
invention also provides an analytical assembly having means to
determine simultaneously the presence in a sample of multiple
different nucleic acid target molecules.
[0016] According to embodiments of the invention, PCR conditions
are provided wherein a single target nucleic acid molecule is
confined and amplified in a volume small enough to produce a
detectable product through fluorescence microscopy. According to
embodiments of the invention, samples of a few nanoliters or less
can be isolated, enclosed and retained under thermocycling
conditions, and a plurality of such samples can be collectively
analyzed to determine the existence and initial concentration of
target nucleic acid molecules and/or sequences. According to some
embodiments of the invention, sample retaining chambers having
volumes of about 10 picoliters or less can be achieved.
[0017] According to embodiments of the invention, methods of
forming small fluid samples, isolating them and protecting them
from evaporation are provided wherein different affinities of a
sample retaining means and a communicating channel are used to
retain sample in the means while a second fluid displaces sample
from the channel. According to some embodiments of the invention,
the resultant isolated samples are then subject to PCR thermal
cycling.
[0018] According to embodiments of the invention, methods are
provided for determining the existence and/or initial concentration
of a target nucleic acid molecule in samples of about 1 microliter
or less. According to some embodiments of the invention, methods
are provided for a clinical diagnosis PCR analysis which can
quickly and inexpensively detect a single target nucleic acid
molecule.
[0019] According to some embodiments of the invention, sample
chambers of about 1 microliter or less are provided that have a
greater affinity for a sample to be retained than for a displacing
fluid. The displacing fluid displaces sample from around the
chambers and isolates the sample portion retained in the
chambers.
[0020] According to embodiments of the invention, nucleic acid
samples are isolated, retained and amplified in microcapillary
devices having volumes of about 100 nanoliters or less, including
microcapillary tubes, planar microcapillaries and linear
microcapillaries. The devices may be provided with absolute,
selective or partial barrier means.
[0021] According to embodiments of the invention, a porous or
microporous material retains samples of about 100 nanoliters or
less, and an assembly is provided which includes a cover for
sealing sample within the porous or microporous material.
[0022] According to embodiments of the present invention, PCR
methods and apparatus are provided wherein the sensitivity of a
"TaqMan" fluorescence assay can be used to enable detection of
single starting nucleic acid molecule in reaction volumes of about
100 nl or less. According to the present invention, assemblies for
retaining PCR reaction volumes of about 10 nl or less are provided,
wherein a single target molecule is sufficient to generate a
fluorescence-detectable concentration of PCR product.
[0023] According to the invention, methods are provided for
carrying out PCR in minute volumes, for example, 10 nl or less,
which allows detection of PCR products generated from a single
target molecule using the "TaqMan" or other fluorescence energy,
transfer systems.
[0024] According to embodiments of the present invention, methods
of detecting and quantifying DNA segments by carrying out
polymerase chain reaction in a plurality of discrete
nanoliter-sized samples are provided. The present invention also
provides methods for determining the number of template molecules
in a sample by conducting replicate polymerase chain reactions on a
set of terminally diluted or serially smaller samples and counting
the number of positive polymerase chain reactions yielding specific
product. The present invention is useful in detecting single
starting molecules and for quantifying the concentration of a
nucleic acid molecule in a sample through PCR. The present
invention also provides methods of detecting and quantifying a
plurality of target DNA sequences.
[0025] The present invention also provides methods and assemblies
for separating and/or analyzing multiple minute portions of a
sample of fluid medium that could be useful for other applications.
Applications of the apparatus of the invention include the
separation of biological samples into multiple minute portions for
the individual analysis of each portion, and can be used in the
fields of fertility, immunology, cytology, gas analysis, and
pharmaceutical, screening.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The invention will be described in connection with various
embodiments exemplified in the accompanying drawings, wherein:
[0027] FIG. 1 is an exploded view of an analytical assembly
according to an embodiment of the present invention, shown in
partial cutaway;
[0028] FIG. 2 is an exploded view of an analytical assembly
according to an embodiment of the present invention, shown in
partial cutaway, comprising sample chambers in the form of wells
formed into patterned layers on the inner surfaces of both a top
plate and a bottom plate;
[0029] FIG. 3 is a cross-sectional view through a longitudinal
central portion of an analytical assembly according an embodiment
of the present invention;
[0030] FIG. 4 is a perspective view of a bottom portion of an
analytical assembly according to an embodiment of the present
invention, the bottom portion comprising sample retaining means in
the form of patches of fluid retaining material formed on a
patterned layer coated on the inner surface of a bottom plate;
[0031] FIG. 5 is a cross-sectional view through a longitudinal
central portion of an analytical assembly according an embodiment
of the present invention;
[0032] FIG. 6A is a perspective view of a bottom portion of an
analytical assembly according to another embodiment of the present
invention;
[0033] FIG. 6B is an enlarged view of portion VIA shown in FIG.
6A;
[0034] FIG. 7 is a top plan view of a microcapillary analytical
assembly according to an embodiment of the present invention;
[0035] FIG. 8 is a histogram showing the maximum values of the
fluorescein:rhodamine intensity ratio in over 1.00 capillary
reactions of terminally diluted genomic DNA carried out in an
assembly according to the present invention and according to a
method according to the invention; and
[0036] FIGS. 9-14 are plots of the fluorescein:rhodamine ratio
along a few representative microcapillaries containing samples
subject to PCR in accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0037] According to embodiments of the present invention, methods
of manipulating a sample of fluid medium are provided. The methods
comprise loading a sample of fluid medium into sample retaining
means of an analytical assembly and displacing excess sample from
areas adjacent to the portion retained by the sample retaining
means. According to embodiments of the invention, sample fluid is
displaced from regions adjacent to the retained sample, without
displacing the retained sample. In some embodiments, a displacing
fluid is used to isolate a retained sample, and the displacing
fluid may be curable to form a retaining chamber entrapping the
fluid sample retained by the sample retaining means.
[0038] The assemblies of the present invention provide samples or
sample portions enclosed in a protective environment which protects
the sample or portion from evaporation and contamination.
Preferably, sample is protected from evaporation at temperatures of
about 95.degree. C. or more, for example, at temperatures achieved
during thermal cycling under conditions for PCR. The isolated,
entrapped or enclosed sample or portion is preferably protected
from contamination and evaporation throughout an amplification
protocol, for example, a PCR thermal cycling protocol.
[0039] According to some embodiments of the invention, an
analytical assembly is provided and comprises a plurality of sample
chambers each confined in at least one dimension by opposing
barriers separated by a first dimension of about 500 microns or
less, preferably by 100 microns or less, and in some embodiments by
about 20 microns. Means are provided for sealing the plurality of
sample chambers to prevent evaporation and contamination of fluid
sample confined within the plurality of sample chambers. Means are
provided for restraining reaction product formed from reactions of
a chemical substance restrained within the plurality of sample
chambers. According to some embodiments, means may be provided for
minimizing diffusion and substantially preventing convection of
amplification reaction product formed from reactions of the fluid
sample restrained within the plurality of sample chambers. If
provided, at least one of the means for restraining and the means
for minimizing diffusion and substantially preventing convection
may preferably comprise a patterned layer which at least partially
defines the plurality of sample chambers. Preferably, the fluid
sample contains at least one target, nucleic acid molecule to be
amplified and constituents for enabling amplification of the target
nucleic acid molecule. The fluid sample is divided into a plurality
of sample portions and the plurality of sample chambers are loaded
which respective portions of the fluid sample. According to some
embodiments of the invention, the sample portions are in fluid
communication with each other, rather than being completely
isolated from each other, and separated by barrier means which may
be in the shape of crosses, lines, semicircles, circles having at
least one opening along the arc thereof, or other geometric shapes.
According to embodiments of the invention, the barrier means may
define sample retaining portions or chambers of the assembly.
According to some embodiments, means for minimizing diffusion and
substantially preventing convection are provided, and may comprise
the herein described barrier means. According to some embodiments,
the barrier means includes physical structures which may extend
between the aforementioned opposing barriers which are separated by
500 microns. The barrier means may form a wall or walls between the
opposing barriers. The barrier means may comprise flow restriction
means. Flow restriction means may be, for example, semi-circular
walls extending from one of the opposing barriers toward the other,
and having the concave side of the semi-circle facing the direction
of fluid flow during loading, and the semicircular arc may have at
least hole or interruption therein through which air may escape
during fluid sample loading. According to embodiments of the
invention, the first dimension, the means for restraining, and, if
provided, the means for minimizing diffusion and substantially
preventing convection are such that the reaction product of a
single target nucleic acid molecule amplified within at least one
sample chamber can attain a concentration of reaction product
molecules sufficient to be detected by a homogeneous detection
assay, for example a concentration of about 10.sup.11 product
molecules per microliter (.mu.l). Preferably, at least one of the
plurality of portions is initially free of the target nucleic acid
molecule, and at least one of the plurality of portions initially
contains at least one target nucleic acid molecule.
[0040] According to some embodiments of the invention, an
analytical assembly is provided comprising a substrate and a cover
in registry with one another and attached to one another and having
facing surfaces spaced a substantially uniform distance apart from
one another. The facing surface of the substrate comprises a first
material having a first affinity to a sample of fluid to be
isolated, for example, an aqueous PCR solution sample. A
flow-through channel is disposed between the first material and the
cover, and at least one sample retaining means is bounded on at
least one side by the first material. The first material may
comprise, for example, a patterned layer of moderately hydrophobic
material, that is, a material having a surface energy of from about
30 dynes/cm to about 50 dynes/cm. The first material may be
deposited on the inner surface of the substrate.
[0041] Herein, the term "affinity" is to be understood to mean a
sample-holding capacity or sample-holding capability. The affinity
of the sample retaining means may be caused by surface energy of
the material contacting or restraining a sample fluid, or it may be
caused by electrostatic force, flow restriction means, temperature
differences or by other means. The "affinity" of a flow-through
channel may be defined by the dimensions of the channel, or by the
material comprising one or more bounding surface of the channel, or
by a combination of dimensions and bounding surface properties. The
sample retaining means may have different affinities to retain a
sample fluid and a displacing fluid. The flow-through channel may
exhibit different affinities to retain a sample fluid and a
displacing.
[0042] According to embodiments of the invention, the first
material preferably does not comprise an extremely hydrophobic
material; for example, the first material preferably does not
comprise a material having a surface energy of less than about 20
dynes/cm. Extremely hydrophobic materials do not tend to be wetted
by aqueous samples or by many displacing fluids such as mineral
oil, two-part adhesives, UV-curable and cyanoacrylate adhesives,
and thus such displacing fluids would tend not to completely
surround, isolate and restrain a sample (e.g. a gaseous sample)
held by the sample retaining means.
[0043] The sample retaining means is in communication with the
flow-through channel such that sample entering the flow-through,
channel can reach and be retained by the sample retaining means.
The cover and first material, should be of such properties and/or
special relationship to allow a sample fluid and a displacing fluid
to enter the flow-through channel, whether by capillary action,
pressure, or other force. The sample retaining means has a second
affinity to the sample of fluid medium, and the second affinity is
greater than the first affinity. The second affinity may be a
property induced in the first material by chemical, optical,
electronic, or electromagnetic means, for example. An exemplary
chemical means to permanently induce an affinity may be, for
examples an O.sub.2 plasma exposure to a portion of a silicone
surface to effectively change the affinity of the surface to retain
an aqueous sample at the portion treated. Embedding ions in a
surface may also be used to permanently induce an increased or
decreased affinity at a location on a surface. An affinity may be
temporarily induced according to some embodiments of the invention,
for example, where a surface charge on a sample retaining or
repelling surface is induced to increase the effective surface
tension of that surface. According to some embodiments of the
invention, a temporary affinity may be reshaped, moved or relocated
during or after a sample portion is retained, for the purpose of
enlarging or joining sample portions in the assembly. According to
embodiments of the invention, the difference of affinities enables
the retaining means to collect a portion of sample from the
flow-through channel and to retain the portion while a second fluid
medium: is introduced to the flow-through channel; isolates sample
retained by the sample retaining means; and displaces non-retained
sample from adjacent to the sample retaining means.
[0044] According to embodiments of the invention, the flow-through
channel is adjacent to the sample retaining means. An entrance
opening is provided for introducing sample into the flow-through
channel. According to embodiments of the invention, an analytical
assembly is also provided which contains isolated sample entrapped
by a substantially immiscible displacing fluid.
[0045] Methods according to embodiments of the invention involve
causing the displacing fluid to flow through the flow-through
channel and displace sample from the flow-through channel without
displacing sample from the sample retaining means. Such methods are
accomplished according to embodiments of the invention by providing
a sample retaining means having a greater affinity for the sample
than for the displacing fluid. Preferably, the flow-through channel
has a much lower affinity for the sample than does the sample
retaining means.
[0046] According to some embodiments of the invention, the sample
of fluid medium is a gaseous sample and the sample retaining means
is extremely hydrophobic, for example, having a surface energy of
from about 30 dynes/cm to about 10 dynes/cm. Because of the extreme
hydrophobicity of the retaining means, displacing fluids tend to be
repelled from the retaining means, and thus avoid displacing
gaseous sample from the retaining means. Displacing fluids can thus
entrap, retain and isolate a sample within an assembly of the
invention.
[0047] According to some embodiments, an organic sample is trapped
in a high surface energy displacing fluid.
[0048] Both the sample to be isolated and the displacing fluid may
be introduced into the flow-through channel by being drawn in under
the influence of capillary forces. Pressurized loading techniques
may also be used, but if displacing fluid is forced into the device
under pressure, the pressure should not be so high as to displace
sample from the sample retaining means. Other means of loading
sample fluid and/or displacing fluid may be used according to the
invention and include electrokinetic or electrostatic loading
techniques, temperature differentials, centrifugal force, vacuum or
suction loading, magnetic attraction loading of magnetic fluids,
and electrophoretic or columbic force loading. An exemplary
magnetic attraction loading technique involves drawing a fluid
containing magnetic particles dispersed therein into the device
under the influence of a magnetic field and using mineral oil
containing dispersed iron particles as a displacing fluid and
drawing the mineral oil into a flow-through channel with a magnetic
field. According to some embodiments, the sample is loaded with
magnetic particles and attracted toward and held by a sample
retaining means adjacent to or including a source of a magnetic
field or a material which is attracted to a magnet, for example, an
iron-containing material.
[0049] Preferably, the displacing fluid is substantially immiscible
with the sample of fluid medium to be isolated. According to
embodiments of the invention wherein a displacing fluid is used,
the displacing fluid may comprise a flowable, curable fluid such as
a curable adhesive selected from the group consisting of:
ultra-violet-curable and other light-curable adhesives; heat,
two-part, or moisture activated adhesives; and cyanoacrylate
adhesives. Exemplary displacing fluids include Norland optical
adhesives available from Norland Products, Inc., New Brunswick,
N.J., cyanoacrylate adhesives disclosed in U.S. Pat. Nos. 5,328,944
and 4,866,198, available from Loctite Corporation, Newington,
Conn., resins, monomers, mineral oil, silicone oil, fluorinated
oils, and other fluids which are preferably substantially
non-miscible with water. According to some embodiments, the
displacing fluid may be transparent, have a refractive index
similar to glass, have low or no fluorescence, have a low
viscosity, and/or be curable.
[0050] Some methods according to embodiments of the invention,
including methods of nucleic acid molecule amplification, including
PCR, may comprise isolating a sample into a plurality of discrete
retained sample portions, and processing and/or analyzing the
portions to determine concentrations of components and other
characteristics of the sample. To carry out methods of the
invention wherein a plurality of sample portions are formed and
isolated, assemblies are provided having a plurality of sample
retaining means. Evaluating multiple portions of a sample can then
be used to determine characteristics of the entire sample.
[0051] While the resolution and accuracy of many analytical
techniques can be improved according to the invention by forming
and analyzing a plurality of portions of a sample, embodiments of
methods of the present invention more generally involve
manipulating a fluid sample. Manipulating may comprise cloning a
segment of DNA, for example, where the sample of fluid medium
comprises a polymerase chain reaction solution, and at least one
segment of DNA to be amplified. Methods of PCR according to the
invention comprise exposing isolated sample retained by the sample
retaining means to a temperature profile which causes polymerase
chain reaction amplification of a target nucleic acid molecule
segment within the sample. According to embodiments of the
invention, isolated and minute PCR samples can be retained under
conditions which protect the sample during temperature profiling to
dehybridize double stranded polynucleotides, anneal primers to the
dehybridized molecules, and to polymerize and thus amplify the
polynucleotide.
[0052] According to embodiments of the invention, PCR methods are
carried out in assemblies according to the invention, and the
methods include manipulating a PCR sample which contains an
effective amount of a probe or system of probes having fluorescent
properties or chemical properties that change upon hybridization to
a nucleic acid target. According to embodiments of the invention, a
normally quenched double labeled fluorescent probe is degraded upon
hybridization to target DNA during PCR and the degradation results
in increased emitted light of a certain wavelength. By measuring
the amount of fluorescence and thus the amount of degraded probe,
methods according to embodiments of the invention can be used to
determine whether a segment of DNA has been amplified and thereby
calculate the concentration of a target DNA segment that existed in
the original sample before PCR. The measured fluorescence of a
certain wavelength may in some cases be used according to the
invention to quantify the amount of a DNA segment which had been
retained by the sample retaining means prior to exposing a retained
sample to a PCR thermal cycling profile or protocol.
[0053] Other detection methods may be used to determine PCR product
and/or reactant concentrations or to make other quantitative or
qualitative evaluations of many types of samples. These other
detection methods include agglutination, turbidity, phosphorescence
detection techniques, light scattering, light absorbance,
fluorescence energy transfer, fluorescence quenching or
dequenching, time-delayed fluorescence, chemiluminescence and
calorimetric evaluation techniques.
[0054] According to some preferred embodiments of the invention,
methods of cloning a segment of DNA are provided wherein a sample
containing a DNA segment to be amplified is divided into a
plurality of sample portions and the portions are simultaneously
subjected to PCR. By providing sample portions of as small as about
10 nanoliters or less, for example 10 picoliters or less, single
molecules of a target DNA segment to be amplified can be detected
according to the invention. For PCR methods according to the
invention which enable amplification of a plurality of sample
portions simultaneously, the sample from which the portions are
derived may comprise a polymerase chain reaction solution, at least
one segment of DNA to be amplified, and a sufficient amount of
primer to carry out a polymerase chain reaction for multiple
cycles, and the method may comprise exposing the sample portions
retained by the plurality of sample retaining means to a
temperature profile which causes polymerase chain reaction and
amplification of a target DNA segment within the portions.
According to embodiments of the invention, the presence of a single
strand of a target DNA segment can be detected in a portion
retained by at least one of a plurality of sample retaining
means.
[0055] Methods according to some embodiments of the present
invention comprise manipulating a sample of fluid medium by loading
the sample into an analytical assembly having a porous sample
retaining means. Porous sample retaining means according to the
invention comprise a porous structure having an exposed porous
surface and a plurality of pores having open upper ends at the
exposed porous surface. Preferably, the exposed porous surface is
moderately hydrophobic yet receptive to adhesive bonding. The pores
of the sample retaining means may have substantially the same
volume and have closed lower ends which may be defined by a
substrate onto which the porous structure may be disposed.
[0056] Preferably, the ratio of pore diameter to depth is from
about 2:1 to about 10:1, for example, 4:1 for embodiments wherein a
porous material is attached to a substrate. According to
embodiments of the invention, the ratio of exposed porous surface
area, that is, the area of the surface not taken up by the
openings, to the area of pore openings is from about 4:1 to about
1:1.5, for example, a ratio of about 1:1.
[0057] The pores have a first affinity to a sample such that when
the sample is disposed upon the exposed porous surface, the sample
is drawn into the plurality of pores. The assembly used according
to these methods of the invention further comprises means to
displace sample from the exposed porous surface without displacing
the sample from the pores. The means to displace may comprise a
displacing fluid or a displacing device such as a coverslip pressed
against the exposed surface. The assembly may also include means
for sealing the sample within the pores to prevent evaporation and
contamination of the sample during heat treatment and analysis of
the sample in the pores.
[0058] Loading sample into the pores of a porous sample retaining
means comprises contacting the sample to the exposed porous surface
and retaining the sample in the plurality of pores. Due to the
affinity the pores exhibit to the sample, particularly to aqueous
PCR samples, the sample is drawn into the pores by capillary force
and retained therein. The methods also include displacing sample
from the exposed porous surface without displacing sample from
within the pores. The methods may also include sealing the open
ends of the pores with the sample disposed therein.
[0059] According to some embodiments of the invention, both the top
and the bottom of the pores may be open for loading and then
sealed.
[0060] According to embodiments of the invention, analytical
assemblies are provided for carrying out methods of manipulating a
sample of fluid medium with a porous sample retaining means.
[0061] The porous sample retaining means in such assemblies may
comprise a microchannel array, a metal, glass, ceramic, cellulosic
or polymeric screen or sieve, or a material having a plurality of
pores formed therein, such as a substantially flat plastic disk
having a plurality of pores ablated, molded, etched or drilled
therein. The pores may be treated or coated with a material to
provide an affinity to a sample. The exposed porous surface may be
treated or coated with a material to render the exposed surface
moderately hydrophobic.
[0062] According to some embodiments, the sample retaining pores
have a volume of from about 1 microliter to about 100 nanoliters or
less, preferably about 10 nanoliters or less, and may have pore
volumes of about 1 picoliter or less for some applications.
According to one embodiment, the pores have volumes of about 10
picoliters.
[0063] After sample is loaded into the pores of the porous sample
retaining means, remaining sample which is not retained by the
pores but rather which remains on the exposed porous surface is
removed or displaced. According to embodiments of the invention, a
sealing means such as a microscope slide coverslip, tape, film or a
device including other components, a silicon film or device, a
device having an array of reactants, or other means is disposed on
the exposed porous surface and displaces sample from the exposed
porous surface, without displacing sample from within the pores.
After displacing sample from the exposed porous surface, the
displacing means may become the sealing means if subsequently held,
adhered or attached to the porous surface, which would then no
longer be exposed. Preferably, the sealing means comprises a
material having a second affinity for the sample which is less than
the first, affinity, for example, the sealing means comprises a
relatively hydrophobic material which contacts the exposed porous
surface, and the pores are defined by a relatively hydrophilic
porous material.
[0064] According to embodiments of the invention using the
aforementioned porous sample retaining means, the means for
displacing sample and the means for sealing may be a single cover
having a hydrophobic surface which contacts the porous surface. The
sealing means may be glued to the previously exposed porous
surface, glued to a substrate on which the porous retaining means
is disposed, or clamped or otherwise attached to the porous
retaining means or a substrate therefor, as for example, with clips
or springs. A surface of the cover, substrate or porous sample
retaining means may provide adhesive or glue properties.
[0065] According to methods of some embodiments of the invention, a
target nucleic acid molecule or segment, is amplified in at least
one of the pores of a porous sample retaining means. According to
such embodiments, the sample to be retained by the retaining means
may comprise a polymerase chain reaction solution, and at least one
target segment of nucleic acid molecule to be amplified. The method
further comprises exposing the sample sealed within the pores to a
temperature profile which causes polymerase chain reaction
amplification of the target segment within at least one of the
pores. In some embodiments of the invention, the sample further
comprises an effective amount of a fluorescent probe which
fluoresces upon degradation caused by the successful amplification
of the target segment in the sample. Such methods may further
comprise measuring the fluorescence emitted from degraded probe
after the polymerase chain reaction and determining whether the
target segment was amplified in at least one of the pores. The
fluorescence measurement may be of the amount of fluorescence, the
lifetime of fluorescence, or another fluorescence property.
[0066] The methods of the present invention using a porous sample
retaining means may further comprise using measured fluorescence to
quantify the amount of target nucleic acid segment which had been
retained in at least one of the pores prior to exposing retained
sample portions to a thermal cycling protocol. Due to the extremely
small volume of sample chambers which can be achieved with a porous
sample retaining means, the presence of a single strand of the
target segment can be detected in the pores. The methods may also
comprise determining the initial concentration of the target
segment which had been in the sample prior to amplification.
[0067] According to yet other embodiments of the present invention,
methods of manipulating a sample of fluid medium are also provided
wherein a sample of fluid medium is loaded into an analytical
assembly comprising a microcapillary device, for example, a
microcapillary tube having an inner diameter of about 500 .mu.m or
less, preferably about 100 .mu.m or less. Loading may comprise
filling the microcapillary tube with a sample by capillary action.
Microcapillary devices provided with a sample retaining means, for
example, a hydrophobic or hydrophilic surface or an electromagnetic
or electrostatic force, may have an inner dimension or inner
diameter of about 500 .mu.m or less. Tubular or linear
microcapillaries not provided with sample retaining means may
preferably have an inner dimension of about 100 .mu.m or less.
Planar microcapillaries not provided with sample retaining means,
for example, comprising the space between two facing plates, may
preferably have an inner dimension of about 20 .mu.m or less.
[0068] According to some embodiments, a first fluid may initially
be disposed into the microcapillary device, for example, a
microcapillary tube. The first fluid may preferably be
substantially immiscible, and more preferably, completely
immiscible with a sample fluid. Loading then comprises disposing a
portion of a sample fluid into the microcapillary tube adjacent the
first fluid, and subsequently disposing a second fluid into the
microcapillary tube adjacent the sample. The second fluid is also
preferably substantially immiscible, and more preferably completely
immiscible, with the sample. The sample is disposed between and
restrained by the first and second fluids. The first and second
fluids may be the same fluid, and can be, for example, mineral oil
or a gas or a curable monomer formulation, if the sample is an
aqueous fluid. The microcapillary device may contain more than one
isolated sample portion, which may be separated by one of the first
and second fluids. Each portion may be about 100 nl or less,
preferably 10 nl or less.
[0069] Some methods of the invention which employ microcapillary
sample retaining means may also comprise sealing both ends of the
microcapillary tube with the sample therein. The sample may fill
the entire capillary tube and be sealed therein, or the sample may
be sealed in the tube sandwiched between first and second fluids.
Means may be employed to load numerous sample portions, such as an
ink jet or fluidic control apparatus.
[0070] According to embodiments of the invention, microcapillary
analytical assemblies are also provided and can be used to isolate
and retain a sample of fluid medium comprising about 100 nanoliters
or less of a fluid medium, preferably about 60 nanoliters or less.
Assemblies are also provided which comprise a plurality of such
microcapillary tubes, each tube having a sample of fluid medium
disposed therein. A plurality of tubes may be attached to a pair of
microscope slide coverslips, or to a tape or pair of tapes, or
otherwise held together.
[0071] The microcapillary sample retaining devices of the present
invention may be used to carry out nucleic acid amplification
methods, according to embodiments of the invention. When used in
PCR applications, the fluid sample may comprise a polymerase chain
reaction solution which may include reagents, enzymes, buffer,
bovine serum albumin and other well known ingredients commonly used
to perform a polymerase chain reaction. Methods according to the
invention which employ the inventive microcapillary devices may
further comprise exposing the sample sealed within the
microcapillary tube, or within a plurality of tubes, to a
temperature profile which causes amplification of the target
segment within the sealed sample.
[0072] As mentioned in connection with other methods and apparatus
according to the invention, the PCR sample in the microcapillary
tube may further comprise an effective amount of a fluorescent
probe which fluoresces upon degradation of the probe caused by the
successful amplification of the target segment. The fluorescence
emitted from the hybridized probe can be measured to determine
whether the target segment was amplified. By using different
dilutions of starting sample or by using replicate samples in
different volumes, the concentration of starting target segment can
be determined.
[0073] According to embodiments of the present invention wherein
PCR is carried out in a microcapillary assembly, fluorescence or
other detection properties are promptly analyzed after PCR thermal
cycling, for example, within about 5 hours; more preferably within
about 1 hour after PCR thermal cycling is complete. Prompt analysis
of the fluorescence emitted maximizes the concentration of measured
degraded probe along regions of the microcapillary tube, before the
degraded probe diffuses along the tube and becomes less detectable.
Preferably the amplification is carried out as rapidly as possible,
for example, in less than one hour.
[0074] Miniaturized assemblies according to embodiments of the
invention have been briefly described above and will be discussed
in greater detail, below. Assemblies according to embodiments of
the invention can take many forms but can generally be classified
into three types, (1) assemblies having flow-through channels and
sample retaining means in communication with the flow-through
channel, (2) assemblies having porous sample retaining means and
means for sealing sample within sample retaining pores, and (3)
assemblies comprising at least one microcapillary tube.
[0075] According to embodiments of the invention, assemblies are
provided for manipulating samples of fluid medium, for example,
methods for isolating small sample volumes, such as sample volumes
of 100 nanoliters or less.
[0076] Some assemblies according to the invention comprise a
substrate and a cover in registry with and attached to one another
and having facing surfaces spaced a substantially uniform distance
apart from one another. The facing surface of the substrate
comprises a first material having a first affinity to a sample of
fluid medium to be contained in the assembly. For example, if an
aqueous fluid is to be manipulated, the first material is
preferably a moderately hydrophobic material. The first material
preferably defines at least a portion of a flow-through channel
which is disposed between the substrate and the cover. The
flow-through channel is in fluid communication with a sample
retaining means, and the retaining means is bounded on at least one
side thereof by the first material. The sample retaining means has
a second affinity to the same sample of fluid medium. Preferably,
the second affinity to the sample fluid is greater than the first
affinity thereto, preferably much greater, enabling the sample
retaining means to retain or collect a portion of a sample of fluid
medium which flows through the flow-through channel and to retain
the portion while a second fluid medium flows through and displaces
sample from the flow-through channel. The result can be a very
small isolated portion of the sample being retained by the sample
retaining means and entrapped, encased or otherwise surrounded by
the second fluid medium in the flow-through channel.
[0077] According to embodiments of the invention wherein the
assembly comprises a flow-through channel and sample retaining
means, the substrate may be a first plate having a patterned layer
of the first material formed thereon. The cover may be a second
plate attached to and substantially parallel to the first plate,
with the first and second plates having facing substantially
parallel planar surfaces. The first and second plates may each be
rigid or flexible, flat or contoured, a sheet, a film, a microscope
slide, a microscope slide coverslip, a glass plate, a tape, a
device including other components, a silicon device, a silicon
film, or the like. According to some embodiments, the plate has a
substantially planar surface on a side adjacent or defining the
sample retaining means. The patterned layer is preferably located
between the planar surfaces and at least partially forms a boundary
for the sample retaining means. For example, when the sample
retaining means is a chamber formed from an opening in, a cavity or
recess in, or a hole through the patterned layer, the closed end of
the chamber may be defined by the substrate surface on which the
patterned layer is disposed or by the patterned layer, and the
patterned layer may define the sidewalls of the sample chamber.
According to embodiments wherein the sample retaining means is a
recessed sample chamber at least partially defined by the patterned
layer, the chamber may comprise a sidewall, a closed lower end, and
an upper end which may be open or closed. The sample chamber has a
communication with the flow-through channel. According to some
embodiments of the invention, the sample chamber also extends into
and is partially defined by a patterned layer formed on the facing
surface of the assembly cover, in which case the sample chamber may
have a closed upper end and the communication to the chamber may be
formed in the chamber sidewall, for example, an annular gap in an
otherwise continuous sidewall. According to other embodiments of
the invention, the cover is substantially planar and the sample
chamber does not extend into the cover or a layer disposed on the
cover, in which case the chamber has an open upper end in
communication with the flow-through channel.
[0078] According to embodiments of the invention wherein the
substrate and cover comprise first and second plates, the closed
lower end of the sample chamber may comprise the first plate. The
sample retaining means may comprise a third material at the lower
end of the sample chamber. The third material may in some cases be
a hydrophilic material deposited on the first plate and defining
the lower end of the chamber. According to some embodiments, the
third material may be very hydrophobic. For aqueous samples in
particular, hydrophilic materials may be used at the lower end of
the sample chamber. According to some embodiments of the present
invention, a sample chamber comprising a recess in a substrate or
substrate coating may preferably have hydrophilic material at a
lower end thereof and hydrophobic material forming the sidewall.
Preferably, the hydrophilic lower end provides an affinity to an
aqueous PCR sample which is sufficient to retain the sample while a
displacing fluid carries away sample adjacent the sample chamber.
The hydrophobic sidewalls prevent sample from being displaced from
the sample chamber.
[0079] Preferred sample chambers according some embodiments of the
invention, for nucleic acid amplification methods to detect single
target nucleic acid molecules, have volumes of from about 1
microliter to about 1 picoliter or less. Printing,
photolithography, etching, ablation and other methods of forming
sample chambers in, for example, printed layers, can provide sample
chambers of ten nanoliters or less, for example, about 100
picoliters.
[0080] Screen printing and photolithography are preferred methods
of forming a patterned layer on the substrate. Screen printing
methods can provide a patterned layer on a 1 inch by 3 inch
microscope slide wherein the layer contains over one thousand
isolated and spaced sample chambers each having a volume of about 1
nanoliter. Such a patterned layer could enable over one thousand
PCR chambers of about 1, nanoliter each, all on the surface of a
1''.times.3'' microscope slide. Photolithographic methods can
provide from about 10,000 to over 100,000 sample chambers of about
100 picoliters each on a 1''.times.3'' substrate.
[0081] According to embodiments of the invention, the first
material or patterned layer material is moderately hydrophobic.
Herein, the term "moderately hydrophobic" refers to a material or
layer that exhibits a contact angle to water of from greater than
about 30.degree. to less than about 85.degree.. According to some
embodiments, a hydrophobic patterned layer is disposed on the
facing surfaces of both the substrate and the cover, the average
contact angle of the two patterned layers is preferably more than
about 30.degree. to less than about 85.degree., and more preferably
each of the two layers is moderately hydrophobic. Patterned layers
exhibiting higher contact angles to water may be employed on the
substrate surface if the cover comprises a hydrophilic facing
surface. Preferably, the patterned layer or first material is of
such a nature that displacing fluid can bond thereto when the
second fluid or displacing fluid is a polymerizable fluid. If the
patterned layer or first material comprises TEFLON, for example, a
curable or polymerizable displacing fluid may not be capable of
bonding thereto and thus may not sufficiently seal an entrapped
sample fluid posing contamination and evaporation risks.
[0082] According to some embodiments of the invention, the
substrate and cover comprise first and second facing planar
surfaces with a first layer on the first surface and a second layer
on the second surface. According to some embodiments, sample
chambers are formed in the first layer and the second layer is
substantially smooth. According to some other embodiments, the
second layer also has sample chambers formed therein, preferably
mirroring the sample chambers formed in the first layer. The second
layer may comprise a moderately hydrophobic material.
[0083] According to some embodiments, the interior surface(s) of
the substrate and/or cover may be altered by chemical or other
means to form a pattern of retaining means having altered surface
energy or structure.
[0084] According to some embodiments of the invention, a sample
chamber comprises a hole or recess formed in a first patterned
layer, and a hole or recess formed in a second patterned layer, the
sidewall comprises both the first and second patterned layers, a
flow-through channel is disposed between the first and second
patterned layers, and the communication to the flow-through channel
interrupts the sidewall.
[0085] According to embodiments comprising first and second
patterned layers, the first and second layers may comprise the same
material. The second patterned layer may have a substantially
smooth surface facing the first patterned layer, and the second
layer may have a substantially uniform layer thickness. In
embodiments wherein a smooth and continuous second layer is
provided on the inner surface of a cover or top plate of an
assembly, the sample chamber may have an open upper end in fluid
communication with the flow-through channel and the sidewall may
completely comprise the first patterned layer.
[0086] The patterned layer may be the retaining means, for example,
hydrophobic or hydrophilic spots on the substrate or cover.
According to some embodiments, the patterned layer may comprise the
retaining means and may comprise a microporous material such as an
epoxy material which is highly filled with micron-sized beads.
[0087] According to embodiments of the invention wherein an
assembly comprises a flow-through channel, the flow-through channel
may have an entrance opening and an exit vent. Assemblies designed
for forced pressure loading may not require an exit vent. In
embodiments of the invention comprising a flow-through channel, the
channel may have a substantially uniform cross-sectioned area
throughout. In other embodiments, the cross-sectional area of the
flow through channel increases or decreases in a direction from the
entrance opening to said exit vent. The changing cross-sectional
area of the flow-through channel can influence the travel of the
sample and/or displacing fluid through the flow-through channel due
to increasing or decreasing resistance of the fluid flow. For
example, in embodiments wherein the flow-through channel increases
in cross-sectional area from adjacent to the entrance opening in
the direction of the exit vent, fluid flow toward the exit vent is
subject to decreased flow resistance compared to embodiments
wherein the cross-sectional area is the same throughout the
flow-through channel. One method of forming a flow-through channel
with an increasing or decreasing cross-sectional area is to space
the cover or top plate further from the substrate or bottom plate
at the entrance end of the assembly than at the exit end. Although
the substrate and cover, or first and second plates, would remain
substantially parallel to each other as defined by the present
invention, they would not be exactly parallel. Another method of
forming an increasing or decreasing flow-through channel
cross-sectional area is to form the patterned layer thicker at one
end of the device than at the opposite end of the device, and to
have the thickness of the layer gradually increase or decrease in
thickness. According to some embodiments wherein holes in a
patterned layer define sample retaining means, the retaining means
may have different sizes and shapes.
[0088] According to some embodiments of the invention, a
hydrophilic pattern is provided to form sample retaining means and
the pattern may be induced by electrets or by internal or external
electrodes to provide a charged surface having higher surface
energy and wettability than a flow-through channel in communication
with the retaining means.
[0089] According to some preferred embodiments of the invention, an
analytical assembly is provided with a plurality of sample
retaining means separated from one another. In some embodiments,
the plurality of sample retaining means comprise sample chambers
formed in a patterned layer disposed between the substrate and
cover or first and second plates. The sample retaining means may
comprise chambers each having a sidewall, an upper end, a closed
lower end, and a communication with a flow-through channel.
[0090] Filled assemblies are also provided according to embodiments
of the invention, and may comprise a sample fluid retained by the
sample retaining means, and a different, second fluid retained in
the flow-through channel. Preferably, the second fluid is
substantially immiscible with the sample fluid. The sample fluid
may comprise a polymerase chain reaction solution and at least one
segment of DNA to be amplified. The second fluid is referred to as
a displacing fluid and may comprise a curable fluid, particularly
curable adhesives, and preferably fluid adhesives selected from the
group consisting of light-curable, heat-curable, two-part-curable,
moisture-curable and cyanoacrylate adhesives. When UV-curable
adhesives are used and cured, UV-blocking spots may be provided on
the cover and/or substrate, aligned with the sample retaining
means, to protect the retained sample from harmful light.
[0091] According to some embodiments of the invention, wherein an
assembly is provided having opposing surfaces, sample retaining
means between the surfaces, and a flow-through channel in
communication with the retaining means, the sample retaining means
may comprise a fibrous or porous material which absorbs a sample of
fluid medium through capillary forces. The fibrous or porous
material may be formed on a patterned layer of a first material
deposited on one of the surfaces. The first material may comprise a
moderately hydrophobic material. A second material layer may be
included on one of the facing surfaces and may comprise a
moderately hydrophobic material. The fibrous or porous material may
be a cellulosic material, a filter paper material, absorbent
textured material, absorbent sintered materials, absorbent, pastes,
microporous membranes, fiberglass, and the like. Preferably, the
fibrous or porous material is a porous membrane having a maximum
pore diameter size of about 1 micron. The fibrous or porous
material, may fill the gap between the substrate and cover or may
be disposed on just one of the interior substrate or cover
surfaces.
[0092] The fibrous or porous material has a wicking rate for a
sample which can be measured in millimeters per second, and sample
flowing through the flow-through channel advances through the
channel at an advancing rate which can be measured in millimeters
per second. Preferably, the wicking rate exceeds the advancing
rate, thus minimizing the possibility of an aqueous sample
entrapping or encircling the sample retaining means before air in
the retaining means can escape and be carried away by the advancing
fluid. In cases where the advancing rate exceeds the wicking rate,
sample tends to be absorbed too slowly by the retaining means and
the advancing fluid in the channel, tends to surround the retaining
means and entrap air before the air has a chance to escape.
[0093] The size and shape of the porous retaining means also
influences whether air will be trapped in the sample retaining
means. For larger sample retaining means, there is a greater chance
that air may be entrapped in the sample retaining means than for
smaller sample retaining means. Therefore, it is preferable to use
higher wicking rates for larger fibrous or porous sample retaining
means than for smaller retaining means. For example, if the
retaining means has a wicking rate of 1 mm/sec and the advancing
rate is also 1 mm/sec, a substantially flat sample retaining means
having a diameter of about 1 mm tends to be wicked by sample
without entrapping air, whereas a retaining means having a diameter
of about 2 mm may not be completely wicked with sample but instead
is more likely to entrap air. Retaining means that are elongated in
the direction of sample flow may be preferred for large
samples.
[0094] According to some embodiments of the invention, sample
retaining means are provided which exhibit an affinity to retain a
sample through application of a generated force. Rather than using
materials having different affinities, the sample retaining means
may be provided with means to generate, for example, an
electrostatic force. Indium oxide or other conductive coating
materials can be strategically placed on or in the substrate, cover
or patterned material to form a region or spot which can be charged
to form an electrostatic attractive force. If a curable displacing
fluid is then used to displace sample from around the charged
region or spot, generation of the force is no longer needed after
the displacing fluid displaces sample and/or cures.
[0095] In some embodiments wherein a generated force is used to
retain sample in a region or at a spot, the generated force may be
a temperature gradient or temperature altering means which provides
a temperature to the sample retaining means which affects the
affinity of the retaining means to a sample, and produces a
different affinity for the sample at the retaining means than at
surrounding regions of the assembly such as in the flow-through
channel.
[0096] According to some embodiments of the invention having a
flow-through channel, the sample retaining means is not a sample
well or recess but rather a sample chamber formed between a patch
of a second material and a cover or patch of a third material. The
second and third materials may be the same, and preferably both the
second and third materials have a greater affinity for a sample of
fluid medium than a first material layer which at least partially
defines the flow-through channel. For example, the second and third
materials preferably have a greater affinity for an aqueous PCR
sample than the affinity the first material exhibits to the same
PCR sample. According to some embodiments, the patch or spot of the
second and/or third material is disposed on the first material.
[0097] According to embodiments of the invention having a
flow-through channel, the substrate is a first, plate having a
patterned layer formed thereon, and the cover is a second plate
attached to and substantially parallel to the first, plate. The
first and second plates have facing substantially parallel planar
surfaces, and the patterned layer is located between the planar
surfaces and at least, partially bounds the sample retaining means.
The sample retaining means comprises a sample retaining patch
disposed on the patterned layer and is spaced a first distance from
the facing surface of the second plate.
[0098] Preferably, the flow-through channel has a bottom surface
which is spaced from the facing surface of the second plate by a
second distance, and the second distance is greater than the first
distance. According to some preferred embodiments, the sample
retaining means comprises a plurality of sample retaining patches
disposed on the patterned layer, spaced from one another, and
spaced a first distance from the facing surface of the second
plate.
[0099] According to some embodiments of the invention, the
substrate comprises a flexible material, for example, a polymeric
film such as polypropylene film, polyethylene film, polycarbonate
film, polyethyleneterephthalate film, silicone film, teflon film,
celluloid film, or other film such as a metal or ceramic film. When
a flexible material is used, it may instead be molded or formed if
not in the form of a tape. The cover may also comprise a flexible
material such as a polymeric film, such that the entire assembly is
substantially flexible. According to such embodiments, a flexible
tape can be constructed and cut to size depending upon the number
of sample retaining means desired to be utilized. An entrance
opening and an exit vent can be used to load and displace sample
fluid, and to load displacing fluid. The entrance opening and/or
exit vent may be in the form of a gap formed between the substrate
and cover at an end or gaps formed at opposite ends of a piece of
tape and in communication with a flow-through channel and a supply
of sample to be apportioned.
[0100] According to embodiments of the invention, a combination is
provided which includes a miniaturized assembly for containing a
sample of fluid medium, a means for displacing a portion of a
sample of fluid medium, and a sealing means, which may be packaged
together as a kit or available separately. The miniaturized
assembly comprises a substrate having a surface and a sample
retaining means disposed on the surface. The sample retaining means
comprises a porous structure having a porous surface and a
plurality of pores having open ends at the surface. The pores may
each have substantially the same volume and closed lower ends. The
pores have a first affinity to a sample of fluid medium such that a
sample of fluid medium disposed upon the porous surface is drawn
into and retained by the plurality of pores. The pores preferably
have an affinity to retain a sample of aqueous medium, particularly
an aqueous PCR sample. The means to displace a sample of fluid
medium from the porous surface without displacing the sample from
within said pores may comprise a cover or coverslip, for example, a
standard microscope slide coverslip. Preferably, the cover or
coverslip has a hydrophobic surface, which may be adhesive, and
which contacts the porous surface and is not wet by aqueous
samples. The cover or coverslip may be permanently attached to the
substrate after displacing sample from the porous surface, or the
cover or coverslip may be removed and replaced with a sealing
device. The sealing device according to this and other embodiments
is preferably transparent so that fluorescence emitted from sample
retained by the sample retaining means can be observed and/or
measured. The means for displacing fluid and the means for sealing
are the same device, for example, a single covering device such as
a single coated microscope coverslip. The porous sample retaining
means may be a metal plastic, glass or ceramic sieve or screen, or
other materials having a plurality of pores formed therein, such as
a substantially flat plastic disk having a plurality of pores
etched, ablated, molded, drilled, poked or otherwise formed
therein. The volume of the pores may be from about 1 microliter to
about 1 picoliter or less. Preferably, the volume of the pores is
about 100 nanoliters or less, and for some applications may be 1
nanoliter or less.
[0101] According to some embodiments of the invention, an
analytical assembly is provided comprising a microcapillary tube
having opposite ends, an inner diameter of about 100 microns or
less, and at least one amplifiable nucleic acid molecule segment
entrapped inside the tube. Microcapillary tubes having inner
diameters of about 500 .mu.m or less may also be used if a sample
restraining means is included in the tube, for example, glass
beads, gels, absorbent particles, barrier means or electrophoretic
means. Tube lengths of from about 1 mm to about 100 mm are
preferred.
[0102] Capillary action is preferably used to introduce a sample
fluid in the microcapillary tube, and after the sample is disposed
in the tube, the tube is sealed at both ends thereof to entrap the
sample inside. The sample may be divided into a plurality of
portions separated by, for example, mineral oil. The entrapped
sample or sample portions is/are thereby protected from
contamination and from evaporation. The sample of fluid medium
disposed in the tube may have a volume of about 100 nanoliters or
less, more preferably about 10 nanoliters or less, and for some
applications, a volume of about 1 nanoliter or less.
[0103] According to some microcapillary embodiments, a first fluid
is disposed in the tube on a side of the sample and adjacent to the
sample, and the first fluid is preferably substantially immiscible
with the sample. A second fluid may be disposed in the tube on the
opposite side of the sample and adjacent to the sample, and the
second fluid is also preferably substantially immiscible with the
sample, such that the sample is disposed between, and restrained
by, the first and second fluids. According to some embodiments, the
sample is an aqueous medium and the first and second fluids are
both mineral oil or polymerizable fluid. According to some
embodiments, the sample preferably comprises about 100 nanoliters
or less of a fluid medium. Multiple isolated sample segments may be
introduced by ink jet or other sample dispensing means.
[0104] The microcapillary assemblies according to embodiments of
the invention may comprise a plurality of sealed microcapillary
tubes having inner diameters of 100 microns or less, with each tube
containing at least one portion of a sample of fluid medium.
Microcapillary tubes having inner diameters of about 500 .mu.m or
less may also be used if a restraining means such as a mineral oil
is used to separate minute sample portions from each other. Tube
lengths of from about 1 mm to about 100 mm are preferred. The tubes
may each contain substantially immiscible fluids on opposite sides
of the respective portion(s) of sample within each tube, such that
the sample portion(s) in each tube is/are disposed between, and
restrained by, the substantially immiscible fluid. The
substantially immiscible fluids are preferably essentially
immiscible with the sample. According to some embodiments, the
sample comprises a polymerase chain reaction solution including
primer, and at least one segment of a nucleic acid molecule to be
amplified.
[0105] According to embodiments wherein microcapillary assemblies
are provided, the tube or tubes used in the assembly may be self
sealing or sealed at either or both ends thereof with a curable
fluid, preferably a curable adhesive. Exemplary curable adhesives
include those selected from the group consisting of light-curable,
heat-curable, two-part-curable, moisture-curable and cyanoacrylate
adhesives. When UV-curable adhesives are used and cured,
UV-blocking spots may be provided on the cover and/or substrate,
aligned with the sample retaining means, to protect the retained
sample from harmful light.
[0106] According to yet other embodiments of the invention, a
method is provided for loading a fluid sample into an assembly for
isolating and retaining a small portion of the sample. Assemblies
for carrying out such methods comprise a sample retaining means,
for example, sample chambers, for retaining the small portion of
the sample. The method of loading a fluid into an assembly may
comprise providing a first fluid to be retained and providing a
displacing fluid. An assembly is provided comprising a substrate
and a cover having facing surfaces spaced from one another, wherein
the facing surface of at least one of the substrate and the cover
comprises a first material. The assembly has a flow-through channel
between the substrate and the cover, and a first fluid retaining
means bounded on at least, one side thereof by the first material.
The channel is in communication with the flow-through channel. The
first fluid retaining means has a first affinity to the first fluid
and a second affinity to the displacing fluid, and the flow-through
channel has a third affinity to the first fluid and a fourth
affinity to the displacing fluid. The first, second, third and
fourth affinities are such that the first fluid retaining meals
retains at least a portion of the first: fluid loaded into the
assembly while displacing fluid displaces first fluid from the
flow-through channel. The displacing fluid preferably can displace
the first fluid from the flow-through channel without substantially
displacing first fluid from the first fluid retaining means. The
method further comprises causing the first fluid to be loaded into
the flow-through channel and be retained by the first fluid
retaining means, and causing the displacing fluid to enter the
flow-through channel and displace first fluid from the flow-through
channel without substantially displacing first fluid from the first
fluid retaining means. According to some embodiments of the
invention, the first affinity is greater than the second affinity.
According to some embodiments, the fourth affinity is greater than
the third affinity. According to some embodiments, the flow-through
channel comprises at least one bounding surface and has dimensions,
and the third and fourth affinities are provided by the dimensions
of the flow-through channel, and the respective affinities of the
at least one bounding surface to the first fluid and to the
displacing fluid.
[0107] According to some embodiments of the invention, a method is
provided which comprises providing an assembly including a
substrate and a cover in registry with and attached or affixed to
one another and having facing surfaces spaced a substantially
uniform distance apart from one another. The facing surface of the
substrate comprises a first material having a first affinity to a
sample of fluid medium to be contained in the assembly. A
flow-through channel is disposed between the substrate and the
cover, and the sample retaining means is bounded on at least one
side thereof by the first material. The first material may be, for
example, a surface of a glass plate or slide, or a patterned layer
such as a hydrophobic material layer deposited on the facing
surface of the substrate. The sample retaining means is in
communication with the flow-through channel and has a second
affinity to the sample of fluid medium, wherein the second affinity
is greater than the first affinity. The different affinities enable
the sample retaining means to collect a portion of a sample of
fluid medium which flows through the flow-through channel and to
retain the portion while a second fluid medium flows through and
displaces the sample from the flow-through channel.
[0108] The method of loading also comprises causing the sample to
flow through the flow-through channel and to be retained by the
sample retaining means, and causing a displacing fluid to flow
through the flow-through channel and displace the sample of fluid
medium from the flow-through channel without displacing sample from
the sample retaining means, wherein the sample retaining means has
a greater affinity for the sample than for the displacing fluid.
The displacing fluid thereby covers, entraps, encircles, and/or
surrounds, and isolates the sample retained by the sample retaining
means on at least one side of the retained sample.
[0109] According to some methods of loading, the displacing fluid
is preferably substantially immiscible with the sample of fluid
medium, and more preferably, is essentially or completely
immiscible with the sample. According to embodiments of the
invention, the sample comprises an aqueous medium. According to
embodiments of the invention, the displacing fluid may comprise a
curable fluid, preferably a curable adhesive.
[0110] According to some methods of loading, the flow-through
channel has an entrance opening for introducing sample to the
channel, and an exit vent for the escape of displaced air, sample
or excess displacing fluid from the channel, and the method further
comprises introducing the displacing fluid to the channel through
the entrance opening and causing sample in the channel to be
displaced by the displacing fluid. The displacing fluid may cause
excess sample to exit the assembly through the exit vent,
particularly in embodiments wherein the displacing fluid is forced
into the assembly by other than capillary forces, for example, by
pressure loading. In some embodiments, the exit vent may comprise a
porous or absorbent material such as paper, or other materials that
are wettable by the sample.
[0111] According to some embodiments, the method further comprises
sealing the entrance opening and exit vent after the displacing
fluid displaces sample from the channel. In some embodiments, the
displacing fluid cures to seal the entrance opening and the exit
vent. Preferably, the displacing fluid cures adjacent to the sample
retaining means to seal the sample within the sample retaining
means. In some embodiments, the sample retaining means retains
about 1 microliter to about 1 picoliter of sample or less,
preferably about 1.0 nanoliters of sample or less, and for some
embodiments, about 1 nanoliter or less.
[0112] According to embodiments of the invention, methods are also
provided for determining the existence and/or quantitation of
multiple types of nucleic acid target molecules. According to some
embodiments, different amplification targeting reagents can be
loaded separately or together with respective different samples to
be amplified. According to some embodiments, different
amplification targeting reagents are preloaded into different
sample retaining means within a single assembly, for example, an
assembly having two parallel but separated flow-through channels
having respective sample retaining means in communication with one
of the channels. According to some embodiments, each sample
retaining means contains a specific primer, pair of primers, and/or
probe and at least one of the sample retaining means contains a
primer or probe that differs from the primer or probe of a second
retaining means. A sample possibly containing more than one
different target sequence to be amplified is then introduced and
retained by the sample retaining means. When isolated, amplified
and quantitated, the existence and quantitation of two or more
different target sequences can be determined, for example by using
a fluorescence energy transfer assay. According to the invention,
the device is permanently sealed with a curable adhesive and a
homogeneous assay is performed. The sealed assay does not require
physical separation of components of the assembly to determine
whether specific target sequences have been amplified.
[0113] The invention will now be described with reference to the
drawing figures which are exemplary in nature and not intended to
limit the scope of the invention in any respect.
[0114] FIG. 1 shows an exploded view of an analytical assembly
according to an embodiment of the present invention, shown in
partial cutaway. The assembly comprises a substrate 20 and a cover
22. In the embodiment depicted, the substrate and cover comprise
substantially rigid plates such as microscope slides, but may
comprise more flexible materials such as microscope slide
coverslips. The substrate includes a bottom plate 23 having an
inner surface 24, and a patterned layer 26 of a first material
disposed on the inner surface. The first material may be a
patterned layer comprising a hydrophobic material, and provides the
substrate 20 with a facing surface 28 which faces the cover 22.
Within the patterned layer 26 of the first material are formed a
plurality of wells or holes therethrough defining sample chambers
30. Each sample chamber 30 has a closed lower end 32, defined by
the inner surface 24 of the bottom plate 23. Each sample chamber
also has a sidewall which extends from the closed lower end 32 up
to the facing surface 28 of the substrate. In some embodiments, the
sample chamber extends up through the flow-through channel.
[0115] The cover 22 comprises a top plate 34 and a facing surface
36 which may comprise the same material as the top plate or a
patterned layer 38 of a second material disposed on the inner
surface of the top plate. The second material may be the same as
the first material, and in some embodiments is preferably
moderately hydrophobic, that is, it preferably has a surface energy
of from about 30 dynes/cm to about 50 dynes/cm. Exemplary materials
for the second material include silanes, methacrylates, epoxies,
acrylates, cellulosics, urethanes, silicones, and materials having
good adhesion to the displacing fluid, for example, materials
having good adhesion to displacing fluids comprising curable
adhesives. The patterned layer 38 may be smooth and of uniform
thickness or it may have a plurality of sample chambers formed
therein complementary to the chambers formed in patterned layer
26.
[0116] When assembled, the substrate 20 and cover 22 are attached
together, with the facing surface 36 of the cover being spaced from
the facing surface 28 of the substrate. The space between the
facing surfaces 36 and 28 defines a flow-through channel through
which sample fluid and displacing fluid may travel. The facing
surfaces 36 and 28 are maintained spaced apart by spacer strips 40,
which may comprise transfer adhesive strips, films or adhesive
layers applied to edge regions of the substrate. In embodiments
wherein a spacer strip is used and comprises an adhesive material,
the spacer may also provide means to hold the substrate and cover
together. Other means of holding the substrate and cover together
may be used and include clips and clamps. According to some
embodiments, hydrophobic spacer materials are preferred.
[0117] As can be seen in FIG. 1, the spacer strips are disposed
along the longitudinal edges of facing surface 28 but are not
included on the lateral edges of the facing surface 28. Thus, a gap
is provided at the lateral edges of the assembled device and can be
employed as an entrance opening and/or an exit vent.
[0118] When assembled, a sample of fluid medium, for example, an
aqueous PCR sample, can be introduced to the flow-through channel
by entering a gap at a lateral edge of the assembly. The sample
flows through and fills the flow-through channel and the sample
chambers, which are in fluid communication with the flow-through
channel. Then, a displacing fluid is caused to enter the
flow-through channel through a gap at a lateral edge of the
assembly, and the displacing fluid flows through and fills the
channel displacing sample from within the channel but without
displacing sample from the sample chambers. The result is a
plurality of discrete, isolated portions of the sample, held by the
sample chambers.
[0119] According to the embodiment of FIG. 1 and other embodiments,
sample chambers having dimensions of a few microns in diameter and
a few microns in depth can be provided, and result in sample
chamber volumes of about 10 picoliters or less in embodiments
wherein sample chambers are provided having diameters of about 0.5
millimeter and depths of about 0.05 millimeter, sample volumes of
about 1.0 nanoliters can be achieved.
[0120] The flow-through channel may have a depth of about 0.1 to
about 500 microns, preferably from about 10 to about 100
microns.
[0121] FIG. 2 is an exploded view of an analytical assembly
according to an embodiment of the present invention, shown in
partial cutaway. The assembly comprises a substrate 50 and a cover
52. In the embodiment depicted, the substrate and cover comprise
substantially rigid plates such as microscope slides, but may
comprise more flexible materials such as microscope slide
coverslips. The substrate includes a bottom plate 53 having an
inner surface 54, and a patterned layer 56 of a first material
disposed on the inner surface. The first material may be a
patterned layer comprising a hydrophobic material, and provides the
substrate 50 with a facing surface 58 which faces the cover 52.
Within the patterned layer 56 of the first material are formed a
plurality of wells or holes therethrough defining bottom portions
60 of sample chambers. Each sample chamber bottom portion 60 has a
closed lower end 62, defined by the inner surface 54 of the bottom
plate 53. Each sample chamber also has a sidewall which extends
from the closed lower end 62 up to the facing surface 58 of the
substrate.
[0122] The cover 52 comprises a top plate 64 and a facing surface
66 which comprises a patterned layer 68 of a second material. The
second material may be the same as the first material, and in some
embodiments is preferably moderately hydrophobic, that is, it
preferably has a surface energy of from about 30 dynes/cm to about
50 dynes/cm. The patterned layer 68 may be smooth and of uniform
thickness or, as shown in FIG. 2, the patterned layer 68 may have a
plurality of upper portions 69 of sample chambers formed therein
which are complementary to the bottom portions 60 formed in
patterned layer 56.
[0123] When assembled, the substrate 50 and cover 52 are attached
together, with the facing surface 66 of the cover being spaced from
the facing surface 58 of the substrate. The space between the
facing surfaces 66 and 58 defines one or more flow-through channels
through which sample fluid and displacing fluid may travel.
Entrance openings 70 in the form of holes through the cover 52 are
provided for the sample fluid and displacing fluid to enter the
flow-through channel. Two entrance openings 70 are provided as the
assembly depicted in FIG. 2 comprises two flow-through channels. An
exit vent (not shown) is provided for each flow-through channel and
may comprise a hole formed through the substrate or cover and in
communication with the flow-through channel. The facing surfaces 66
and 58 are maintained spaced apart by spacer strips 72, which may
comprise transfer adhesive strips or patterned layer applied to
edge regions of the substrate. In embodiments wherein a spacer
strip is used and comprises an adhesive material, the spacer may
also provide means to hold the substrate and cover together. Other
means of holding the substrate and cover together may be used and
include clips and clamps. According to some embodiments,
hydrophobic spacer materials are preferred.
[0124] According to some embodiments of the invention, for example,
some embodiments similar to that of FIG. 2, a centrally located
entrance opening in the top plate can be provided and a sample
fluid and/or displacing fluid can be loaded into the assembly by
capillary force, centrifugal force, or other loading
techniques.
[0125] As can be seen in FIG. 2, the spacer strips 72 are disposed
along the entire peripheral edge of facing surface 58. A spacer
strip 74 may also be included to divide the assembly into different
portions. As shown in FIG. 2, spacer strip 74 divides the assembly
into first and second halves, 76 and 78, respectively.
[0126] When assembled, the lower and upper portions of the sample
chambers complement one another to form a plurality a sample
chambers, each having a closed lower end, a closed upper end, and a
communication to a flow-through channel, the communication of each
chamber being formed in the sidewall of the chamber. A sample of
fluid medium, for example, an aqueous PCR sample, can be introduced
to the flow-through channels through entrance openings 70. The
sample flows through and fills the flow-through channels and both
the lower and upper portions of the sample chambers, which are in
fluid communication with the respective flow-through channels.
Then, a displacing fluid is caused to enter the flow-through
channels through the entrance openings 70, and the displacing fluid
flows through and fills the channels displacing sample from within
the channels but without displacing sample from the sample
chambers. The result is a plurality of discrete, isolated portions
of the sample, held by the sample chambers. According to some
embodiments of the invention, two different sample fluids are used,
one in each half (76, 78) of the assembly.
[0127] FIG. 3 is a cross-sectional view through a longitudinal
central portion of an analytical assembly according to another
embodiment of the present invention. The assembly of FIG. 3
comprises a substrate 90 and a cover 92. In the embodiment
depicted, the substrate and cover comprise substantially rigid
plates such as microscope slides, but may comprise more flexible
materials such as films or microscope slide coverslips. The
substrate includes a bottom plate 93 having an inner surface 94,
and a patterned layer 96 of a first material disposed on the inner
surface. The first material, may be a patterned layer comprising a
hydrophobic material, and provides the substrate 90 with a facing
surface 98 which faces the cover 92. Within the patterned layer 96
of the first material, are formed a plurality of wells or holes
therethrough defining bottom portions 100 of sample chambers. Each
sample chamber bottom portion 100 has a closed lower end 102,
defined by the inner surface 94 of the bottom plate 93. Each sample
chamber also has a sidewall, which extends from the closed lower
end 102 up to the facing surface 98 of the substrate.
[0128] The cover 92 comprises a top plate 104 and a facing surface
106 which comprises a patterned layer 108 of a second material. The
second material may be the same as the first material, and in some
embodiments is preferably moderately hydrophobic, that is, it
preferably has a surface energy of from about 20 dynes/cm to about
30 dynes/cm. The patterned layer 108 may be smooth and of uniform
thickness or, as shown in FIG. 3, the patterned layer 108 may have
a plurality of upper portions 109 of sample chambers formed therein
which are complementary to the bottom portions 100 formed in
patterned layer 96.
[0129] The substrate 90 and cover 92 are attached together, with
the facing surface 106 of the cover being spaced from the facing
surface 98 of the substrate. The space between the facing surfaces
106 and 98 defines one or more flow-through channels 110 through
which sample fluid and displacing fluid may travel. An entrance
opening 112 in communication with the flow-through channel 110, in
the form of a gap formed at a lateral end of the assembly, is
provided for the sample fluid and displacing fluid to enter the
flow-through channel 110. An exit vent 114 is provided for sample
to exit the channel 110 as the sample is displaced from the channel
by the displacing fluid. The entrance opening and exit vent may
instead be in the form of holes formed through the substrate and/or
cover. The facing surfaces 106 and 98 are maintained spaced apart
by spacer strips 116, which may comprise transfer adhesive strips
or a patterned layer applied to edge regions of the substrate. In
embodiments wherein a spacer strip is used and comprises an
adhesive material, the spacer may also provide means to hold the
substrate and cover together. Other means of holding the substrate
and cover together may be used and include clips and clamps.
[0130] As can be seen in FIG. 3, the spacer strips are disposed
along the longitudinal edges between the facing surfaces but are
not included on the lateral edges of the of facing surfaces. Thus,
gaps used as the entrance opening and exit vent are provided at the
lateral edges of the device.
[0131] As can be seen in FIG. 3, the lower portions 100 and upper
portions 109 of the sample chambers complement one another to form
a plurality a sample chambers 118, each having a closed lower end,
a closed upper end, and a communication to a flow-through channel,
the communication of each chamber being formed in the sidewall of
the chamber. A sample of fluid medium, for example, an aqueous PCR
sample, can be introduced to the flow-through channel 110 through
entrance openings 112. The sample flows through and fills the
flow-through channel and both the lower and upper portions of the
sample chambers. Then, a displacing fluid is caused to enter the
flow-through channel through the entrance openings, and the
displacing fluid flows through and fills the channel displacing
sample from within the channel but not displacing sample from the
sample chambers. The result is a plurality of discrete, isolated
portions of the sample, held or retained by the sample chambers.
The sample fluid retained in the sample chambers may form a
cylinder extending from the closed lower, through and interrupting
the flow-through channel, and up to the closed upper end.
[0132] FIG. 4 is a perspective view of a bottom portion of an
analytical assembly according to an embodiment of the present
invention, with the cover removed. The bottom portion of the
assembly comprises a substrate 120, depicted in the figure as a
coated substantially rigid plate such as microscope slide. The
substrate includes a bottom plate 122 having an inner surface 124,
and a patterned layer 126 of a first material disposed on the inner
surface. The patterned layer has a substantially uniform thickness.
The first material may comprise a hydrophobic material, and
provides the substrate 120 with a facing surface 128 which faces a
cover (not shown), for example, the cover used in the embodiment of
FIG. 1. On the patterned layer 126 are formed a plurality of
patches 130 of sample retaining material, preferably capable of
retaining a fluid sample volume of from about 1 microliter to about
1 picoliter or less. Adhesive strips, a patterned layer of pressure
sensitive adhesive, or a patterned layer of a curable adhesive,
132, may be used to attach a cover to the bottom portion.
[0133] According to some embodiments, the patch occupies a volume
of about 100 nanoliters or less. The patch may be an absorbent
material which absorbs and retains sample, or the patch may be made
of material which has an affinity sufficient enough to retain, for
example, an aqueous PCR sample. According to some embodiments, the
patches of material comprise a hydrophilic material. According to
embodiments of the invention wherein the sample retaining patches
comprise a substantially non-absorbent material which has a
retaining affinity for a sample, the patch defines a closed lower
end of a sample chamber defined between the exposed surface of the
patch and the facing surface of a cover. In some embodiments, a
patch of porous material may fill a volume between the substrate
and the cover and the sample chamber is defined by the void volume
of the porous patch.
[0134] FIG. 5 is a cross-sectional view through a longitudinal
central portion of an analytical assembly according an embodiment
of the present invention wherein sample chambers comprise absorbent
material patches or comprise the volume between complementary
opposing patches of sample retaining materials.
[0135] The assembly of FIG. 5 comprises a substrate 134 and a cover
136 attached to one another. In the embodiment depicted, the
substrate and cover comprise substantially rigid plates such as
microscope slides. The substrate includes a bottom plate 138 having
an inner surface 140, and a patterned layer 142 of a first material
disposed on the inner surface. The first material may be a
hydrophobic material, and provides the substrate 134 with a facing
surface 144 which faces the cover 136. On the patterned layer 142
are formed a plurality of sample retaining patches 146 which define
sample chambers by absorbency or surface energy or other retentive
property.
[0136] The cover 136 comprises a top plate 148 and a patterned
layer 150 of a second material, defining a facing surface 152. The
second material may be the same as the first material, and in some
embodiments is preferably a hydrophilic material if aqueous samples
are to be retained and isolated. The patterned layer 150 may be
smooth and of uniform thickness or, as shown in FIG. 5, the
patterned layer 150 may have a plurality of sample retaining
patches 154 complementary to and mirroring patches 146.
[0137] The substrate 134 and cover 136 are attached together, with
the facing surface 152 of the cover being spaced from the facing
surface 144 of the substrate. The space between the facing surfaces
152 and 144 defines one or more flow-through channels 156 through
which sample fluid and displacing fluid may travel. An entrance
opening 158 comprising a hole formed through the cover and in
communication with the flow-through channel 156. An exit vent 160
comprising a hole through the substrate 134 is provided for sample
to exit the channel 156 as the sample is displaced from the channel
by the displacing fluid.
[0138] The facing surfaces 152 and 144 are maintained spaced apart
by spacer strips 162, which may comprise transfer adhesive strips
applied to edge regions of the substrate. In embodiments wherein a
spacer strip is used and comprises an adhesive material, the spacer
may also provide means to hold the substrate and cover together.
Other means of holding the substrate and cover together may be used
and include clips and clamps.
[0139] According to some embodiments, the patches comprise an
absorbent sample retaining material. According to some embodiments,
the patches retain sample by surface energy, and have an affinity
for a sample, for example, a hydrophilic material patch which
retains an aqueous sample on a surface 164 thereof. In some
embodiments, the affinity may be induced by optical or
electromagnetic means. The sample chamber may comprise the volume
166 between the facing surfaces of each complementary pair of
patches, wherein the sample chambers have no sidewalls but are
defined as the volume between the two complementary patch surfaces.
The affinity of the two opposing patch surfaces to a retained
sample is sufficient to support a column of sample between the two
patches while a displacing flows through the channel adjacent the
column. In some embodiments, patches of porous material extending
from the substrate to the cover may interrupt the flow-through
channel and the sample chamber may be defined as the void volume of
a porous patch.
[0140] Sample flows through and fills the flow-through channel and
the sample chambers, which are in fluid communication with the
flow-through channel. A displacing fluid can subsequently be caused
to enter the flow-through channel through the entrance opening,
displacing sample from within the channel but not displacing sample
from the sample chambers. The result is a plurality of discrete,
isolated portions of the sample, held or retained or absorbed by
the sample chambers.
[0141] FIG. 6A is a perspective view of another embodiment of the
present invention, and FIG. 6B shows an enlargement of portion VIB
from FIG. 6A. As can be seen in FIGS. 6A and 6B, a bottom support
plate 174 supports a sample retaining means 167 comprising a
plurality of pores or cavities 168 having open ends in
communication with an exposed surface 170 on which a sample of
fluid is applied. The pores have a first affinity to a sample of
fluid medium such that when the sample is disposed upon the exposed
porous surface 170 the sample is drawn into and retained by the
plurality of pores 168. For example, through capillary or other
force, the sample may wet and fill the pores 168. The pores 168
have substantially the same volume as one another and each has a
closed lower end 169. Preferably the pores 168 each have a volume
of about 1 .mu.l or less, more preferably, about 10 nl or less, and
for some applications about 1 nl or less. The closed lower ends 169
of the pores are defined or bound by a layer of coating material
172 deposited on a bottom plate 174. The coating material may be a
hydrophilic material if aqueous samples are to be retained in the
pores.
[0142] A cover 171 is provided on one of the sample retaining means
shown in FIG. 6A. The cover 171 can be used to displace sample
fluid from the porous surface 170 of the retaining means and for
sealing sample within the pores 168. The cover 171 displaces sample
fluid from the porous surface without displacing sample from within
the pores. Preferably, the cover 171 comprises a hydrophobic
coating 173 on the surface of the cover which contacts the exposed
surface 170 of the sample retaining means. The coating 173 contacts
sample and can be used to squeeze excess sample off of the surface
170. A means for sealing the sample of fluid within the pores is
also provided, and in the embodiment shown in FIG. 6B the means for
displacing excess sample and the means for sealing sample in the
pores are the same means, that is, the cover 171. Preferably, the
coating 173 is adhesive as well, as hydrophobic, although an
adhesive and/or hydrophobic coating may instead or additionally be
provided in the exposed surface 170 of the sample retaining means.
Sealing the sample in the pores prevents evaporation and
contamination of the sealed sample.
[0143] The displacing means and the sealing means, for example the
cover 170 shown in FIGS. 6A and 6B, may be pressed against the
exposed surface 170 by any of a variety of means. Clamps, clips or
springs may be used to attach the sealing means to the exposed
surface 170, and/or to force the displacing means against the
exposed surface.
[0144] According to some embodiments of the invention, other means
may be used as displacing means for a device similar to, or the
same as, that shown in FIGS. 6A and 6B. For example, according to
some embodiments, a drop or bead of curable fluid, for example, a
curable adhesive, can be forced across an exposed surface of a
sample retaining means having one or more sample retaining cavity,
recess, hole or pore formed in the surface. Gravity, pressurized
gas, or mechanical means, for example, can be used to force the
curable fluid across the exposed surface, displacing excess sample
from the surface without displacing sample from within the sample
retaining cavity. According to some embodiments of the invention,
as the curable fluid traverses the exposed surface a thin layer of
the fluid is deposited on the surface and coats the top of a sample
portion retained in the cavity. Upon curing, the curable fluid
forms a seal for the sample retained within the cavity such that
the sample retained is isolated from other cavities and from excess
sample. The sealed sample is protected from contamination and
evaporation. Preferably, the curable fluid seal is sufficient to
prevent contamination and evaporation of the entrapped sample
during thermal cycling conditions generally used in PCR
amplification methods.
[0145] According to some embodiments of the invention, separate
displacing means and sealing means are provided for isolating a
sample portion within a sample retaining cavity. For example,
according to embodiments of the invention, a displacing means is
provided for removing excess sample from an exposed surface of a
device having a sample retaining cavity associated therewith. The
sample retaining cavity may be formed on, formed in, in contact
with or adjacent to a substrate. The cavity may be defined by at
least one sidewall, and the sidewall may comprise a hydrophilic
material. The displacing means may comprise a wiping device such as
a squeegy, a wiper, a blade or other scraping or rubbing device
which can physically move excess sample away from the open upper
end of a sample retaining cavity and preferably off of the exposed
surface of the sample retaining means. Preferred displacing means
may comprise a wiping device made of an elastomeric material, for
example, a stiff silicone rubber blade. Preferably, the wiping
device comprises a hydrophobic material to which aqueous sample
fluids will not cling.
[0146] According to some embodiments, the displacing means
comprises an opening or channel through which a sample retaining
means snugly fits, and a wiping device is provided adjacent to the
opening or channel such that the wiping device wipes excess sample
from an exposed surface of the sample retaining means as the
retaining means is forced through the opening or channel.
[0147] According to some embodiments of the invention, sealing
means are applied to the sample retaining cavity immediately after
the displacing means displaces excess sample from adjacent the
sample retaining cavity. When retained sample portions having
volumes of about 1 .mu.l or less are formed, they tend to evaporate
rapidly and thus require prompt sealing to protect sample
integrity. According to some embodiments of the invention, a wiping
device is used to displace excess sample fluid from an exposed
surface of a sample retaining means, and the wiping device is
provided with a trailing edge that pulls a bead or drop of a
curable sealing fluid across the exposed surface as a leading edge
of the wiping device displaces excess sample.
EXAMPLE 1 AND CONTROL 1
[0148] Conventional PCR was performed in 0.2 ml polypropylene
ependorf tubes to set standards. Microcapillary PCR was then
performed according to the present invention on the same sample
material in quartz glass microcapillaries. The PCR sample
containing the nucleic acid sequence to be amplified was prepared
and included materials from a "TaqMan" kit available from
Perkin-Elmer, Applied Biosystems Division, Foster City, Calif. The
kit contained human DNA at 10 ng/.mu.l, the forward primer
5'-TCACCCACACTGTGCCCATCTACGA-3' (SEQUENCE ID NO:1) and the reverse
primer 5'-CAGCGGAACCGCTCATTGCCAATGG-3' (SEQUENCE ID NO:2) that
amplify a 295 bp segment of the human .beta. actin gene, and a dual
fluor-labeled probe comprising
5'-[6FAM]-ATGCCC-[TAMRA]-CCCCCATGCCATCCTGCGT-3' (SEQUENCE ID NO:3)
that is complementary to bases 31 to 56 of the PCR product. The
designation FAM represents 6-carboxyfluorescein, and TAMPA
represents 6-carboxytetramethyl-rhodamine. With reference to the
Control 1 and Example 1, the term "FAM" is referred to herein as
"fluorescein" and the term "TAMRA," is referred to herein as
"rhodamine".
[0149] The PCR sample comprised Taq polymerase available from
Boehringer Mannheim, Indianapolis, Ind., and anti-Taq antibody from
Clonetech, Palo Alto, Calif. The sample also comprised
concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, about 0.01%
by weight gelatin, 500 .mu.g/ml, to 5 mg/ml bovine serum albumin
(BSA), 3.5 mM MgCl.sub.2, 0.2 mM each of dATP, dCTP, dGTP and dUTP,
0.3 .mu.M forward and reverse primers, 0.2 .mu.M dual-fluor-labeled
probe, 0.5 manufacturer's units (u) Taq polymerase per 10 .mu.l PCR
mixture, 0.1 .mu.l anti-Taq antibody per 1.0 .mu.l PCR mixture, and
varying amounts of template DNA. The specific activity of Taq
polymerase was about 250,000 units per mg which, at a molecular
weight of 100,000 Daltons, translates to about 10.sup.9 molecules
per .mu.l. Since .beta. actin is a single copy gene, it was
estimated that there is one copy of .beta. actin template per 3 pg
of human genomic DNA. In some PCRs the dual-fluor-labeled probe was
replaced with the fluorescent, DNA-staining dye SYBR.TM. Green I
(product # S-7567) available from Molecular Probes, Eugene, Oreg.,
used a 10.sup.-4 dilution from the stock supplied by the
manufacturer.
[0150] The conventional PCRs (CONTROL 1) performed in the 0.2 ml
polypropylene tubes were thermocycled in a model 9600 thermocycler
from Perkin Elmer using 92.degree. C. for 15 seconds, 54.degree. C.
for 15 seconds, and 72.degree. C. for 15 seconds, for 40
cycles.
[0151] Microcapillary PCR was performed according to the present
invention in quartz glass microcapillaries from Polymicro
Technologies (Phoenix, Ariz.). These capillaries had inner
diameters ranging from 20 .mu.m to 75 .mu.m and outer diameters of
250 .mu.m to 375 .mu.m. The capillaries come with either a
polyimide or Teflon external coating to make them flexible. Because
the polyimide coating is opaque and fluorescent, it had to be
removed before use. The coating was removed by flaming a segment of
polyimide capillary with a Bunson burner for several seconds and
then gently wiping off the burned coating. Flaming and wiping was
repeated as necessary until the capillary was clear. The resulting
bare capillaries were very fragile. The Teflon coated
microcapillaries were easier to work with since the optically clear
and non-fluorescent Teflon coating did not need to be removed. Both
types of capillaries gave equivalent results in PCR.
[0152] Hundreds of microcapillary tubes (EXAMPLE 2) were filled by
touching an open end to a drop of the PCR sample which wicked in by
capillary action. The microcapillaries were then sealed and
supported by gluing the two ends thereof to two respective
coverslips, leaving an unsupported segment in the middle, thus
minimizing thermal mass. A UV-curable fluid glue was used to seal
the end of the tube and to glue the tube ends to coverslips.
Assemblies comprising a plurality of microcapillaries were formed
by sealing the ends of each tube in the assembly to the same pair
of coverslips.
[0153] The glue was available as optical adhesive #81 from Norland
Products, New Brunswick, N.J. The glue was cured by exposure to 366
nm UV light. A source of 366 nm wavelength light is UV lamp model
UVL-21., available from UVP Inc., San Gabriel, Calif. The lamp was
held about 1 cm from the sample for 30 seconds. The PCR mixture was
shielded from UV light by laying a small piece of opaque paper over
the center section of the capillary during UV exposure.
[0154] FIG. 7 is a top plan view of an exemplary microcapillary
device used in accordance with Example 1 of the present invention.
As shown in FIG. 7, an assembly 176 is provided having four
microcapillary tubes 178 having inner diameters of about 100
microns each, and two support tubes 180 having outer diameters of
about 1.5 mm. The ends of the tubes 178 and 180 were adhered to two
respective plates or bases 182 with UV-curable adhesive 184. One
end of each tube microcapillary and support tube shared a
respective plate 182. In the embodiment shown in FIG. 7, the plates
comprised standard 1''.times.1'' microscope cover slips and the
tubes were each about 4 cm long. All the tubes were arranged
substantially parallel to one another with the microcapillary tubes
178 each being positioned between the two larger diameter support
tubes.
[0155] The sample holding assembly 176 was attached with SCOTCH
tape to the sample holder of a Rapidcycler air oven available from
Idaho Technologies, Idaho Falls, Id., and cycled through a protocol
comprising cycles of 92.degree. C. for 5 seconds, 54.degree. C. for
5 seconds, and 72.degree. C. for 15 seconds, for 40 cycles. The
cycling protocol took about 30 minutes in the Rapidcycler.
[0156] After PCR cycling, fluorescence of the samples was measured
with a Zeiss Axiovert 410 laser scanning microscope using a
20.times. -0.5 NA objective, 15 mW external argon laser
approximately 5% of the power of which was focused to a spot size
of 1 .mu.m.sup.2, and band pass filters of 515-565 nm for
fluorescein and SYBR.TM. Green, and >590 nm for rhodamine (power
loss and spot size estimates provided from the manufacturer).
Average pixel intensity was measured in regions of about 20
.mu.m.times.50 .mu.m overlying the capillary image. Fluorescence
intensity was examined visually along the 2.5 cm lengths of
capillaries by manually translating the stage; quantitative
measurements were made every 2-5 mm or more often if variability in
the fluorescence signal was observed.
[0157] Results
[0158] When PCRs were performed in 20 .mu.l volumes in ependorf
tubes in a model 9600 Thermocycler (Perkin Elmer, Norwalk, Conn.),
the .beta. actin primers amplified an approximately 300 bp segment
from human DNA as expected. PCRs performed in the presence of the
TaqMan probe were transferred to capillary tubes and analyzed by
fluorescence microscopy. Typical, values for average pixel
intensity were about 130 (relative fluorescence units) for
fluorescein aid about 60 for rhodamine, with values of background
emission from empty capillaries of about 20 at both wavelengths. In
different experiments the fluorescein:rhodamine (F/R) intensity
ratio varied from about 1.0:1.0 to about 2.0:1.0 in samples
containing PCR product. For negative control PCRS containing no
template DNA, no Taq polymerase, or no reverse primer, the
rhodamine emission was about the same (about 60), while the
fluorescein emission was reduced to about 30, giving a F/R
intensity ratio of about 0.5. The absolute values of fluorescein
and rhodamine emission varied between experiments and with small
changes in machine settings (laser power, attenuation, brightness,
contrast) whereas the F/R intensity ratio was fairly constant.
Therefore, the F/R intensity ratio was used as a measure of whether
the .beta. actin product had been amplified.
[0159] The yield of PCR product in conventional reactions in
polypropylene tubes was estimated by ethidium bromide staining of
product in agarose gels, and by adding a known amount of
.sup.32P-dCTP to a PCR and counting radioactivity in the purified
PCR product. Both methods gave an estimate of about 10.sup.11
product molecules/.mu.l of PCR. This corresponds to a product
concentration of about 0.16 .mu.M, which implies that about half of
the PCR primers were converted to product.
[0160] To assess the extent of degradation of TaqMan probe
following PCR, the effect of mung bean nuclease on the F/R
intensity ratio was examined. Treatment of probe with mung bean
nuclease for 10 minutes at 37.degree. C. raised the F/R intensity
ratio from 0.5 to 5. This presumably represents complete
degradation since further incubation did not increase the ratio. An
F/R intensity ratio of 1.5, characteristic of positive PCRs,
therefore suggests that about 30 of probe was degraded. This
corresponds to a concentration of degraded probe of 0.06 .mu.M and
implies that about one third of the probe that could have
hybridized to PCR product was degraded.
[0161] When PCRs were performed in small diameter glass
capillaries, the volume of the reaction was too small to detect PCR
product by standard gel electrophoresis. While products might have
been detectable by capillary electrophoresis, it was of interest to
see whether the TaqMan assay could be used as a detection method.
The F/R intensity ratio was therefore used as a surrogate measure
of amplification. This ratio was about 0.5 in negative control
reactions (no template, no enzyme, or no reverse primer) and was
usually greater than 1 in samples where product was expected. In
capillaries containing terminal dilutions of genomic DNA template,
the ratio sometimes varied with position along the capillary, which
was attributed to localized accumulation of degraded probe,
discussed in more detail below. In cases where the intensity ratio
varied, the maximum value of the F/R intensity ratio in the
capillary was used as the measure of whether the target sequence
had been amplified.
[0162] A histogram of the maximum values of the F/R intensity ratio
in over 100 capillary reactions of terminally diluted genomic DNA
is shown in FIG. 8, along with the corresponding values for
negative control reactions. The negative controls had a mean ratio
of 0.5 with a range of 0.4 to 0.9. The experimental samples had a
bimodal distribution with one arm of the distribution paralleling
that, of the negative control samples. This suggests that the
experimental samples consisted of positive and negative samples.
Since the nadir of the experimental sample distribution occurred at
about an F/R intensity ratio of 1, we chose this value as a
"cut-off" to distinguish positive from negative samples. This
"cut-off" is consistent with the F/R values of 1 to 2 in PCRs
carried out in conventional volumes in ependorf tubes.
[0163] Using a "cut-off" of F/R.gtoreq.1, the sensitivity of the
detection system was estimated by mixing exonuclease-digested probe
with undegraded probe. An F/R ratio of .gtoreq.1 was obtained when
.gtoreq.0.02 .mu.M degraded probe was mixed with 0.2 .mu.M
undegraded probe. This corresponds to about 10.sup.8 molecules of
degraded probe in a 10 nl volume. Using the confocal feature of the
microscope, it was determined that the signal decreased rapidly
when the depth of field dropped below 20 .mu.m. Thus, an estimate
of the lower limit of detection for this system is about 10.sup.5
molecules of degraded probe in a volume of 20 .mu.m.times.20
.mu.m.times.20 .mu.m, that is, about 10 picoliters (pl).
[0164] As has been noted by others performing PCR in glass tubes,
for example in the publication of Wittwer et al., Rapid Cycle DNA
Amplification: Time and Temperature Optimization, BioTechniques,
Vol. 10, pp. 76-83 (1991), it was important to include bovine serum
albumin (BSA) in the PCRs. Presumably, BSA blocks non-specific
sticking of DNA to glass. When BSA was not included, the F/R
intensity ratio was about 0.5. Generally, a 500 .mu.g/ml final
concentration of BSA was used in the PCRs although for some batches
of BSA the concentration was increased to 5 mg/ml.
[0165] Human DNA was diluted so that PCRs contained 0-14 haploid
genome equivalents (0-42 pg)/capillary. Reactions were scored as
positive if the maximum F/R intensity ratio along the tube was 1.0
or greater. The results for a series of PCRs in capillaries with
internal diameters of 20 to 75 are shown in Table 1 below and shown
graphically in FIG. 8.
TABLE-US-00001 TABLE 1 Replicate PCRs in microcapillaries with
terminal dilutions of genomic DNA Haploid Probability of Capillary
genome .gtoreq.1 template Fraction diameter equivalents per
capillary positive Number of (microns) per capillary, m 1-e.sup.-m
PCRs PCRs 20 0 0.00 0.00 2 0.2 0.18 0.10 10 0.5 0.39 0.33 15 25 0
0.00 0.00 4 0.4 0.33 0.28 18 0.8 0.55 0.50 8 1.5 0.78 1.00 3 3 0.95
1.00 3 30 0 0.00 0.00 8 0.5 0.39 0.52 23 1 0.63 0.92 13 1.5 0.78
1.00 10 4 0.98 1.00 3 50 0 0.00 0.00 13 0.4 0.33 0.00 3 0.8 0.55
0.67 27 1.5 0.78 0.82 28 3 0.95 1.00 2 6 1.00 0.89 9 13 1.00 1.00 7
75 0 0.00 0.00 7 0.8 0.55 0.50 6 1.7 0.82 0.67 12 3.4 0.97 1.00 9 7
1.00 1.00 5 14 1.00 1.00 6
[0166] Capillaries containing more than one haploid genome
equivalent generally had F/R intensity ratios greater than 1. In
capillaries containing less than one haploid genome equivalent, the
fraction of capillaries with F/R intensity ratio .gtoreq. 1 was
roughly proportional to the fraction of capillaries expected to
contain 0.1 or more template molecules. This fraction was
calculated from the Poisson distribution as 1-e.sup.-m where m=the
amount of DNA/capillary/3 pg. The results provide strong support
for the hypothesis that reactions were positive when capillaries
contained 1 or more template molecules.
EXAMPLE 2
[0167] Similar results were obtained with another preparation of
human genomic DNA obtained from Promega: at 8 haploid genome
equivalents (24 pg) per capillary, 4 of 4 capillaries gave maximum
F/R intensity ratios .gtoreq. 1; at 0.7 haploid genome equivalents
(2 pg) per capillary, 3 of 4 capillaries were positive; at 0.1
haploid genome equivalents (0.4 pg) per capillary, 0 of 4
capillaries were positive.
EXAMPLE 3
[0168] The inhomogeneity of F/R intensity ratio along the length of
capillaries containing about 1 template molecule suggested that
residual localization of degraded probe may be observed as a result
of localized accumulation of PCR product. To investigate this
possibility, amplifications in 2.5 cm long sections of capillaries
containing about 0.5 haploid genome equivalent per capillary were
performed. A plot of F/P intensity ratio along a few representative
capillaries is shown in FIGS. 9-14. Some capillaries had a single
peak while others had two. Two peaks indicate two areas where PCR
product and degraded probe had accumulated. The half-widths of the
peaks (measured at half-height) were about 3-6 mm. When capillaries
were left overnight, the distributions broadened and flattened.
Inhomogeneities in F/R intensity ratio were not seen when
capillaries were examined before PCR, or after PCR in capillaries
containing no template DNA or about 75 initial template molecules.
Representative experiments are shown in Table 2 below. The results
indicate that high variability in the F/R intensity ratio is
specific for capillaries with about 1 target molecule and decreases
with time.
TABLE-US-00002 TABLE 2 NUMBER OF HAPLOID CAPILLARIES STANDARD GENE
WITH AVERAGE DEVIATION EQUIVALENTS TIME WHEN MAXIMUM F/R F/R OF F/R
PER FLUORESCENCE NUMBER OF INTENSITY INTENSITY INTENSITY GROUP
CAPILLARY ANALYZED CAPILLARIES RATIO > 1 RATIO RATIO A 0 1 HOUR
AFTER PCR 9 0 0.50 0.05 B 75 1 HOUR AFTER PCR 10 10 1.75 0.10 C 1
BEFORE PCR 5 0 0.51 0.04 D 1 1 HOUR AFTER PCR 5 4 0.83 0.46 E 1 24
HOURS AFTER PCR 5 3 0.89 0.23 F 1 48 HOURS AFTER PCR 5 1 0.82 0.23
G 1 BEFORE PCR 5 0 0.46 0.03 H 1 1 HOUR AFTER PCR 5 5 1.22 0.43 I 1
24 HOURS AFTER PCR 5 5 1.14 0.25 J 1 48 HOURS AFTER PCR 5 5 1.11
0.21
[0169] These results support a theory that the inhomogeneities were
not due to smudges blocking light transmission, thermal variations
during PCR, or photobleaching. It was also determined that the 30
seconds of UV irradiation used to cure the sealing glue at the ends
of the capillaries did not alter the F/R ratio. Photobleaching of
fluorescein (but not the rhodamine) was detectable with repeating
laser scanning at the highest power, with 10 scans reducing the
fluorescein signal about 1.0%; however, only 1 or 2 scans at this
power were performed at any one location when collecting data, and
thus photobleaching does not explain the inhomogeneities. To see if
convection after PCR might be broadening peaks, 0.2% by weight to
about 0.8% by weight low-melt agarose was added to some PCRs but no
effect of the agarose was noted. A few of the capillaries
fortuitously contained air bubbles that divided the sample into two
or more segments. In several of these cases, the F/R intensity
ratio was .gtoreq.1 on one side of a bubble and 0.5 on the other
side, consistent with blocked diffusion of degraded probe.
[0170] To confirm the results of the TaqMan assay, the fluorescent
dye SYBR.TM. Green I was substituted for the TaqMan probe. Because
the fluorescence of SYBR.TM. Green I increases many fold in the
presence of double stranded DNA, it can be used to detect double
stranded PCR product, although it does not distinguish spurious
product such as "primer dimer" from desired product. The SYBR.TM.
Green I fluorescence assay has to be performed at elevated
temperature to reduce background fluorescence from non-specific
annealing of primers. To do this, segments of capillaries were
placed, after PCR, in about 1 ml of mineral oil in a special 35 mm
petri dish, the bottom of which was made of optically clear,
conducting glass coated with a thin layer of indium tin oxide
available from Bioptechs, of Butler, Pa. By applying 3-4 volts
across the bottom of the dish, the temperature in the oil, was
raised to about 70.degree. C. Because only a portion of the bottom
of the petri, dish was flat and accessible in the microscope, the
capillaries had to be cut after PCR into approximately 1 cm
segments in order to be imaged.
[0171] Using this device, PCR product derived from single template
molecules could be detected. For example, the fluorescence
intensity was 155-194 in 7 capillary segments derived from a PCR
containing 30 haploid genome equivalents per cm of capillary
length, compared to a fluorescence intensity of 40-57 in 7
capillary segments containing no template DNA. The variability in
fluorescence at 2-3 mm intervals along these capillaries was about
20%. In contrast, in 7 capillary segments derived from PCRs
containing 0.3 haploid genome equivalents per cm of capillary, the
fluorescence intensity varied from 49 to 136, with 4 capillary
segments having fluorescence intensity <73 at all tested
positions along their lengths, 2 capillary segments having
fluorescence intensity >100 at all positions, and one capillary
having a fluorescence intensity of 68 at one end increasing to 122
at the other end thereof. These results provide additional evidence
that PCR products derived from single molecules can be detected and
remain localized in microcapillaries for several hours after
PCR.
EXAMPLE 4
[0172] A device substantially similar to that depicted in FIG. 3,
having a substrate and a cover comprising microscope slide
coverslips, and provided with first and second patterned layers
comprising cured Norland 68 UV-curable adhesive coated respectively
thereon by screen printing, was filled by capillary action with a
Beta-actin polymerase chain reaction solution. The substrate and
coverslip were spaced apart and held together with adhesive strips
and the first and second patterned layers had complementary sample
chamber portions formed therein having radii of about 1 mm. A
displacing fluid comprising uncured Norland 81 UV-curable adhesive
was then loaded into the flow-through channel by capillary action
and displaced the polymerase chain reaction solution from the
flow-through channel but not from the sample chambers. The device
was then exposed to UV light to cure the displacing fluid, and the
sample portions were shielded from the light with a transparency
laid on top of the device and having inked spots aligned with the
sample chambers. The device was then thermocycled and analyzed by
fluorescence microscopy as described in connection with Example 1.
Six of six sample chambers estimated to contain an average of 3
copies of genomic DNA template had F/R intensity ratios greater
than 1 and four of six sample chambers estimated to contain an
average of 0.3 template copies had F/R intensity ratios of greater
than 1.
Discussion
[0173] The results presented above provide strong evidence that the
TaqMan assay can easily detect as little as 1 template molecule
when the volume of the reaction is on the order of 10 nl. For 50
.mu.m inner diameter capillaries, the reaction volume is about 20
nl per 1 cm length of capillary. Using terminal dilutions of two
preparations of genomic DNA, good correlation has been found
according to the invention between the number of capillaries giving
positive reactions and the number of capillaries calculated to
contain 1 or more template molecules. The inference of single
molecule sensitivity is further supported by the observation of
peaks of elevated F/R emission along the length of capillaries
estimated to contain 1 or 2 template molecules. Presumably these
peaks results from localized accumulation of PCR product and
corresponding degraded probe.
[0174] The localized accumulations of PCR product and degraded
probe remained detectable for several hours after PCR. It is
believed that the narrowness of the capillaries effectively
eliminates convection so that molecular movement is dominated by
diffusion. Molecules the size of completely degraded probe (e.g.
rhodamine-dGTP) have diffusion constants of about
3-5.times.10.sup.-6 cm.sup.2/sec in water at room temperature as
reported in the publication of Chang, PHYSICAL CHEMISTRY with
application to Biological Systems, MacMillan Publishing Co., New
York, page 87. The diffusion constant increases with temperature as
D is proportional to kT/.eta., where T is measured in degrees
Kelvin and .eta., the viscosity, decreases with temperature. The
viscosity of water decreases about 3-fold as temperature increases
from 25.degree. C. (298.degree. K) to 92.degree. C. (365.degree.
K); thus, D would increase about 3.25-fold over this temperature
range. The root mean square distance traveled by a molecule with
diffusion constant D in time t is (2Dt).sup.1/2 or about 2-5 mm in
2 hours for molecules the size of completely degraded probe at
temperatures between 25.degree. C. and 92.degree. C. The PCR
product, based on its molecular weight, should have a diffusion
constant of about 0.45.times.10.sup.-6 cm.sup.2/sec and should
diffuse about 3 times less far than degraded probe in the same
time. These calculations indicate that the widths of the observed
fluorescent peaks are consistent with diffusion-mediated spreading
of PCR product and degraded probe.
[0175] The width of peaks might be slightly larger than predicted
by diffusion, due to the tendency of PCR to saturate in regions
where the concentration of PCR product is high. So long as all of
the amplified molecules are replicated each cycle, the progeny from
a single starting template will have the same average displacement
(i.e., root mean square displacement) as a collection of
independent molecules, that is, they will appear to diffuse with a
root mean square displacement that is proportional to the square
root of the time. However, as PCR begins to saturate, molecules
near the center of the distribution where concentration is high
have a lower probability of being replicated than molecules near
the "edge" of the distribution where concentration is low. This
unequal probability of replication tends to make the distribution
broader.
[0176] The diffusion model suggests that detection of single target
molecules by TaqMan assay would be difficult using conventional
size capillaries. A 5 mm segment (characteristic diffusion distance
for degraded probe) in a 0.8 mm inner diameter capillary contains
about 2.5 .mu.l. After spreading in 1/50th of this volume (about 50
nl), the fluorescent signal obtained from single starting molecules
in 50 .mu.m inner diameter capillaries was sometimes not above the
background (see Table 2, average F/R intensity at 24 and 48 hours
and number of capillaries in which maximum F/R>1). Thus, PCR
would have to be significantly more efficient than achieved to
detect single molecules in volumes greater than 1 .mu.l.
[0177] While limited diffusion of product and degraded probe was
important for our ability to detect single starting molecules in
capillaries, diffusion of reactants present in the original
reaction mixture is usually not limiting for PCR; for example, at
the conventional concentration of Taq polymerase used here, the
average distance between polymerase molecules is about 1 .mu.m, and
polymerase molecules (MW about 100,000 Daltons) diffuse this
distance in about 0.01 second. Thus, all portions of the reaction
should be sampled by a polymerase molecule many times each
second.
[0178] The data also shows that single DNA molecules or segments
can be detected with the "TaqMan" system when PCRS are confined to
volumes of 100 nanoliters or less, preferably 60 nanoliters or
less, by using capillaries with small diameters and relying on the
fortuitously slow rate of diffusion. Many PCR reactions with single
molecule sensitivity can be performed simultaneously in small
spaces by confining PCR's to small regions in 3 dimensions as
described in other embodiments of the present invention. The
devices of the invention can be used to measure the number of
template molecules in a sample simply by counting the number of
positive reactions in replicate PCRs containing terminal dilutions
of sample. Due to the closed system environment which prevents
carryover contamination, and the ability to automate fluorescence
detection, devices according to the present invention and methods
for using the devices have significant potential for clinical uses
of PCR. An assay based on presence versus absence of PCR product in
replicate reactions may be more robust with respect to small
changes in amplification efficiency than quantitative competitive
assays or time-to-reach-threshold level assays that require
assumptions about relative or absolute amplification rates.
[0179] Although the present invention has been described in
connection with preferred embodiments, it will be appreciated by
those skilled in the art that additions, modifications,
substitutions and deletions not specifically described may be made
without departing from the spirit and scope of the invention
defined in the appended claims.
Sequence CWU 1
1
3125DNAArtificialPolymerase Chain Reaction Primer for the Human
Beta Actin Gene. 1tcacccacac tgtgcccatc tacga
25225DNAArtificialPolymerase Chain Reaction Primer for the Human
Beta Actin Gene. 2cagcggaacc gctcattgcc aatgg
25325DNAArtificialDual Fluor-Labeled Probe. 3ntgcccnccc catgccatcc
tgcgt 25
* * * * *