U.S. patent application number 11/225247 was filed with the patent office on 2006-01-19 for apparatus for analysis of a nucleic acid amplification reaction.
This patent application is currently assigned to Cepheid. Invention is credited to David A. Borkholder, Lee A. Christel, William A. McMillan, Steven J. Young.
Application Number | 20060014200 11/225247 |
Document ID | / |
Family ID | 24245208 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060014200 |
Kind Code |
A1 |
McMillan; William A. ; et
al. |
January 19, 2006 |
Apparatus for analysis of a nucleic acid amplification reaction
Abstract
An apparatus for determining a threshold value (e.g., a
threshold cycle number or a time value) in a nucleic acid
amplification reaction comprises a detection mechanism for
measuring, at a plurality of different times during the
amplification reaction, at least one signal whose intensity is
related to the quantity of a nucleic acid sequence being amplified
in the reaction. A controller in communication with the detection
mechanism is programmed to store signal values defining a growth
curve for the nucleic acid sequence, determine a derivative of the
growth curve, and calculate a cycle number or time value associated
with a characteristic of the derivative.
Inventors: |
McMillan; William A.;
(Cupertino, CA) ; Christel; Lee A.; (Palo Alto,
CA) ; Borkholder; David A.; (San Jose, CA) ;
Young; Steven J.; (Los Gatos, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Cepheid
Sunnyvale
CA
|
Family ID: |
24245208 |
Appl. No.: |
11/225247 |
Filed: |
September 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10027404 |
Dec 19, 2001 |
6942971 |
|
|
11225247 |
Sep 12, 2005 |
|
|
|
09808674 |
Mar 14, 2001 |
6713297 |
|
|
10027404 |
Dec 19, 2001 |
|
|
|
09562195 |
May 1, 2000 |
6783934 |
|
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09808674 |
Mar 14, 2001 |
|
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|
Current U.S.
Class: |
435/6.11 ;
435/287.2 |
Current CPC
Class: |
G01N 2035/00366
20130101; C12Q 1/6851 20130101; G01N 21/278 20130101; C12Q 1/6851
20130101; G01N 21/6428 20130101; G01N 2021/6419 20130101; G01N
21/645 20130101; G01N 2035/00376 20130101; G01N 2021/6463 20130101;
C12Q 2545/101 20130101; C12Q 2545/113 20130101; C12Q 2545/107
20130101; C12Q 2545/113 20130101; B01L 7/52 20130101; C12Q 2537/165
20130101; G01N 21/274 20130101; G01N 2201/062 20130101; G01N
2021/6417 20130101; G01N 2021/6471 20130101; G01N 2021/6441
20130101; G01N 2021/6432 20130101; C12Q 1/6851 20130101; G01N
2201/0627 20130101; G01N 2021/6439 20130101; G01N 2035/0097
20130101; G01N 2021/6421 20130101; C12Q 1/6851 20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Claims
1. An apparatus for determining a threshold time value in a nucleic
acid amplification reaction, the apparatus comprising: a) a
detection mechanism for measuring, at a plurality of different
times during the amplification reaction, at least one signal whose
intensity is related to the quantity of a nucleic acid sequence
being amplified in the reaction; and b) a controller in
communication with the detection mechanism, wherein the controller
is programmed to perform the steps of: i) deriving a growth curve
from the measurements of the signal; ii) calculating a derivative
of the growth curve; iii) identifying a characteristic of the
derivative; and iv) determining a time value associated with the
characteristic of the derivative.
2. The apparatus of claim 1, wherein the controller is programmed
to calculate a second derivative of the growth curve, and wherein
the characteristic comprises a positive peak of the second
derivative.
3. The apparatus of claim 1, wherein the controller is programmed
to calculate a second derivative of the growth curve, and wherein
the characteristic comprises a negative peak of the second
derivative.
4. The apparatus of claim 1, wherein the controller is programmed
to calculate a second derivative of the growth curve, and wherein
the characteristic comprises a zero crossing of the second
derivative.
5. The apparatus of claim 1, wherein the controller is programmed
to calculate a first derivative of the growth curve, and wherein
the characteristic comprises a positive peak of the first
derivative.
6. The apparatus of claim 1, wherein the controller is programmed
to calculate second derivative values of the growth curve at a
plurality of different measurement times in the reaction to yield a
plurality of second derivative data points, the characteristic
comprises a positive peak of the second derivative, and the
controller is further programmed to determine the time value
associated with the positive peak by: i) fitting a second order
curve to the second derivative data points; and ii) calculating the
time value as the location of a peak of the second order curve.
Description
CONTINUING APPLICATION DATA
[0001] This application is a continuation of U.S. Ser. No.
09/808,674 filed Mar. 14, 2001 which application is a division of
U.S. Ser. No. 09/562,195 filed May 1, 2000. All of these
applications are incorporated by reference herein for all
purposes.
COPYRIGHT AUTHORIZATION
[0002] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by any one of
the patent disclosure as it appears in the U.S. Patent and
Trademark Office patent files or records, but otherwise reserves
all copyright rights whatsoever.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention relates to an apparatus for analysis of a
nucleic acid amplification reaction.
BACKGROUND OF THE INVENTION
[0004] Quantitative nucleic sequence analysis plays an increasingly
important role in the fields of biological and medical research.
For example, quantitative gene analysis has been used to determine
the genome quantity of a particular gene, as in the case of the
human HER-2 oncogene, which is amplified in approximately 30% of
human breast cancers. Gene and genome quantitation have also been
used in determining and monitoring the levels of human
immunodeficiency virus (HIV) in a patient throughout the different
phases of the HIV infection and disease. It has been suggested that
higher levels of circulating HIV and failure to effectively control
virus replication after infection may be associated with a negative
disease prognosis. Accordingly, an accurate determination of
nucleic acid levels early in an infection may serve as a useful
tool in diagnosing illness, while the ability to correctly monitor
the changing levels of viral nucleic acid in one patient throughout
the course of an illness may provide clinicians with critical
information regarding the effectiveness of treatment and
progression of disease.
[0005] Several methods have been described for the quantitative
analysis of nucleic acid sequences. The polymerase chain reaction
(PCR) and reverse-transcriptase PCR (RT-PCR) permit the analysis of
small starting quantities of nucleic acid (e.g., as little as one
cell equivalent). Early methods for quantitation involved measuring
PCR product at the end of temperature thermal cycling and relating
this level to the starting DNA concentration. Unfortunately, the
absolute amount of product generated does not always bear a
consistent relationship to the amount of target sequence present at
the initiation of the reaction, particularly for clinical samples.
Such an endpoint analysis reveals the presence or absence of
starting nucleic acid, but generally does not provide an accurate
measure of the number of DNA targets. Both the kinetics and
efficiency of amplification of a target sequence are dependent on
the starting quantity of that sequence, and on the sequence match
of the primers and target template, and may also be affected by
inhibitors present in the sample. Consequently, endpoint
measurements have very poor reproducibility.
[0006] Another method, quantitative competitive PCR (QC-PCR), has
been developed and used widely for PCR quantitation. QC-PCR relies
on the inclusion of a known amount of an internal control
competitor in each reaction mixture. To obtain relative
quantitation, the unknown target PCR product is compared with the
known competitor PCR product, usually via gel electrophoresis. The
relative amount of target-specific and competitor DNA is measured,
and this ratio is used to calculate the starting number of target
templates. The larger the ratio of target specific product to
competitor specific product, the higher the starting DNA
concentration. Success of a QC-PCR assay relies on the development
of an internal control that amplifies with the same efficiency as
the target molecule. However, the design of the competitor and the
validation of amplification efficiencies require much effort. In
the QC-PCR method of RNA quantitation, a competitive RNA template
matched to the target sequence of interest, but different from it
by virtue of an introduced internal deletion, is used in a
competitive titration of the reverse transcription and PCR steps,
providing stringent internal control. Increasing amounts of known
copy numbers of competitive template are added to replication
portions of the test sample, and quantitation is based on
determination of the relative (not absolute) amounts of the
differently sized amplified products derived from the wild-type and
competitive templates, after electrophoretic separation.
[0007] In addition to requiring time-consuming and burdensome
downstream processing such as hybridization or gel electrophoresis,
these assays have limited sensitivity to a range of target nucleic
acid concentrations. For example, in competitor assays, the
sensitivity to template concentration differences may be
compromised when either the target or added competitor DNA is
greatly in excess of the other. The dynamic range of the assays
that measure the amount of end product can also be limited in that
the chosen number of cycles of some reactions may have reached a
plateau level of product prior to other reactions. Differences in
starting template levels in these reactions may therefore not be
well reflected. Furthermore, small differences in the measured
amount of product may result in widely varying estimates of the
starting template concentration, leading to inaccuracies due to
variable reaction conditions, variations in sampling, or the
presence of inhibitors.
[0008] To reduce the amount of post-amplification analysis required
to determine a starting nucleic acid quantity in a sample,
additional methods have been developed to measure nucleic acid
amplification in real-time. These methods generally take advantage
of fluorescent labels (e.g., fluorescent dyes) that indicate the
amount of nucleic acid being amplified, and utilize the
relationship between the number of cycles required to achieve a
chosen level of fluorescence signal and the concentration of
amplifiable targets present at the initiation of the PCR process.
For example, European Patent Application No. 94112728 (Publication
number EP/0640828) describes a quantitative assay for an
amplifiable nucleic acid target sequence which correlates the
number of thermal cycles required to reach a certain concentration
of target sequence to the amount of target. DNA present at the
beginning of the PCR process. In this assay system, a set of
reaction mixtures are prepared for amplification, with one
preparation including an unknown concentration of target sequence
in a test sample and others containing known concentrations
(standards) of the sequence. The reaction mixtures also contain a
fluorescent dye that fluoresces when bound to double-stranded
DNA.
[0009] The reaction mixtures are thermally cycled in separate
reaction vessels for a number of cycles to achieve a sufficient
amplification of the targets. The fluorescence emitted from the
reaction mixtures is monitored in real-time as the amplification
reactions occur, and the number of cycles necessary for each
reaction mixture to fluoresce to an arbitrary cutoff level
(arbitrary fluorescent value, or AFV) is determined. The AFV is
chosen to be in a region of the amplification curves that is
parallel among the different standards (e.g., from 0.1 to 0.5 times
the maximum fluorescence value obtained by the standard using the
highest initial known target nucleic acid concentration). The
number of cycles necessary for each of the standards to reach the
AFV is determined, and a regression line is fitted to the data that
relates the initial target nucleic acid amount to the number of
cycles (i.e., the threshold cycle number) needed to reach the AFV.
To determine the unknown starting quantity of the target nucleic
acid sequence in the sample, the number of cycles needed to reach
the AFV is determined for the sample. This threshold cycle number
(which can be fractional) is entered into the equation of the
fitted regression line and the equation returns a value that is the
initial amount of the target nucleic acid sequence in the
sample.
[0010] The primary disadvantage of this method for determining an
unknown starting quantity of a target nucleic acid sequence in a
sample is that differences in background signal, noise, or reaction
efficiency between the reaction mixtures being amplified in
different reaction vessels may bias the calculation of the
threshold cycle numbers. Consequently, several of the data points
used to generate the regression line may deviate significantly from
linearity, resulting in inaccurate quantitation of the unknown
starting quantity of the target nucleic acid sequence in the
sample. Small differences in the selection of threshold cycle
numbers used in quantitation algorithms may have a substantial
effect on the ultimate accuracy of quantitation. Thus, there
remains a need to provide an objective and automatic method of
selecting threshold values that will allow users of amplification
methods to determine the initial concentrations of target nucleic
acid sequences more accurately and reliably than present
methods.
SUMMARY
[0011] It is therefore an object of the present invention to
provide an improved apparatus for determining a threshold value in
a nucleic acid amplification reaction. The threshold value may be a
threshold cycle number in a thermal cycling amplification reaction,
or the threshold value may be a time value (e.g., an elapsed time
of amplification) in an isothermal nucleic acid amplification
reaction. It is another object of the present invention to provide
an improved apparatus for determining an unknown starting quantity
of a nucleic acid sequence in a test sample.
[0012] According to a first embodiment, the invention provides an
apparatus for determining a threshold cycle number (which may be
fractional) in a nucleic acid amplification reaction. The apparatus
comprises a detection mechanism for measuring, at a plurality of
different times during the amplification reaction, at least one
signal whose intensity is related to the quantity of a nucleic acid
sequence being amplified in the reaction. The apparatus also
includes a controller (e.g., a computer or processor) in
communication with the detection mechanism. The controller is
programmed to perform the steps of deriving a growth curve from the
measurements of the signal; calculating a derivative of the growth
curve; identifying a characteristic of the derivative; and
determining a cycle number associated with the characteristic of
the derivative. The step of calculating a derivative of the growth
curve preferably comprises calculating second derivative values of
the growth curve at a number of different cycles in the reaction to
yield a plurality of second derivative data points. The
characteristic of the derivative is preferably a positive peak of
the second derivative, and the step of determining the cycle number
associated with the positive peak preferably comprises fitting a
second order curve to the second derivative data points and
calculating the threshold cycle number as the location, in cycles,
of a peak of the second order curve. Alternatively, the
characteristic of the derivative used to determine the threshold
cycle number may comprise a negative peak of the second derivative,
a zero crossing of the second derivative, or a positive peak of the
first derivative.
[0013] According to a second embodiment, the invention provides an
apparatus for determining a threshold time value in a nucleic acid
amplification reaction. The method is particularly useful for
determining a threshold time value (e.g., an elapsed time of
amplification required to reach a threshold level) in isothermal
nucleic acid amplification reactions. The apparatus comprises a
detection mechanism for measuring, at a plurality of different
times during the amplification reaction, at least one signal whose
intensity is related to the quantity of a nucleic acid sequence
being amplified in the reaction. The apparatus also includes a
controller (e.g., a computer or processor) in communication with
the detection mechanism. The controller is programmed to perform
the steps of deriving a growth curve from the measurements of the
signal; calculating a derivative of the growth curve; identifying a
characteristic of the derivative; and determining a time value
associated with the characteristic of the derivative. The step of
calculating a derivative of the growth curve preferably comprises
calculating second derivative values of the growth curve at a
number of different times in the reaction to yield a plurality of
second derivative data points. The characteristic of the derivative
is preferably a positive peak of the second derivative, and the
step of determining the time value associated with the positive
peak preferably comprises fitting a second order curve to the
second derivative data points and calculating the threshold time
value as the location of a peak of the second order curve.
Alternatively, the characteristic of the derivative used to
determine the threshold time value may comprise a negative peak of
the second derivative, a zero crossing of the second derivative, or
a positive peak of the first derivative.
[0014] Using derivatives of growth curves to determine threshold
values provides for highly reproducible threshold values even when
there is significant variation (e.g., in terms of timing, optics,
or noise due to other sources) between the reaction sites at which
the various test and calibration samples are amplified. The
threshold value for each target nucleic acid sequence being
amplified in a particular reaction is based on the data from that
reaction, not from all of the reactions in a batch so that a single
discrepant reaction in the batch will not bias the calculation of
threshold values for target nucleic acid sequences at other
reaction sites.
[0015] According to another embodiment, the invention provides an
apparatus for determining an unknown starting quantity of a target
nucleic acid sequence in a test sample. The apparatus comprises
means for amplifying the unknown starting quantity of the target
nucleic acid sequence in the test sample and for amplifying a
plurality of known starting quantities of a calibration nucleic
acid sequence in respective calibration samples. The apparatus also
includes at least one detection mechanism for measuring, at a
plurality of different times during amplification of the nucleic
acid sequences, signals indicative of the quantities of the nucleic
acid sequences being amplified in the test and calibration samples.
The apparatus further includes at least one controller (e.g.,
computer or processor) in communication with the detection
mechanism. The controller is programmed to determine a respective
threshold value for each of the known starting quantities of the
calibration nucleic acid sequence in the calibration samples and
for the target nucleic acid sequence in the test sample. Each
threshold value is determined for a nucleic acid sequence in a
respective sample by deriving a growth curve for the nucleic acid
sequence from the measured signals; calculating a derivative of the
growth curve; identifying a characteristic of the derivative; and
determining the threshold value associated with the characteristic
of the derivative. The controller is also programmed to derive a
calibration curve from the threshold values determined for the
known starting quantities of the nucleic acid sequence in the
calibration samples and to determine the starting quantity of the
target nucleic acid sequence in the test sample using the
calibration curve and the threshold value determined for the target
sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a partially exploded, isometric view of a reaction
vessel in which the major walls of the reaction chamber are removed
to show the interior of the chamber.
[0017] FIG. 2 is a front view of the vessel of FIG. 1.
[0018] FIG. 3 is a top view of a plunger cap of the vessel of FIG.
1.
[0019] FIG. 4 is another front view of the vessel of FIG. 1.
[0020] FIG. 5 is a side view of the vessel of FIG. 1 inserted into
a thermal sleeve formed by opposing plates.
[0021] FIG. 6 is a front view of one of the plates of FIG. 5.
[0022] FIGS. 7A-7D are schematic, cross-sectional views of a
plunger being inserted into a channel of the reaction vessel of
FIG. 1.
[0023] FIG. 8 is a schematic, front view of a heat-exchanging
module according to the present invention having a thermal sleeve,
a pair of optics assemblies, and a cooling system. The reaction
vessel of FIG. 1 is inserted into the thermal sleeve.
[0024] FIG. 9 is an exploded view of a support structure for
holding the plates of FIG. 5.
[0025] FIGS. 10-11 are assembled views of the support structure of
FIG. 9.
[0026] FIG. 12 is an isometric view of the reaction vessel of FIG.
1 inserted between the plates of FIG. 5.
[0027] FIG. 13 is an isometric view showing the exterior of one the
optics assemblies of FIG. 8.
[0028] FIG. 14 is an isometric view of the optics assembly of FIG.
13, the plates of FIG. 5 in contact with the optics assembly, and
the vessel of FIG. 1 positioned above the plates.
[0029] FIGS. 15A and 15B are graphs showing the excitation and
emission spectra, respectively, of four dyes often used to label
nucleic acid sequences.
[0030] FIG. 15C shows the effects of filtering the outputs of green
and blue LEDs to provide distinct excitation wavelength ranges.
[0031] FIG. 15D shows the effects of filtering light emitted from
each of the four dyes of FIG. 15B to form distinct emission
wavelength ranges.
[0032] FIG. 16 is a plan view of an optical excitation assembly of
the module of FIG. 8.
[0033] FIG. 17 is an exploded view of the excitation assembly of
FIG. 16.
[0034] FIG. 18 is a plan view of an optical detection assembly of
the module of FIG. 8.
[0035] FIG. 19 is an exploded view of the detection assembly of
FIG. 18.
[0036] FIG. 20 is an isometric view of a multi-site reactor system
according to the present invention.
[0037] FIG. 21 is a schematic, block diagram of another multi-site
reactor system having multiple thermal cycling instruments
daisy-chained to a computer and a power source.
[0038] FIG. 22 is a schematic, block diagram of a base instrument
of the system of FIG. 20.
[0039] FIG. 23 is a schematic, block diagram of the electronic
components of the module of FIG. 8.
[0040] FIG. 24A is a graph showing a growth curve for a thermal
cycling nucleic acid amplification reaction. A threshold cycle
number is calculated as the location of the positive peak of the
second derivative of the growth curve.
[0041] FIG. 24B is a graph showing a growth curve for an isothermal
nucleic acid amplification reaction. A threshold time value is
calculated as the location of the positive peak of the second
derivative of the growth curve.
[0042] FIG. 25 is a flow chart illustrating steps executed to
calculate a threshold value in a nucleic acid amplification
reaction according to a preferred embodiment of the invention.
[0043] FIG. 26 is a flow chart illustrating the steps executed in a
"background subtraction" routine.
[0044] FIG. 27 is a graph of fluorescent signal value as a function
of cycle number for cycles M to N.
[0045] FIGS. 28A-28C illustrate five data points defining one
segment of a growth curve.
[0046] FIG. 29 is a flow chart illustrating the step executed to
calculate a noise-based threshold level to be exceeded by the
positive peak of the second derivative of a growth curve.
[0047] FIG. 30 is a graph illustrating the fitting of a second
order curve to three data points.
[0048] FIG. 31 is a flow chart showing the steps performed to
calculate a peak height and threshold value of the second order
curve of FIG. 30.
[0049] FIG. 32A is a graph showing a growth curve for a thermal
cycling nucleic acid amplification reaction. A threshold cycle
number is calculated as the location of the zero-crossing of the
second derivative of the growth curve.
[0050] FIG. 32B is a graph showing a growth curve for an isothermal
nucleic acid amplification reaction. A threshold time value is
calculated as the location of the zero-crossing of the second
derivative of the growth curve.
[0051] FIG. 33 is a flow chart illustrating steps executed to
calculate a threshold value in a nucleic acid amplification
reaction according to a second embodiment of the invention.
[0052] FIG. 34 is a flow chart illustrating the step executed to
calculate a noise-based threshold level to be exceeded by a primary
optical signal.
[0053] FIG. 35A is a graph showing a growth curve for a thermal
cycling nucleic acid amplification reaction. A threshold cycle
number is calculated as the location of a negative peak of the
second derivative of the growth curve.
[0054] FIG. 35B is a graph showing a growth curve for an isothermal
nucleic acid amplification reaction. A threshold time value is
calculated as the location of a negative peak of the second
derivative of the growth curve.
[0055] FIG. 36 is a flow chart illustrating steps executed to
calculate a threshold value in a nucleic acid amplification
reaction according to a third embodiment of the invention.
[0056] FIG. 37A is a graph showing a growth curve for a thermal
cycling nucleic acid amplification reaction. A threshold cycle
number is calculated as the location of the positive peak of the
first derivative of the growth curve.
[0057] FIG. 37B is a graph showing a growth curve for an isothermal
nucleic acid amplification reaction. A threshold time value is
calculated as the location of a positive peak of the first
derivative of the growth curve.
[0058] FIG. 38 is a flow chart illustrating steps executed to
calculate a threshold value in a nucleic acid amplification
reaction according to a fourth embodiment of the invention.
[0059] FIG. 39 is a flow chart illustrating the step executed to
calculate a noise-based threshold level to be exceeded by a
positive peak of a first derivative of a growth curve.
[0060] FIG. 40 is a setup table containing the known starting
quantities of nucleic acid sequences in calibration samples
(standards).
[0061] FIG. 41 is a table containing the threshold values
determined for the nucleic acid sequences in the calibration
samples of FIG. 40.
[0062] FIG. 42 is a table of averages computed from the table of
FIG. 41.
[0063] FIG. 43 is a calibration curve derived from the values in
the table of FIG. 42.
[0064] FIG. 44 is a table of starting quantities of different
target nucleic acid sequences in a test sample determined using the
calibration curve of FIG. 43.
[0065] FIG. 45 is a setup table containing the known starting
quantities of nucleic acid sequences in calibration samples
(standards) according to another embodiment of the invention. Each
sample includes a quantitative internal control (QIC).
[0066] FIG. 46 is a table containing the threshold values
determined for the nucleic acid sequences and for the quantitative
internal controls in the calibration samples of FIG. 40.
[0067] FIG. 47 is a table of normalized threshold values.
[0068] FIG. 48 is a table of averages computed from the table of
FIG. 47.
[0069] FIG. 49 is a calibration curve derived from the values in
the table of FIG. 48.
[0070] FIG. 50 is a table of starting quantities of different
target nucleic acid sequences in a test sample computed using the
calibration curve of FIG. 49.
[0071] FIG. 51 is a setup table containing the known starting
quantities of two different calibration nucleic acid sequences
(internal standards) at each reaction site.
[0072] FIG. 52 is a table containing the threshold values
determined for the calibration nucleic acid sequences of FIG.
51.
[0073] FIG. 53 is a table of threshold values and known starting
quantities of the calibration nucleic acid sequences amplified at
one of the reaction sites specified in FIG. 52.
[0074] FIG. 54 is a calibration curve derived from the values in
the table of FIG. 53.
DETAILED DESCRIPTION
[0075] The present invention provides methods, apparatus, and
computer program products for determining quantities of target
nucleic acid sequences in samples. FIG. 1 shows a partially
exploded view of a reaction vessel 12 for holding a sample for
nucleic acid amplification and detection. FIG. 2 shows a front view
of the vessel 12. The vessel 12 includes a reaction chamber 17 for
holding a reaction mixture (e.g., the sample mixed with reagents
and one or more fluorescent dyes) for thermal processing and
optical interrogation. The vessel 12 is designed for optimal heat
transfer to and from the mixture and for efficient optical viewing
of the mixture. The thin shape of the vessel contributes to optimal
thermal kinetics by providing large surfaces for thermal
conduction. In addition, the side walls of the vessel 12 provide
optical windows into the chamber 17 so that the entire reaction
mixture can be optically interrogated in real-time as the nucleic
acid amplification reaction occurs.
[0076] In more detail to FIGS. 1-2, the reaction vessel 12 includes
a rigid frame 16 that defines the side walls 19A, 19B, 20A, 20B of
the reaction chamber 17. The rigid frame 16 also includes a port 14
and a channel 28 that connects the port 14 to the chamber 17. The
vessel also includes thin, flexible sheets attached to opposite
sides of the rigid frame 16 to form opposing major walls 18 of the
chamber. (The major walls 18 are shown in FIG. 1 exploded from the
rigid frame 16 for illustrative clarity). The reaction chamber 17
is thus defined by the rigid side walls 19A, 19B, 20A, 20B of the
frame 16 and by the flexible major walls 18 which are sealed to
opposite sides of the frame.
[0077] The major walls 18 facilitate optimal thermal conductance to
the reaction mixture contained in the chamber 17. Each of the walls
18 is sufficiently flexible to contact and conform to a respective
thermal surface, thus providing for optimal thermal contact and
heat transfer between the thermal surface and the reaction mixture
contained in the chamber 17. Furthermore, the flexible walls 18
continue to conform to the thermal surfaces if the shape of the
surfaces changes due to thermal expansion or contraction during the
course of the heat-exchanging operation.
[0078] As shown in FIG. 5, the thermal surfaces for contacting the
flexible walls 18 are preferably formed by a pair of opposing
plates 50A, 50B positioned to receive the chamber 17 between them.
When the chamber 17 of the vessel is inserted between the plates
50A, 50B, the inner surfaces of the plates contact the walls 18 and
the flexible walls conform to the surfaces of the plates. The
plates are preferably spaced a distance from each other equal to
the thickness T of the chamber 17 as defined by the thickness of
the frame 16. In this position, minimal or no gaps are found
between the plate surfaces and the walls 18. The plates may be
heated and cooled by various thermal elements to induce temperature
changes within the chamber 17, as is described in greater detail
below.
[0079] The walls 18 are preferably flexible films of polymeric
material such as polypropylene, polyethylene, polyester, or other
polymers. The films may either be layered, e.g., laminates, or the
films may be homogeneous. Layered films are preferred because they
generally have better strength and structural integrity than
homogeneous films. In particular, layered polypropylene films are
presently preferred because polypropylene is not inhibitory to PCR.
Alternatively, the walls 18 may comprise any other material that
may be formed into a thin, flexible sheet and that permits rapid
heat transfer. For good thermal conductance, the thickness of each
wall 18 is preferably between about 0.003 to 0.5 mm, more
preferably between 0.01 to 0.15 mm, and most preferably between
0.025 to 0.08 mm.
[0080] Referring again to FIGS. 1-2, the reaction vessel 12 also
includes a plunger 22 that is inserted into the channel 28 after
filling the chamber 17 with the reaction mixture. The plunger 22
compresses gas in the vessel 12 thereby increasing pressure in the
chamber 17 and outwardly expanding the flexible walls 18. The gas
compressed by the plunger 22 is typically air filling the channel
28. The pressurization of the chamber 17 is important because it
forces the walls 18 against the surfaces of the plates 50A, 50B
(see FIG. 5) and ensures that the walls 18 fully contact and
conform to the inner surfaces of the plates, thus guaranteeing
optimal thermal conductance between the plates 50A, 50B and the
chamber 17.
[0081] Referring again to FIGS. 1-2, the plunger may comprise any
device capable of establishing a seal with the walls of the channel
28 and of compressing gas in the vessel. Such devices include, but
are not limited to, pistons, plugs, or stoppers. The plunger 22 of
the preferred embodiment includes a stem 30 and a piston 32 on the
stem. When the plunger 22 is inserted into the channel 28, the
piston 32 establishes a seal with the inner walls of the channel
and compresses air in the channel. The piston 32 is preferably a
cup integrally formed (e.g., molded) with the stem 30.
Alternatively, the piston 32 may be a separate elastomeric piece
attached to the stem.
[0082] The plunger 22 also preferably includes an alignment ring 34
encircling the stem for maintaining the plunger 22 in coaxial
alignment with the channel 28 as the plunger is inserted into the
channel. The alignment ring 34 is preferably integrally formed
(e.g., molded) with the stem 30. The stem 30 may optionally
includes support ribs 44 for stiffening and strengthening the stem.
The plunger 22 also includes a plunger cap 36 attached to the stem
30. As shown in FIG. 2, the cap 36 includes a snap ring 38 and the
vessel includes an annular recess 23 encircling the port 14 for
receiving the snap ring 38. The cap 36 may optionally include a
lever portion 40 which is lifted to remove the plunger 22 from the
channel 28.
[0083] Referring to FIG. 7A, the rigid frame 16 has an inner
surface 41 defining the channel 28. The inner surface 41 preferably
has one or more pressure control grooves 42 formed therein. In the
preferred embodiment, the inner surface has four pressure control
grooves (only three shown in the view of FIG. 7A) spaced
equidistantly about the circumference of the channel 28. The
pressure control grooves 42 extend from the port 14 to a
predetermined depth D.sub.1 in the channel 28. The pressure control
grooves 42 allow gas to escape from the channel 28 and thus prevent
pressurization of the chamber 17 until the piston 32 reaches the
depth D.sub.1 in the channel. When the piston 32 reaches the depth
D.sub.1, the piston establishes an annular seal with the walls of
the channel 28 and begins to compress air trapped in the channel.
The compression of the trapped air causes the desired
pressurization of the chamber 17.
[0084] The stroke of the plunger 22 into the channel 28 is fully
illustrated in FIGS. 7A-7D. As shown in FIG. 7A, prior to inserting
the plunger 22 into the channel 28, the chamber 17 is filled with
the desired reaction mixture R. Specific methods for filling the
chamber (e.g., pipetting) are discussed in detail below. The
reaction mixture R fills the vessel 12 to a liquid surface level S.
Also prior to inserting the plunger 22 into the channel 28, the
channel 28 contains air having pressure equal to the pressure of
the atmosphere external to the vessel, hereinafter called ambient
pressure. The ambient pressure is usually standard atmospheric
pressure, e.g., about 14.7 pounds per square inch (psi). As shown
in FIG. 7B, when the plunger 22 is first inserted into the channel
28, the piston 32 begins to displace the air in the channel. The
displaced air escapes from the channel 28 through the pressure
control grooves 42.
[0085] Referring now to FIG. 7C, when the piston 32 reaches the
depth D.sub.1 at which the pressure control grooves end, the piston
32 establishes an annular seal with the walls of the channel 28 and
begins to compress air trapped in the channel between the piston 32
and the surface level S of the reaction mixture. The reaction
mixture is usually a liquid and therefore substantially
incompressible by the piston. The air trapped in the channel 28,
however, may be compressed to increase pressure in the chamber. As
shown in FIG. 7D, as the plunger 22 is inserted further into the
channel 28, the alignment ring 34 keeps the plunger 22 coaxially
aligned with the channel 28 as the piston 32 continues to compress
air trapped in the channel. When the plunger 22 is fully inserted
in the channel 28, the snap ring 38 snaps into the annular recess
23, ending the plunger stroke.
[0086] When the plunger 22 is fully inserted, the piston 32 seals
the channel 28 at a depth D.sub.2 which is lower than the depth
D.sub.1 at which the pressure control grooves 42 terminate. The
distance D.sub.3 traveled by the piston 32 between depths D.sub.1
and D.sub.2, i.e. the distance of the pressure stroke, determines
the amount of pressurization of the chamber 17. Referring again to
FIG. 5, the pressure in the chamber 17 should be sufficiently high
to ensure that the flexible major walls 18 of the chamber outwardly
expand to contact and conform to the surfaces of the plates 50A,
50B. The pressure should not be so great, however, that the
flexible walls 18 burst, become unattached from the rigid frame 16,
or deform the frame or plates.
[0087] It is presently preferred to pressurize the chamber to a
pressure in the range of 2 to 50 psi above ambient pressure. This
range is presently preferred because 2 psi is generally enough
pressure to ensure conformity between the flexible walls 18 and the
surfaces of the plates 50A, 50B, while pressures above 50 psi may
cause bursting of the walls 18 or deformation of the frame 16 or
plates 50A, 50B. More preferably, the chamber 17 is pressurized to
a pressure in the range of 8 to 15 psi above ambient pressure. This
range is more preferred because it is safely within the practical
limits described above, i.e. pressures of 8 to 15 psi are usually
more than enough to ensure that the flexible walls 18 contact and
conform to the surfaces of the plates 50A, 50B, but are
significantly lower than the pressures that might burst the walls
18 or deform the frame 16.
[0088] Referring again to FIG. 7D, the desired pressurization of
the chamber 17 may be achieved by proper design of the plunger 22,
channel 28, and pressure control grooves 42 and by use of the
equation: P.sub.1*V.sub.1=P.sub.2*V.sub.2;
[0089] where:
[0090] P.sub.1 is equal to the pressure in the vessel 12 prior to
insertion of the plunger 22;
[0091] V.sub.1 is equal to the volume of the channel 28 between the
liquid surface level S and the depth D.sub.1 to which the pressure
control grooves 42 extend;
[0092] P.sub.2 is equal to the desired final pressure in the.
chamber 17 after insertion of the plunger 22 into the channel 28;
and
[0093] V.sub.2 is equal to the volume of the channel 28 between the
liquid surface level S and the depth D.sub.2 at which the piston 32
establishes a seal with the walls of the channel 28 when the
plunger 22 is fully inserted into the channel.
[0094] To ensure the desired pressurization P.sub.2 of the chamber
17, one should size the channel 28 and pressure stroke distance
D.sub.3. such that the ratio of the volumes V.sub.1:V.sub.2 is
equal to the ratio of the pressures P.sub.2:P.sub.1. An engineer
having ordinary skill in the art will be able to select suitable
values for the volumes V.sub.1 and V.sub.2 using the description
and equation given above. For example, in the presently preferred
embodiment, the initial pressure P.sub.1 in the vessel is equal to
standard atmospheric pressure of about 14.7 psi, the volume V.sub.1
is equal to 110 .mu.l, the depth D.sub.1 is equal to 0.2 inches,
the depth D.sub.2 is equal to 0.28 inches to give a pressure stroke
distance D.sub.3 of 0.08 inches, and the volume V.sub.2 is equal to
60 .mu.l to give a final pressure P.sub.2 of about 26.7 psi (the
desired 12 psi above ambient pressure). This is just one example of
suitable dimensions for the vessel 12 and is not intended to limit
the scope of the invention. Many other suitable values may be
selected.
[0095] In selecting suitable dimensions for the channel 28 and
pressure stroke distance D.sub.3 (and thus the volumes V.sub.1,
V.sub.2), there is no theoretical limit to how large or small the
dimensions may be. It is only important that the ratio of the
volumes V.sub.1:V.sub.2 yield the desired final desired pressure
P.sub.2 in the chamber. As a practical matter, however, it is
presently preferred to design the vessel such that the distance
D.sub.3 of the pressure stroke is at least 0.05 inches, i.e., so
that the plunger 22 when fully inserted into the channel 28 extends
to a depth D.sub.2 that is at least 0.05 inches below the depth
D.sub.1 at which the pressure control grooves end. This minimum
length of the pressure stroke is preferred to reduce or make
negligible the effect that any manufacturing or operating errors
may have on the pressurization of the chamber. For example, the
length of the pressure stroke may differ slightly from vessel to
vessel due to manufacturing deviations, or the volume of air
compressed may vary due to operator error in filling the vessel
(e.g., different fill levels). If the vessel is designed to have a
sufficiently long pressure stroke, however, such variances will
have a lesser or negligible effect on the ratio of volumes
V.sub.1:V.sub.2 and suitable pressurization of the chamber will
still occur. In addition, to provide a safety margin for
manufacturing or operator errors, one should select a pressure
stroke sufficient to achieve a final pressure P.sub.2 that is
safely higher (e.g., at least 3 psi higher) than the minimum
pressure needed to force the flexible walls of the chamber against
the inner surfaces of the plates. With such a safety margin, any
deviations in the final pressure due to manufacturing deviations or
errors in filling the chamber will have a negligible effect and
suitable pressurization of the chamber 17 will still occur. As
stated above, the plunger stroke is preferably designed to increase
pressure in the chamber 17 to a pressure in the range of 8 to 15
psi above ambient pressure to provide the safety margin.
[0096] The pressure control grooves 42 provide several important
advantages. First, the pressure control grooves 42 provide a simple
mechanism for precisely and accurately controlling the pressure
stroke of the plunger 22, and hence the pressurization of the
chamber 17. Second, the pressure control grooves 42 allow the
plunger 22 to become fully aligned with the channel 28 before the
pressure stroke begins and thus prevent the plunger from becoming
misaligned or cocked in the channel. This ensures a highly
consistent pressure stroke. Although it is possible for the vessel
to have only one pressure control groove, it is preferable for the
vessel to have multiple pressure control grooves (e.g., 2 to 6
grooves) spaced equidistantly about the circumference of the
channel 28. Referring again to FIG. 7A, the pressure control
grooves 42 preferably cut about 0.01 to 0.03 inches into the
surface 41 defining the channel 28. This range is preferred so that
the pressure control grooves 42 are large enough to allow air to
escape from the channel 28, but do not cut so deeply into the
surface 41 that they degrade the structural integrity of the frame
16.
[0097] Although the pressure control grooves 42 are preferred, it
is also possible to construct the vessel 12 without the pressure
control grooves and still achieve the desired pressurization of the
chamber 17. One disadvantage of this embodiment is that the plunger
22 may become misaligned or cocked in the channel 28 during the
pressure stroke so that less consistent results are achieved. In
embodiments in which the vessel lacks pressure control grooves, the
pressure stroke of the plunger 22 begins when the piston 32 enters
the channel 28 and establishes a seal with the walls of the
channel. In these embodiments, the volume V.sub.1 (for use in the
equation above) is equal to the volume of the channel 28 between
the liquid surface level S and the port 14 where the piston 32
first establishes a seal with the walls of the channel. To ensure
the desired pressurization P.sub.2 of the chamber 17, one should
size the channel 28 and length of the pressure stroke such that the
ratio of the volumes V.sub.1:V.sub.2 is equal to the ratio of the
pressures P.sub.2:P.sub.1. As described previously, the minimum
length of the pressure stroke is preferably 0.05 inches to minimize
the effect of any manufacturing or operational deviations.
[0098] Referring again to FIG. 2, the vessel 12 also preferably
includes optical windows for in situ optical interrogation of the
reaction mixture in the chamber 17. In the preferred embodiment,
the optical windows are the side walls 19A, 19B of the rigid frame
16. The side walls 19A, 19B are optically transmissive to permit
excitation of the reaction mixture in the chamber 17 through the
side wall 19A and detection of light emitted from the chamber 17
through the side wall 19B. Arrows A represent illumination beams
entering the chamber 17 through the side wall 19A and arrows B
represent emitted light (e.g., fluorescent signals from fluorescent
probes labeling target nucleic acid sequences in the reaction
mixture) exiting the chamber 17 through the side wall 19B.
[0099] The side walls 19A, 19B are preferably angularly offset from
each other. It is usually preferred that the walls 19A, 19B are
offset from each other by an angle of about 90.degree.. A
90.degree. angle between excitation and detection paths assures
that a minimum amount of excitation radiation entering through the
wall 19A will exit through wall 19B. In addition, the 90.degree.
angle permits a maximum amount of emitted light to be collected
through wall 19B. The walls 19A, 19B are preferably joined to each
other to form a "V" shaped intersection at the bottom of the
chamber 17. Alternatively, the angled walls 19A, 19B need not be
directly joined to each other, but may be separated by an
intermediary portion, such as another wall or various mechanical or
fluidic features which do not interfere with the thermal and
optical performance of the vessel. For example, the walls 19A, 19B
may meet at a port which leads to another processing area in
communication with the chamber 17, such as an integrated capillary
electrophoresis area. In the presently preferred embodiment, a
locating tab 27 extends from the frame 16 below the intersection of
walls 19A, 19B. The locating tab 27 is used to properly position
the vessel 12 in a heat-exchanging module described below with
reference to FIG. 8.
[0100] Optimum optical sensitivity may be attained by maximizing
the optical path length of the light beams exciting the labeled
analytes in the reaction mixture and the emitted light that is
detected, as represented by the equation: I.sub.o/I.sub.i=C*L*A,
where I.sub.o is the illumination output of the emitted light in
volts, photons or the like, C is the concentration of analyte to be
detected, I.sub.i is the input illumination, L is the path length,
and A is the intrinsic absorptivity of the dye used to label the
target sequence.
[0101] The thin, flat reaction vessel 12 of the present invention
optimizes detection sensitivity by providing maximum optical path
length per unit analyte volume. Referring to FIGS. 4-5, the vessel
12 is preferably constructed such that each of the sides walls 19A,
19B, 20A, 20B of the chamber 17 has a length L in the range of 1 to
15 mm, the chamber has a width W in the range of 1.4 to 20 mm, the
chamber has a thickness T in the range of 0.5 to 5 mm, and the
ratio of the width W of the chamber to the thickness T of the
chamber is at least 2:1. These parameters are presently preferred
to provide a vessel having a relatively large average optical path
length through the chamber, i.e. 1 to 15 mm on average, while still
keeping the chamber sufficiently thin to allow for extremely rapid
heating and cooling of the reaction mixture contained therein. The
average optical path length of the chamber 17 is the distance from
the center of the side wall 19A to the center of the chamber 17
plus the distance from the center of the chamber 17 to the center
of the side wall 19B. As used herein, the thickness T of the
chamber 17 is defined as the thickness of the chamber prior to the
outward expansion of the major walls, i.e. the thickness T of the
chamber is defined by the thickness of the frame 16.
[0102] More preferably, the vessel 12 is constructed such that each
of the sides walls 19A, 19B, 20A, 20B of the chamber 17 has a
length L in the range of 5 to 12 mm, the chamber has a width W in
the range of 7 to 17 mm, the chamber has a thickness T in the range
of 0.5 to 2 mm, and the ratio of the width W of the chamber to the
thickness T of the chamber is at least 4:1. These ranges are more
preferable because they provide a vessel having both a larger
average optical path length (i.e., 5 to 12 mm) and a volume
capacity in the range of 12 to 100 .mu.l while still maintaining a
chamber sufficiently thin to permit extremely rapid heating and
cooling of a reaction mixture. The relatively large volume capacity
provides for increased sensitivity in the detection of low
concentration nucleic acids.
[0103] In the preferred embodiment, the reaction vessel 12 has a
diamond-shaped chamber 17 defined by the side walls 19A, 19B, 20A,
20B, each of the side walls has a length of about 10 mm, the
chamber has a width of about 14 mm, the chamber has a thickness T
of 1 mm as defined by the thickness of the frame 16, and the
chamber has a volume capacity of about 100 .mu.l. This reaction
vessel provides a relatively large average optical path length of
10 mm through the chamber 17. Additionally, the thin chamber allows
for extremely rapid heating and/or cooling of the reaction mixture
contained therein. The diamond-shape of the chamber 17 helps
prevent air bubbles from forming in the chamber as it is filled
with the reaction mixture and also aids in optical interrogation of
the mixture.
[0104] The frame 16 is preferably made of an optically transmissive
material, e.g., a polycarbonate or clarified polypropylene, so that
the side walls 19A, 19B are optically transmissive. As used herein,
the term optically transmissive means that one or more wavelengths
of light may be transmitted through the walls. In the preferred
embodiment, the optically transmissive walls 19A, 19B are
substantially transparent. In addition, one or more optical
elements may be present on the optically transmissive side walls
19A, 19B. The optical elements may be designed, for example, to
maximize the total volume of solution which is illuminated by a
light source, to focus excitation light on a specific region of the
chamber 17, or to collect as much fluorescence signal from as large
a fraction of the chamber volume as possible. In alternative
embodiments, the optical elements may comprise gratings for
selecting specific wavelengths, filters for allowing only certain
wavelengths to pass, or colored lenses to provide filtering
functions. The wall surfaces may be coated or comprise materials
such as liquid crystal for augmenting the absorption of certain
wavelengths. In the presently preferred embodiment, the optically
transmissive walls 19A, 19B are substantially clear, flat windows
having a thickness of about 1 mm.
[0105] As shown in FIG. 2, the side walls 20A, 20B preferably
includes reflective faces 21 which internally reflect light trying
to exit the chamber 17 through the side walls 20A, 20B. The
reflective faces 21 are arranged such that adjacent faces are
angularly offset from each other by about 90.degree.. In addition,
the frame 16 defines open spaces between the side walls 20A, 20B
and support ribs 15. The open spaces are occupied by ambient air
that has a different refractive index than the material composing
the frame (e.g., plastic). Due to the difference in the refractive
indexes, the reflective faces 21 are effective for internally
reflecting light trying to exit the chamber through the walls 20A,
20B and provide for increased detection of optical signal through
the walls 19A, 19B. In the preferred embodiment, the optically
transmissive side walls 19A, 19B define the bottom portion of the
diamond-shaped chamber 17, and the retro-reflective side walls 20A,
20B define the top portion of the chamber.
[0106] The reaction vessel 12 may be used in manual operations
performed by human technicians or in automated operations performed
by machines, e.g. pick-and-place machines. As shown in FIG. 1, for
the manual embodiments, the vessel 12 preferably includes finger
grips 26 and a leash 24 that conveniently attaches the plunger 22
to the body of the vessel 12. As shown in FIG. 3, for automated
embodiments, the plunger cap 36 preferably includes a tapered
engagement aperture 46 for receiving and establishing a fit with a
robotic arm or machine tip (not shown in FIG. 3), thus enabling the
machine tip to pick and place the plunger in the channel. The
engagement aperture 46 preferably has tapered side walls for
establishing a friction fit with the machine tip. Alternatively,
the engagement aperture may be designed to establish a vacuum fit
with the machine tip. The plunger cap 36 may optionally include
alignment apertures 48A, 48B used by the machine tip to properly
align the plunger cap 36 as the plunger is inserted into the
channel.
[0107] A preferred method for fabricating the reaction vessel 12
will now be described with reference to FIGS. 1-2. The reaction
vessel 12 may be fabricated by first molding the rigid frame 16
using known injection molding techniques. The frame 16 is
preferably molded as a single piece of polymeric material, e.g.,
clarified polypropylene. After the frame 16 is produced, thin,
flexible sheets are cut to size and sealed to opposite sides of the
frame 16 to form the major walls 18 of the chamber 17.
[0108] The major walls 18 are preferably cast or extruded films of
polymeric material, e.g., polypropylene films, that are cut to size
and attached to the frame 16 using the following procedure. A first
piece of film is placed over one side of the bottom portion of the
frame 16. The frame 16 preferably includes a tack bar 47 for
aligning the top edge of the film. The film is placed over the
bottom portion of the frame 16 such that the top edge of the film
is aligned with the tack bar 47 and such that the film completely
covers the bottom portion of the frame 16 below the tack bar 47.
The film should be larger than the bottom portion of the frame 16
so that it may be easily held and stretched flat across the frame.
The film is then cut to size to match the outline of the frame by
clamping to the frame the portion of the film that covers the frame
and cutting away the portions of the film that extend past the
perimeter of the frame using, e.g., a laser or die. The film is
then tack welded to the frame, preferably using a laser.
[0109] The film is then sealed to the frame 16, preferably by heat
sealing. Heat sealing is presently preferred because it produces a
strong seal without introducing potential contaminants to the
vessel as the use of adhesive or solvent bonding techniques might
do. Heat sealing is also simple and inexpensive. At a minimum, the
film should be completely sealed to the surfaces of the side walls
19A, 19B, 20A, 20B. More preferably, the film is additionally
sealed to the surfaces of the support ribs 15 and tack bar 47. The
heat sealing may be performed using, e.g., a heated platen. An
identical procedure may be used to cut and seal a second sheet to
the opposite side of the frame 16 to complete the chamber 17.
[0110] Many variations to this fabrication procedure are possible.
For example, in an alternative embodiment, the film is stretched
across the bottom portion of the frame 16 and then sealed to the
frame prior to cutting the film to size. After sealing the film to
the frame, the portions of the film that extend past the perimeter
of the frame are cut away using, e.g., a laser or die.
[0111] The plunger 22 is also preferably molded from polymeric
material, preferably polypropylene, using known injection molding
techniques. As shown in FIG. 1, the frame 16, plunger 22, and leash
24 connecting the plunger to the frame may all be formed in the
same mold to form a one-piece part. This embodiment of the vessel
is especially suitable for manual use in which a human operator
fills the vessel and inserts the plunger 22 into the channel 28.
The leash 24 ensures that the plunger 22 is not lost or dropped on
the floor. Alternatively, as shown in FIG. 2, the plunger 22 may be
molded separately from the frame 16 so that the plunger and frame
are separate pieces. This embodiment is especially suitable for
automated use of the vessel in which the plunger 22 is picked and
placed into the channel 28 by an automated machine.
[0112] Referring again to FIG. 5, the plates 50A, 50B may be made
of various thermally conductive materials including ceramics or
metals. Suitable ceramic materials include aluminum nitride,
aluminum oxide, beryllium oxide, and silicon nitride. Other
materials from which the plates may be made include, e.g., gallium
arsenide, silicon, silicon nitride, silicon dioxide, quartz, glass,
diamond, polyacrylics, polyamides, polycarbonates, polyesters,
polyimides, vinyl polymers, and halogenated vinyl polymers, such as
polytetrafluoroethylenes. Other possible plate materials include
chrome/aluminum, superalloys, zircaloy, aluminum, steel, gold,
silver, copper, tungsten, molybdenum, tantalum, brass, sapphire, or
any of the other numerous ceramic, metal, or polymeric materials
available in the art.
[0113] Ceramic plates are presently preferred because their inside
surfaces may be conveniently machined to very high smoothness for
high wear resistance, high chemical resistance, and good thermal
contact to the flexible walls of the reaction vessel. Ceramic
plates can also be made very thin, preferably between about 0.6 and
1.3 mm, for low thermal mass to provide for extremely rapid
temperature changes. A plate made from ceramic is also both a good
thermal conductor and an electrical insulator, so that the
temperature of the plate may be well controlled using a resistive
heating element coupled to the plate.
[0114] Various thermal elements may be employed to heat and/or cool
the plates 50A, 50B and thus control the temperature of the
reaction mixture in the chamber 17. In general, suitable heating
elements for heating the plate include conductive heaters,
convection heaters, or radiation heaters. Examples of conductive
heaters include resistive or inductive heating elements coupled to
the plates, e.g., resistors or thermoelectric devices. Suitable
convection heaters include forced air heaters or fluid
heat-exchangers for flowing fluids past the plates. Suitable
radiation heaters include infrared or microwave heaters. Similarly,
various cooling elements may be used to cool the plates. For
example, various convection cooling elements may be employed such
as a fan, peltier device, refrigeration device, or jet nozzle for
flowing cooling fluids past the surfaces of the plates.
Alternatively, various conductive cooling elements may be used,
such as a heat sink, e.g. a cooled metal block, in direct contact
with the plates.
[0115] Referring to FIG. 6, in the preferred embodiment, each plate
50 has a resistive heating element 56 disposed on its outer
surface. The resistive heating element 56 is preferably a thick or
thin film and may be directly screen printed onto each plate 50,
particularly plates comprising a ceramic material, such as aluminum
nitride or aluminum oxide. Screen-printing provides high
reliability and low cross-section for efficient transfer of heat
into the reaction chamber. Thick or thin film resistors of varying
geometric patterns may be deposited on the outer surfaces of the
plates to provide more uniform heating, for example by having
denser resistors at the extremities and thinner resistors in the
middle. Although it is presently preferred to deposit a heating
element on the outer surface of each plate, a heating element may
alternatively be baked inside of each plate, particularly if the
plates are ceramic. The heating element 56 may comprise metals,
tungsten, polysilicon, or other materials that heat when a voltage
difference is applied across the material.
[0116] The heating element 56 has two ends which are connected to
respective contacts 54 which are in turn connected to a voltage
source (not shown in FIG. 6) to cause a current to flow through the
heating element. Each plate 50 also preferably includes a
temperature sensor 52, such as a thermocouple, thermistor, or RTD,
which is connected by two traces 53 to respective contacts 54. The
temperature sensor 52 may be used to monitor the temperature of the
plate 50 in a controlled feedback loop.
[0117] It is important that the plates have a low thermal mass to
enable rapid heating and cooling of the plates. In particular, it
is presently preferred that each of the plates has a thermal mass
less than about 5 J/.degree. C., more preferably less than 3
J/.degree. C., and most preferably less than 1 J/.degree. C. As
used herein, the term thermal mass of a plate is defined as the
specific heat of the plate multiplied by the mass of the plate. In
addition, each plate should be large enough to cover a respective
major wall of the reaction chamber. In the presently preferred
embodiment, for example, each of the plates has a width X in the
range of 2 to 22 mm, a length Y in the range of 2 to 22 mm, and a
thickness in the range of 0.5 to 5 mm. The width X and length Y of
each plate is selected to be slightly larger than the width and
length of the reaction chamber. Moreover, each plate preferably has
an angled bottom portion matching the geometry of the bottom
portion of the reaction chamber, as is described below with
reference to FIG. 12. Also in the preferred embodiment, each of the
plates is made of aluminum nitride having a specific heat of about
0.75 J/g.degree. C. The mass of each plate is preferably in the
range of 0.005 to 5.0 g so that each plate has a thermal mass in
the range of 0.00375 to 3.75 J/.degree. C.
[0118] FIG. 8 is a schematic side view of a heat-exchanging module
60 into which the reaction vessel 12 is inserted for thermal
processing and optical interrogation. The module 60 preferably
includes a housing 62 for holding the various components of the
module. The module 60 also includes the thermally conductive plates
50 described above. The housing 62 includes a slot (not shown in
FIG. 8) above the plates 50 so that the reaction chamber of the
vessel 12 may be inserted through the slot and between the plates.
The heat-exchanging module 60 also preferably includes a cooling
system, such as a fan 66. The fan 66 is positioned to blow cooling
air past the surfaces of the plates 50 to cool the plates and hence
cool the reaction mixture in the vessel 12. The housing 62
preferably defines channels for directing the cooling air past the
plates 50 and out of the module 60.
[0119] The heat-exchanging module 60 further includes an optical
excitation assembly 68 and an optical detection assembly 70 for
optically interrogating the reaction mixture contained in the
vessel 12. The excitation assembly 68 includes a first circuit
board 72 for holding its electronic components, and the detection
assembly 68 includes a second circuit board 74 for holding its
electronic components. The excitation assembly 68 includes one or
more light sources, such as LEDs, for exciting fluorescent probes
in the vessel 12. The excitation assembly 68 also includes one or
more lenses for collimating the light from the light sources, as
well as filters for selecting the excitation wavelength ranges of
interest. The detection assembly 70 includes one or more detectors,
such as photodiodes, for detecting the light emitted from the
vessel 12. The detection assembly 70 also includes one or more
lenses for focusing and collimating the emitted light, as well as
filters for selecting the emission wavelength ranges of interest.
The specific components of the optics assemblies 68, 70 are
described in greater detail below with reference to FIGS.
16-19.
[0120] The optics assemblies 68, 70 are positioned in the housing
62 such that when the chamber of the vessel 12 is inserted between
the plates 50, the first optics assembly 68 is in optical
communication with the chamber 17 through the optically
transmissive side wall 19A (see FIG. 2) and the second optics
assembly 70 is in optical communication with the chamber through
the optically transmissive side wall 19B (FIG. 2). In the preferred
embodiment, the optics assemblies 68, 70 are placed into optical
communication with the optically transmissive side walls by simply
locating the optics assemblies 68, 70 next to the bottom edges of
the plates 50 so that when the chamber of the vessel is placed
between the plates, the optics assemblies 68, 70 directly contact,
or are in close proximity to, the side walls.
[0121] As shown in FIG. 12, the vessel 12 preferably has an angled
bottom portion (e.g., triangular) formed by the optically
transmissive side walls 19A, 19B. Each of the plates 50A, 50B has a
correspondingly shaped bottom portion. The bottom portion of the
first plate 50A has a first bottom edge 98A and a second bottom
edge 98B. Similarly, the bottom portion of the second plate 50B has
a first bottom edge 99A and a second bottom edge 99B. The first and
second bottom edges of each plate are preferably angularly offset
from each other by the same angle that the side walls 19A, 19B are
offset from each other (e.g., 90.degree.). Additionally, the plates
50A, 50B are preferably positioned to receive the chamber of the
vessel 12 between them such that the first side wall 19A is
positioned substantially adjacent and parallel to each of the first
bottom edges 98A, 99A and such that the second side wall 19B is
positioned substantially adjacent and parallel to each of the
second bottom edges 98B, 99B. This arrangement provides for easy
optical access to the optically transmissive side walls 19A, 19B
and hence to the chamber of the vessel 12.
[0122] The side walls 19A, 19B may be positioned flush with the
edges of the plates 50A, 50B, or more preferably, the side walls
19A, 19B may be positioned such that they protrude slightly past
the edges of the plates. As is explained below with reference to
FIGS. 16-19, each optics assembly preferably includes a lens that
physically contacts a respective one of the side walls 19A, 19B. It
is preferred that the side walls 19A, 19B protrude slightly (e.g.,
0.02 to 0.3 mm) past the edges of the plates 50A, 50B so that the
plates do not physically contact and damage the lenses. A gel or
fluid may optionally be used to establish or improve optical
communication between each optics assembly and the side walls 19A,
19B. The gel or fluid should have a refractive index close to the
refractive indexes of the elements that it is coupling.
[0123] Referring again to FIG. 8, the optics assemblies 68, 70 are
preferably arranged to provide a 90.degree. angle between
excitation and detection paths. The 90.degree. angle between
excitation and detection paths assures that a minimum amount of
excitation radiation entering through the first side wall of the
chamber exits through the second side wall. Also, the 90.degree.
angle permits a maximum amount of emitted radiation to be collected
through the second side wall. In the preferred embodiment, the
vessel 12 includes a locating tab 27 (see FIG. 2) that fits into a
slot formed between the optics assemblies 68, 70 to ensure proper
positioning of the vessel 12 for optical detection. For improved
detection, the module 60 also preferably includes a light-tight lid
(not shown) that is placed over the top of the vessel 12 and made
light-tight to the housing 62 after the vessel is inserted between
the plates 50.
[0124] Although it is presently preferred to locate the optics
assemblies 68, 70 next to the bottom edges of the plates 50, many
other arrangements are possible. For example, optical communication
may be established between the optics assemblies 68, 70 and the
walls of the vessel 12 via optical fibers, light pipes, wave
guides, or similar devices. One advantage of these devices is that
they eliminate the need to locate the optics assemblies 68, 70
physically adjacent to the plates 50. This leaves more room around
the plates in which to circulate cooling air or refrigerant, so
that cooling may be improved.
[0125] The heat-exchanging module 60 also includes a PC board 76
for holding the electronic components of the module and an edge
connector 80 for connecting the module 60 to a base instrument, as
will be described below with reference to FIG. 22. The heating
elements and temperature sensors on the plates 50, as well as the
optical boards 72, 74, are connected to the PC board 76 by flex
cables (not shown in FIG. 8 for clarity of illustration). The
module 60 may also include a grounding trace 78 for shielding the
optical detection circuit. The module 60 also preferably includes
an indicator, such as an LED 64, for indicating to a user the
current status of the module such as "ready to load sample", "ready
to load reagent," "heating," "cooling," "finished," or "fault".
[0126] The housing 62 may be molded from a rigid, high-performance
plastic, or other conventional material. The primary functions of
the housing 62 are to provide a frame for holding the plates 50,
optics assemblies 68, 70, fan 66, and PC board 76. The housing 62
also preferably provides flow channels and ports for directing
cooling air from the fan 66 across the surfaces of the plates 50
and out of the housing. In the preferred embodiment, the housing 62
comprises complementary pieces (only one piece shown in the
schematic side view of FIG. 8) that fit together to enclose the
components of the module 60 between them.
[0127] The opposing plates 50 are positioned to receive the chamber
of the vessel 12 between them such that the flexible major walls of
the chamber contact and conform to the inner surfaces of the
plates. It is presently preferred that the plates 50 be held in an
opposing relationship to each other using, e.g., brackets,
supports, or retainers. Alternatively, the plates 50 may be
spring-biased towards each other as described in International
Publication Number WO 98/38487, the disclosure of which is
incorporated by reference herein. In another embodiment of the
invention, one of the plates is held in a fixed position, and the
second plate is spring-biased towards the first plate. If one or
more springs are used to bias the plates towards each other, the
springs should be sufficiently stiff to ensure that the plates are
pressed against the flexible walls of the vessel with sufficient
force to cause the walls to conform to the inner surfaces of the
plates.
[0128] FIGS. 9-10 illustrate a preferred support structure 81 for
holding the plates 50A, 50B in an opposing relationship to each
other. FIG. 9 shows an exploded view of the structure, and FIG. 10
shows an assembled view of the structure. For clarity of
illustration, the support structure 81 and plates 50A, 50B are
shown upside down relative to their normal orientation in the
heat-exchanging module of FIG. 8. Referring to FIG. 9, the support
structure 81 includes a mounting plate 82 having a slot 83 formed
therein. The slot 83 is sufficiently large to enable the chamber of
the vessel to be inserted through it. Spacing posts 84A, 84B extend
from the mounting plate 82 on opposite sides of the slot 83.
Spacing post 84A has indentations 86 formed on opposite sides
thereof (only one side visible in the isometric view of FIG. 9),
and spacing post 84B has indentations 87 formed on opposite sides
thereof (only one side visible in the isometric view of FIG. 9).
The indentations 86, 87 in the spacing posts are for receiving the
edges of the plates 50A, 50B. To assemble the structure, the plates
50A, 50B are placed against opposite sides of the spacing posts
84A, 84B such that the edges of the plates are positioned in the
indentations 86, 87. The edges of the plates are then held in the
indentations using a suitable retention means. In the preferred
embodiment, the plates are retained by retention clips 88A, 88B.
Alternatively, the plates 50A, 50B may be retained by adhesive
bonds, screws, bolts, clamps, or any other suitable means.
[0129] The mounting plate 82 and spacing posts 84A, 84B are
preferably integrally formed as a single molded piece of plastic.
The plastic should be a high temperature plastic, such as
polyetherimide, which will not deform of melt when the plates 50A,
50B are heated. The retention clips 84A, 84B are preferably
stainless steel. The mounting plate 82 may optionally include
indentations 92A, 92B for receiving flex cables 90A, 90B,
respectively, that connect the heating elements and temperature
sensors disposed on the plates 50A, 50B to the PC board 76 of the
heat-exchanging module 60 (FIG. 8). The portion of the flex cables
90A adjacent the plate 50A is held in the indentation 92A by a
piece of tape 94A, and the portion of the flex cables 90B adjacent
the plate 50B is held in the indentation 92B by a piece of tape
94B.
[0130] FIG. 11 is an isometric view of the assembled support
structure 81. The mounting plate 82 preferably includes tabs 96
extending from opposite sides thereof for securing the structure 81
to the housing of the heat-exchanging module. Referring again to
FIG. 8, the housing 62 preferably includes slots for receiving the
tabs to hold the mounting plate 82 securely in place.
Alternatively, the mounting plate 82 may be attached to the housing
62 using, e.g., adhesive bonding, screws, bolts, clamps, or any
other conventional means of attachment.
[0131] Referring again to FIG. 9, the support structure 81
preferably holds the plates 50A, 50B so that their inner surfaces
are angled very slightly towards each other. In the preferred
embodiment, each of the spacing posts 84A, 84B has a wall 89 that
is slightly tapered so that when the plates 50A, 50B are pressed
against opposite sides of the wall, the inner surfaces of the
plates are angled slightly towards each other. As best shown in
FIG. 5, the inner surfaces of the plates 50A, 50B angle towards
each other to form a slightly V-shaped slot into which the chamber
17 is inserted. The amount by which the inner surfaces are angled
towards each other is very slight, preferably about 1.degree. from
parallel. The surfaces are angled towards each other so that, prior
to the insertion of the chamber 17 between the plates 50A, 50B, the
bottoms of the plates are slightly closer to each other than the
tops. This slight angling of the inner surfaces enables the chamber
17 of the vessel to be inserted between the plates and withdrawn
from the plates more easily. Alternatively, the inner surfaces of
the plates 50A, 50B could be held parallel to each other, but
insertion and removal of the vessel 12 would be more difficult.
[0132] In addition, the inner surfaces of the plates 50A, 50B are
preferably spaced from each other a distance equal to the thickness
of the frame 16. In embodiments in which the inner surfaces are
angled towards each other, the centers of the inner surfaces are
preferably spaced a distance equal to the thickness of the frame 16
and the bottoms of the plates are initially spaced a distance that
is slightly less than the thickness of the frame 16. When the
chamber 17 is inserted between the plates 50A, 50B, the rigid frame
16 forces the bottom portions of the plates apart so that the
chamber 17 is firmly sandwiched between the plates. The distance
that the plates 50A, 50B are wedged apart by the frame 16 is
usually very small, e.g., about 0.035 mm if the thickness of the
frame is 1 mm and the inner surfaces are angled towards each other
by 1.degree..
[0133] Referring again to FIG. 10, the retention clips 88A, 88B
should be sufficiently flexible to accommodate this slight outward
movement of the plates 50A, 50B, yet sufficiently stiff to hold the
plates within the recesses in the spacing posts 84A, 84B during
insertion and removal of the vessel. The wedging of the vessel
between the plates 50A, 50B provides an initial preload against the
chamber and ensures that the flexible major walls of the chamber,
when pressurized, establish good thermal contact with the inner
surfaces of the plates.
[0134] Referring again to FIG. 8, to limit the amount that the
plates 50 can spread apart due to the pressurization of the vessel
12, stops may be molded into the housings of optics assemblies 68,
70. As shown in FIG. 13, the housing 221 of the optics assembly 70
includes claw-like stops 247A, 247B that extend outwardly from the
housing. As shown in FIG. 14, the housing 221 is positioned such
that the bottom edges of the plates 50A, 50B are inserted between
the stops 247A, 247B. The stops 247A, 247B thus prevent the plates
50A, 50B from spreading farther than a predetermined maximum
distance from each other. Although not shown in FIG. 14 for
illustrative clarity, the optics assembly 68 (see FIG. 8) has a
housing with corresponding stops for preventing the other halves of
the plates from spreading farther than the predetermined maximum
distance from each other. Referring again to FIG. 14, the maximum
distance that stops 247A, 247B permit the inner surfaces of the
plates 50A, 50B to be spaced from each other should closely match
the thickness of the frame 16. Preferably, the maximum spacing of
the inner surfaces of the plates 50A, 50B is slightly larger than
the thickness of the frame 16 to accommodate tolerance variations
in the vessel 12 and plates 50A, 50B. For example, the maximum
spacing is preferably about 0.1 to 0.3 mm greater than the
thickness of the frame 16.
[0135] Referring again to FIG. 8, the module 60 includes one or
more detection mechanisms for detecting and measuring a signal
related to the quantity of a target nucleic acid sequence in the
vessel 12. Preferably, the sample in the vessel 12 contains a
fluorescent indicator, and the signal is a fluorescent signal whose
intensity is proportional to the quantity of the target nucleic
acid sequence in the vessel 12. Although fluorescent signals are
presently preferred, it is to be understood that other types of
signals are known and may be used in the practice of the present
invention. To illustrate, indicators of nucleic acid concentration
may be provided by labels that produce signals detectable by
fluorescence, radioactivity, colorimetry, X-ray diffraction or
absorption, magnetism, or enzymatic activity. Suitable labels
include, for example, fluorophores, chromophores, radioactive
isotopes, electron-dense reagents, enzymes, and ligands having
specific binding partners (e.g., biotin-avidin). Electrical signals
may also be used to detect the presence of a target nucleic acid
sequence. For example, measurements of electrical conductance,
inductance, resistance, or capacitance may be used to indicate the
quantity of the target nucleic acid sequence in the sample.
[0136] Labeling of nucleic acid sequences may be achieved by a
number of means, including by chemical modification of a nucleic
acid primer or probe. Suitable fluorescent labels may include
non-covalently binding labels (e.g., intercalating dyes) such as
ethidium bromide, propidium bromide, chromomycin, acridine orange,
and the like. However, in the practice of the present invention the
use of covalently-binding fluorescent agents is preferred. Such
covalently-binding fluorescent labels include fluorescein and
derivatives thereof such as FAM, HEX, TET and JOE (all of which can
be obtained from PE Biosystems, Foster City, Calif.); rhodamine and
derivatives such as Texas Red (Molecular Probes, Eugene, Oreg.);
ROX and TAMRA (PE Biosystems, Foster City, Calif.); Lucifer Yellow;
coumarin derivatives and the like. Another preferred indicator of
nucleic acid concentration is fluorescence energy-transfer (FET),
in which a fluorescent reporter (or "donor") label and a quencher
(or "acceptor") label are used in tandem to produce a detectable
signal that is proportional to the amount of amplified nucleic acid
product (e.g., in the form of double-stranded nucleic acid) present
in the reaction mixture. Yet another detection method useful in the
practice of the present invention is fluorescence polarization (FP)
detection of nucleic acid amplification. Further, although
fluorescence excitation and emission detection is a preferred
embodiment, optical detection methods such as those used in direct
absorption and/or transmission with on-axis geometries are also
within the scope of the present invention. The quantity of a target
nucleic acid sequence may also be measured using time decay
fluorescence. Additionally, the concentration of a target nucleic
acid sequence may be indicated by phosphorescent signals,
chemiluminescent signals, or electrochemiluminescent signals.
[0137] FIGS. 15A and 15B show the fluorescent excitation and
emission spectra, respectively, of four fluorescent dyes (FAM, TET,
TAMRA, and ROX) commonly used to label target nucleic acid
sequences. As shown in FIG. 15A, the excitation spectra curves for
FAM, TET, TAMRA, and ROX are typically very broad at the base, but
sharper at the peaks. As shown in FIG. 15B, the relative emission
spectra curves for the same dyes are also very broad at the base
and sharper at the peaks. Thus, these dyes have strongly
overlapping characteristics in both their excitation and emission
spectra. The overlapping characteristics have traditionally made it
difficult to distinguish the fluorescent signal of one dye from
another when multiple dyes are used to label different nucleic acid
sequences in a reaction mixture.
[0138] According to the present invention, multiple light sources
are used to provide excitation beams to the dyes in multiple
excitation wavelength ranges. Each light source provides excitation
light in a wavelength range matched to the peak excitation range of
a respective one of the dyes. In the preferred embodiment, the
light sources are blue and green LEDs. FIG. 15C shows the effects
of filtering the outputs of blue and green LEDs to provide
substantially distinct excitation wavelength ranges. Typical blue
and green LEDs have substantial overlap in the range of around 480
nm through 530 nm. By the filtering regime of the present
invention, the blue LED light is filtered to a range of about 450
to 495 nm to match the relative excitation peak for FAM. The green
LED light is filtered to a first range of 495 to 527 nm
corresponding to the excitation peak for TET, a second range of 527
to 555 nm corresponding to the excitation peak for TAMRA, and a
third range of 555 to 593 nm corresponding to the excitation peak
for ROX.
[0139] FIG. 15D shows the effects of filtering light emitted
(fluorescent emission) from each of the four dyes to form distinct
emission wavelength ranges. As shown previously in FIG. 15B, the
fluorescent emissions of the dyes before filtering are spherically
diffuse with overlapping spectral bandwidths, making it difficult
to distinguish the fluorescent output of one dye from another. As
shown in FIG. 15D, by filtering the fluorescent emissions of the
dyes into substantially distinct wavelength ranges, a series of
relatively narrow peaks (detection windows) are obtained, making it
possible to distinguish the fluorescent outputs of different dyes,
thus enabling the detection of a number of different
fluorescently-labeled nucleic acid sequences in a reaction
mixture.
[0140] FIG. 16 is a schematic, plan view of the optical excitation
assembly 68. The assembly 68 is positioned adjacent the reaction
vessel 12 to transmit excitation beams to the reaction mixture
contained in the chamber 17. FIG. 17 is an exploded view of the
excitation assembly. As shown in FIGS. 16-17, the excitation
assembly 68 includes a housing 219 for holding various components
of the assembly. The housing 219 includes stops 245A, 245B for
limiting the maximum spacing of the thermal plates, as previously
discussed with reference to FIGS. 8 and 14. The housing 219
preferably comprises one or more molded pieces of plastic. In the
preferred embodiment, the housing 219 is a multi-part housing
comprised of three housing elements 220A, 220B, and 220C. The upper
and lower housing elements 220A and 220C are preferably
complementary pieces that couple together and snap-fit into housing
element 220B. In this embodiment, the housing elements 220A and
220C are held together by screws 214. In alternative embodiments,
the entire housing 219 may be a one-piece housing that holds a
slide-in optics package.
[0141] The lower housing element 220C includes an optical window
235 into which is placed a cylindrical rod lens 215 for focusing
excitation beams into the chamber 17. In general, the optical
window 235 may simply comprise an opening in the housing through
which excitation beams may be transmitted to the chamber 17. The
optical window may optionally include an optically transmissive or
transparent piece of glass or plastic serving as a window pane, or
as in the preferred embodiment, a lens for focusing excitation
beams. The lens 215 preferably directly contacts one of the
optically transmissive side walls of the chamber 17.
[0142] The optics assembly 68 also includes four light sources,
preferably LEDs 100A, 100B, 100C, and 100D, for transmitting
excitation beams through the lens 215 to the reaction mixture
contained in the chamber 17. In general, each light source may
comprise a laser, a light bulb, or an LED. In the preferred
embodiment, each light source comprises a pair of directional LEDs.
In particular, the four light sources shown in FIGS. 16-17 are
preferably a first pair of green LEDs 100A, a second pair of green
LEDs 100B, a pair of blue LEDs 100C, and a third pair of green LEDs
100D. The LEDs receive power through leads 201 which are connected
to a power source (not shown in FIGS. 16-17). The LEDs are mounted
to the optical circuit board 72 which is attached to the back of
the housing element 220B so that the LEDs are rigidly fixed in the
housing. The optical circuit board 72 is connected to the main PC
board of the heat-exchanging module (shown in FIG. 8) via the flex
cable 103.
[0143] The optics assembly 68 further includes a set of filters and
lenses arranged in the housing 219 for filtering the excitation
beams generated by the LEDs so that each of the beams transmitted
to the chamber 17 has a distinct excitation wavelength range. As
shown in FIG. 17, the lower housing element 220C preferably
includes walls 202 that create separate excitation channels in the
housing to reduce potential cross-talk between the different pairs
of LEDs. The walls 202 preferably include slots for receiving and
rigidly holding the filters and lenses. The filters and lenses may
also be fixed in the housing by means of an adhesive used alone, or
more preferably, with an adhesive used in combination with slots in
the housing.
[0144] Referring to FIG. 16, the filters in the optics assembly 68
may be selected to provide excitation beams to the reaction mixture
in the chamber 17 in any desired excitation wavelength ranges. The
optics assembly 68 may therefore be used with any fluorescent,
phosphorescent, chemiluminescent, or electrochemiluminescent labels
of interest. For purposes of illustration, one specific embodiment
of the assembly 68 will now be described in which the assembly is
designed to provide excitation beams corresponding to the peak
excitation wavelength ranges FAM, TAMRA, TET, and ROX.
[0145] In this embodiment, a pair of 593 nm low pass filters 203
are positioned in front of green LEDs 100A, a pair of 555 nm low
pass filters 204 are positioned in front of green LEDs 100B, a pair
of 495 nm low pass filters 205 are positioned in front of blue LEDs
100C, and a pair of 527 nm low pass filters 206 are positioned in
front of green LEDs 100D. Although it is presently preferred to
position a pair of low pass filters in front of each pair of LEDs
for double filtering of excitation beams, a single filter may be
used in alternative embodiments. In addition, a lens 207 is
preferably positioned in front of each pair of filters for
collimating the filtered excitation beams. The optics assembly 68
also includes a 495 nm high pass reflector 208, a 527 nm high pass
reflector 209, a mirror 210, a 555 nm low pass reflector 211, and a
593 nm low pass reflector 212. The reflecting filters and mirrors
208-212 are angularly offset by 30.degree. from the low pass
filters 203-206.
[0146] The excitation assembly 68 transmits excitation beams to the
chamber 17 in four distinct excitation wavelength ranges as
follows. When the green LEDs 100A are activated, they generate an
excitation beam that passes through the pair of 593 nm low pass
filters 203 and through the lens 207. The excitation beam then
reflects off of the 593 nm low pass reflector 212, passes through
the 555 nm low pass reflector 211, reflects off of the 527 nm high
pass reflector 209, and passes through the lens 215 into the
reaction chamber 17. The excitation beam from the LEDs 100A is thus
filtered to a wavelength range of 555 to 593 nm corresponding to
the peak excitation range for ROX. When the green LEDs 100B are
activated, they generate an excitation beam that passes through the
pair of 555 nm low pass filters 204, reflects off of the 555 nm low
pass reflector 211, reflects off of the 527 nm high pass reflector
209, and passes through the lens 215 into the reaction chamber 17.
The excitation beam from LEDs 100B is thus filtered to a wavelength
range of 527 to 555 nm corresponding to the peak excitation range
for TAMRA.
[0147] When the blue LEDs 100C are activated, they generate an
excitation beam that passes through the pair of 495 nm low pass
filters 205, through the 495 nm high pass reflector 208, through
the 527 nm high pass reflector 209, and through the lens 215 into
the reaction chamber 17. The excitation beam from LEDs 100C is thus
filtered to a wavelength below 495 nm corresponding to the peak
excitation range for FAM. When the green LEDs 100D are activated,
they generate an excitation beam that passes through the pair of
527 nm low pass filters 206, reflects off of the mirror 210,
reflects off of the 495nm high pass reflector 208, passes through
the 527 nm high pass reflector 209, and passes through the lens 215
into the reaction chamber 17. The excitation beam from LEDs 100D is
thus filtered to a wavelength range of 495 to 527 nm corresponding
to the peak excitation range for TET. In operation, the LEDs 100A,
100B, 100C, 100D are sequentially activated to excite the different
fluorescent labels contained in the chamber 17 with excitation
beams in substantially distinct wavelength ranges.
[0148] FIG. 18 is a schematic, plan view of the optical detection
assembly 70. The assembly 70 is positioned adjacent the reaction
vessel 12 to receive light emitted from the chamber 17. FIG. 19 is
an exploded view of the detection assembly 70. As shown in FIGS.
18-19, the assembly 70 includes a housing 221 for holding various
components of the assembly. The housing 221 includes the stops
247A, 247B previously described with reference to FIGS. 13-14. The
housing 221 preferably comprises one or more molded plastic pieces.
In the preferred embodiment, the housing 221 is a multi-part
housing comprised of upper and lower housing elements 234A and
234B. The housing elements 234A, 234B are complementary, mating
pieces that are held together by screws 214. In alternative
embodiments, the entire housing 221 may be a one-piece housing that
holds a slide-in optics package.
[0149] The lower housing element 234B includes an optical window
237 into which is placed a cylindrical rod lens 232 for collimating
light emitted from the chamber 17. In general, the optical window
may simply comprise an opening in the housing through which the
emitted light may be received. The optical window may optionally
include an optically transmissive or transparent piece of glass or
plastic serving as a window pane, or as in the preferred
embodiment, the lens 232 for collimating light emitted from the
chamber 17. The lens 232 preferably directly contacts one of the
optically transmissive side walls of the chamber 17.
[0150] The optics assembly 70 also includes four detectors 102A,
102B. 102C, and 102D for detecting light emitted from the chamber
17 that is received through the lens 232. In general, each detector
may be a photomultiplier tube, CCD, photodiode, or other known
detector. In the preferred embodiment, each detector is a PIN
photodiode. The detectors 102A, 102B, 102C, and 102D are preferably
rigidly fixed in recesses formed in the lower housing element 234B.
The detectors are electrically connected by leads 245 to the
optical circuit board 74 (see FIG. 8) which is preferably mounted
to the underside of the lower housing element 234B.
[0151] The optics assembly 70 further includes a set of filters and
lenses arranged in the housing 221 for separating light emitted
from the chamber 17 into different emission wavelength ranges and
for directing the light in each of the emission wavelength ranges
to a respective one of the detectors. As shown in FIG. 19, the
lower housing element 234B preferably includes walls 247 that
create separate detection channels in the housing, with one of the
detectors positioned at the end of each channel. The walls 247
preferably include slots for receiving and rigidly holding the
filters and lenses. The filters and lenses may also be rigidly
fixed in the housing 221 by an adhesive used alone, or more
preferably, with an adhesive used in combination with slots in the
housing.
[0152] Referring to FIG. 18, the filters in the optics assembly 70
may be selected to block light emitted from the chamber 17 outside
of any desired emission wavelength ranges. The optics assembly 70
may therefore be used with any fluorescent, phosphorescent,
chemiluminescent, or electrochemiluminescent labels of interest.
For purposes of illustration, one specific embodiment of the
assembly 70 will now be described in which the assembly is designed
to detect light emitted from the chamber 17 in the peak emission
wavelength ranges of FAM, TAMRA, TET, and ROX.
[0153] In this embodiment, the set of filters preferably includes a
515 nm Schott Glass.RTM. filter 222A positioned in front of the
first detector 102A, a 550 nm Schott Glass.RTM. filter 222B
positioned in front of the second detector 102B, a 570 nm Schott
Glass.RTM. filter 222C positioned in front of the third detector
102C, and a 620 nm Schott Glass.RTM. filter 222D positioned in
front of the fourth detector 102D. These Schott Glass.RTM. filters
are commercially available from Schott Glass Technologies, Inc. of
Duryea, Pa. The optics assembly 70 also includes a pair of 505 nm
high pass filters 223 positioned in front of the first detector
102A, a pair of 537 nm high pass filters 224 positioned in front of
the second detector 102B, a pair of 565 nm high pass filters 225
positioned in front of the third detector 102C, and a pair of 605
nm high pass filters 226 positioned in front of the fourth detector
102D.
[0154] Although it is presently preferred to position a pair of
high pass filters in front of each detector for double filtering of
light, a single filter may be used in alternative embodiments. In
addition, a lens 242 is preferably positioned in each detection
channel between the pair of high pass filters and the Schott
Glass.RTM. filter for collimating the filtered light. The optics
assembly 70 further includes a 605 nm high pass reflector 227, a
mirror 228, a 565 nm low pass reflector 229, a 537 nm high pass
reflector 230, and a 505 nm high pass reflector 231. The reflecting
filters and mirrors 227-231 are preferably angularly offset by
30.degree. from the high pass filters 223-226. As shown in FIG. 19,
the detection assembly 70 also preferably includes a first aperture
238 positioned between each detector and Schott Glass.RTM. filter
222 and an aperture 240 positioned between each lens 242 and Schott
Glass.RTM. filter 222. The apertures 238, 240 reduce the amount of
stray or off-axis light that reaches the detectors 102A, 102B,
102C, and 102D.
[0155] Referring again to FIG. 18, the detection assembly 70
detects light emitted from the chamber 17 in four emission
wavelength ranges as follows. The emitted light passes through the
lens 232 and strikes the 565 nm low pass reflector 229. The portion
of the light having a wavelength in the range of about 505 to 537
nm (corresponding to the peak emission wavelength range of FAM)
reflects from the 565 nm low pass reflector 229, passes through the
537 nm high pass reflector 230, reflects from the 505 nm high pass
reflector 231, passes through the pair of 505 nm high pass filters
223, through the lens 242, through the 515 nm Schott Glass.RTM.
filter 222A, and is detected by the first detector 102A. Meanwhile,
the portion of the light having a wavelength in the range of about
537 to 565 nm (corresponding to the peak emission wavelength range
of TET) reflects from the 565 nm low pass reflector 229, reflects
from the 537 nm high pass reflector 230, passes through the pair of
537 nm high pass filters 224, through the lens 242, through the 550
nm Schott Glass.RTM. filter 222B, and is detected by the second
detector 102B.
[0156] Further, the portion of the light having a wavelength in the
range of about 565 to 605 nm (corresponding to the peak emission
wavelength range of TAMRA) passes through the 565 nm low pass
reflector 229, through the 605 nm high pass reflector 227, through
the pair of 565 nm high pass filters 225, through the lens 242,
through the 570 nm Schott Glass.RTM. filter 222C, and is detected
by the third detector 102C. The portion of the light having a
wavelength over 605 nm (corresponding to the peak emission
wavelength range of ROX) passes through the 565 nm low pass
reflector 229, reflects from the 605 nm high pass reflector 227,
reflects from the mirror 228, passes through the pair of 605 nm
high pass filters 226, through the lens 242, through the 620 nm
Schott Glass.RTM. filter 222D, and is detected by the fourth
detector 102D. In operation, the outputs of detectors 102A, 102B,
102C, and 102D are analyzed to determine the starting quantities or
concentrations of one or more target nucleic acid sequences in the
reaction mixture, as will be described in greater detail below.
[0157] FIG. 20 shows a multi-site reactor system 106 according to
the present invention. The reactor system 106 comprises a thermal
cycler 108 and a controller 112, such as a personal or network
computer. The thermal cycler 108 includes a base instrument 110 for
receiving multiple heat-exchanging modules 60 (previously described
with reference to FIG. 8). The base instrument 110 has a main logic
board with edge connectors 114 for establishing electrical
connections to the modules 60. The base instrument 110 also
preferably includes a fan 116 for cooling its electronic
components. The base instrument 110 may be connected to the
controller 112 using any suitable data connection, such as a
universal serial bus (USB), ethernet connection, or serial line. It
is presently preferred to use a USB that connects to the serial
port of controller 112. Alternatively, the controller may be built
into the base instrument 110.
[0158] The term "thermal cycling" is herein intended to mean at
least one change of temperature, i.e. increase or decrease of
temperature, in a reaction mixture. Therefore, samples undergoing
thermal cycling may shift from one temperature to another and then
stabilize at that temperature, transition to a second temperature
or return to the starting temperature. The temperature cycle may be
performed only once or may be repeated as many times as required to
study or complete the particular chemical reaction of interest. Due
to space limitations in patent drawings, the thermal cycler 108
shown in FIG. 20 includes only sixteen reaction sites provided by
the sixteen heat-exchanging modules 60 arranged in two rows of
eight modules each. It is to be understood, however, that the
thermal cycler can include any number of desired reaction sites,
i.e., it can be configured as a multi-hundred site instrument for
simultaneously processing hundreds of samples. Alternatively, it
may be configured as a small, hand held, battery-operated
instrument having, e.g., 1 to 4 reaction sites.
[0159] Each of the reaction sites in the thermal cycler 108 is
provided by a respective one of the heat-exchanging modules 60. The
modules 60 are preferably independently controllable so that
different chemical reactions can be run simultaneously in the
thermal cycler 108. The thermal cycler 108 is preferably modular so
that each heat-exchanging module 60 can be individually removed
from the base instrument 110 for servicing, repair, or replacement.
This modularity reduces downtime since all the modules 60 are not
off line to repair one, and the instrument 110 can be upgraded and
enlarged to add more modules as needed. The modularity of the
thermal cycler 108 also means that individual modules 60 can be
precisely calibrated, and module-specific schedules or corrections
can be included in the control programs, e.g., as a series of
module-specific calibration or adjustment charts.
[0160] In embodiments in which the base instrument 110 operates on
external power, e.g. 110 V AC, the instrument preferably includes
two power connections 122, 124. Power is received though the first
connection 122 and output through the second connection 124.
Similarly, the instrument 110 preferably includes network interface
inlet and outlet ports 118, 120 for receiving a data connection
through inlet port 118 and outputting data to another base
instrument through outlet port 120. As shown in the block diagram
of FIG. 21, this arrangement permits multiple thermal cyclers 108A,
108B, 108C, 108D to be daisy-chained from one controller 112 and
one external power source 128.
[0161] FIG. 22 is a schematic, block diagram of the base instrument
110. The base instrument includes a power supply 134 for supplying
power to the instrument and to each module 60. The power supply 134
may comprise an AC/DC converter for receiving power from an
external source and converting it to direct current, e.g., for
receiving 110V AC and converting it to 12V DC. Alternatively, the
power supply 134 may comprise a battery, e.g., a 12V battery. The
base instrument 110 also includes a microprocessor or
microcontroller 130 containing firmware for controlling the
operation of the base instrument 110 and modules 60. The
microcontroller 130 communicates through a network interface 132 to
the controller computer via a USB. Due to current limitations of
processing power, it is currently preferred to include at least one
microcontroller in the base instrument per sixteen modules 60. Thus
if the base instrument has a thirty-two module capacity, at least
two microcontrollers should be installed in the instrument 110 to
control the modules.
[0162] The base instrument 110 further includes a heater power
source and control circuit 136, a power distributor 138, a data bus
140, and a module selection control circuit 142. Due to space
limitations in patent drawings, control circuit 136, power
distributor 138, data bus 140, and control circuit 142 are shown
only once in the block diagram of FIG. 22. However, the base
instrument 110 actually contains one set of these four functional
components 136, 138, 140, 142 for each heat-exchanging module 60.
Thus, in the embodiment of FIG. 22, the base instrument 110
includes sixteen control circuits 136, power distributors 138, data
buses 140, and control circuits 142. Similarly, the base instrument
110 also includes a different edge connector 131 for connecting to
each of the modules 60, so that the instrument includes sixteen
edge connectors for the embodiment shown in FIG. 22. The edge
connectors are preferably 120 pin card edge connectors that provide
cableless connection from the base instrument 110 to each of the
modules 60. Each control circuit 136, power distributor 138, data
bus 140, and control circuit 142 is connected to a respective one
of the edge connectors and to the microcontroller 130.
[0163] Each heater power and source control circuit 136 is a power
regulator for regulating the amount of power supplied to the
heating element(s) of a respective one of the modules 60. The
source control circuit 136 is preferably a DC/DC converter that
receives a +12V input from the power supply 134 and outputs a
variable voltage between 0 and -24V. The voltage is varied in
accordance with signals received from the microcontroller 130. Each
power distributor 138 provides -5v, +5V, +12V, and GND to a
respective module 60. The power distributor thus supplies power for
the electronic components of the module. Each data bus 140 provides
parallel and serial connections between the microcontroller 130 and
the digital devices of a respective one of the modules 60. Each
module selection controller 94 allows the microcontroller 130 to
address an individual module 60 in order to read or write control
or status information.
[0164] FIG. 23 is a schematic, block diagram of the electronic
components of a heat-exchanging module 60. Each module includes an
edge connector 80 for cableless connection to a corresponding edge
connector of the base instrument. The module also includes heater
plates 50A, 50B each having a resistive heating element as
described above. The plates 50A, 50B are wired in parallel to
receive power input 146 from the base instrument. The plates 50A,
50B also include temperature sensors 52, e.g. thermistors, that
output analog temperature signals to an analog-to-digital converter
154. The converter 154 converts the analog signals to digital
signals and routes them to the microcontroller in the base
instrument through the edge connector 80. The heat-exchanging
module also includes a cooling system, such as a fan 66, for
cooling the plates 50A, 50B. The fan 66 receives power from the
base instrument and is activated by switching a power switch 164.
The power switch 164 is in turn controlled by a control logic block
162 that receives control signals from the microcontroller in the
base instrument.
[0165] The module further includes four light sources, such as LEDs
100, for excitation of labeled nucleic acid sequences in the
reaction mixture and four detectors 102, preferably photodiodes,
for detecting fluorescent signals from the reaction mixture. The
module also includes an adjustable current source 150 for supplying
a variable amount of current (e.g., in the range of 0 to 30 mA) to
each LED to vary the brightness of the LED. A digital-to-analog
converter 152 is connected between the adjustable current source
150 and the microcontroller of the base instrument to permit the
microcontroller to adjust the current source digitally. The
adjustable current source 150 may be used to ensure that each LED
has about the same brightness when activated. Due to manufacturing
variances, many LEDs have different brightnesses when provided with
the same amount of current. The brightness of each LED may be
tested during manufacture of the heat-exchanging module and
calibration data stored in a memory 160 of the module. The
calibration data indicates the correct amount of current to provide
to each LED. The microcontroller reads the calibration data from
the memory 160 and controls the current source 150 accordingly. The
microcontroller may also control the current source 150 to adjust
the brightness of the LEDs 100 in response to optical feedback
received from the detectors 102.
[0166] The module additionally includes a signal conditioning/gain
select/offset adjust block 156 comprised of amplifiers, switches,
electronic filters, and a digital-to-analog converter. The block
156 adjusts the signals from the detectors 102 to increase gain,
offset, and reduce noise. The microcontroller in the base
instrument controls block 156 through a digital output register
158. The output register 158 receives data from the microcontroller
and outputs control voltages to the block 156. The block 156
outputs the adjusted detector signals to the microcontroller
through the analog-to-digital converter 154 and the edge connector
80. The module also includes the memory 160, preferably a serial
EEPROM, for storing data specific to the module, such as
calibration data for the LEDs 100, thermal plates 50A, 50B, and
temperature sensors 52, as well as calibration data for a
deconvolution algorithm described in detail below.
[0167] Referring again to FIG. 20, the controller 112 is programmed
to perform the functions described in the operation section below.
These functions include providing a user interface to enable a user
to specify desired thermal processing parameters (e.g., set point
temperatures and hold times at each temperature), thermal
processing of samples according to the selected parameters,
detection and measurement of optical signals emitted from the
samples, and recording, manipulating, and analyzing the optical
data. The creation of software and/or firmware for performing these
functions can be performed by a computer programmer having ordinary
skill in the art upon consideration of the following description.
In addition, Appendix A lists exemplary source code for performing
various functions described below relating to the manipulation and
analysis of optical signals. The code is written in the Java
programming language. The software and/or firmware may reside
solely in the controller 112 or may be distributed between the
controller and one or more microprocessors in the thermal cycler
108. Alternatively, the controller 112 may simply comprise one or
more processors built into the thermal cycler 108.
[0168] In operation, the reactor system 106 is used to determine an
unknown starting quantity of one or more target nucleic acid
sequences in one or more test samples. The nucleic acid sequences
in the samples may be amplified according to any known nucleic acid
amplification method, including both thermal cycling amplification
methods and isothermal amplification methods. Suitable thermal
cycling methods useful in the practice of the present invention
include, but are not limited to, the Polymerase Chain Reaction
(PCR; U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,965,188); Reverse
Transcriptase PCR (RT-PCR); DNA Ligase Chain Reaction (LCR;
International Patent Application No. WO 89/09835); and
transcription-based amplification (D. Y. Kwoh et al. 1989, Proc.
Natl. Acad. Sci. USA 86, 1173-1177). Suitable isothermal
amplification methods useful in the practice of the present
invention include, but are not limited to, Rolling Circle
Amplification; Strand Displacement Amplification (SDA; Walker et
al. 1992, Proc. Natl. Acad. Sci. USA 89, 392-396); Q-.beta.
replicase (Lizardi et al. 1988, Bio/Technology 6, 1197-1202);
Nucleic Acid-Based Sequence Amplification (NASBA; R. Sooknanan and
L. Malek 1995, Bio/Technology 13, 563-65); and Self-Sustained
Sequence Replication (3SR; Guatelli et al. 1990, Proc. Natl. Acad.
Sci. USA 87, 1874-1878).
[0169] According to a first mode of operation, the thermal cycler
108 is used to amplify an unknown starting quantity of a target
nucleic acid sequence in a test sample and a plurality of different
known quantities of a calibration nucleic acid sequence in
respective calibration samples (i.e., standards). Preferably, the
nucleic acid sequences that are amplified in the calibration and
test samples are the same or similar. Referring again to FIG. 2,
each sample is placed into a separate reaction vessel by aspirating
the sample into a pipette (not shown), inserting the pipette tip
through the channel 28 into the chamber 17, and dispensing the
sample into the chamber. It is presently preferred that the chamber
17 be filled from the bottom up by initially inserting the pipette
tip close to the bottom of the chamber 17 and by slowly retracting
the pipette tip as the chamber 17 is filled. Filling the chamber 17
in this manner reduces the likelihood that air bubbles will form in
the chamber. Such air bubbles could have a negative effect on
subsequent optical detection.
[0170] The sample may be mixed with chemicals necessary for the
intended reaction (e.g., PCR reagents and fluorescent probes for
labeling the nucleic acid sequences to be amplified) prior to being
added to the chamber 17. Alternatively, the sample may be
introduced to the chemicals in the chamber 17, e.g., by adding the
chemicals to the chamber before or after the sample to form the
desired reaction mixture in the chamber. In one embodiment, the
reagents and fluorescent probes for the intended reaction are
placed in the chamber 17 when the vessel is manufactured. The
reagents are preferably placed in the chamber 17 in dried or
lyophilized form so that they are adequately preserved until the
vessel is used. After the chamber 17 is filled with the desired
reaction mixture, the plunger 22 is inserted into the channel 28 to
seal and pressurize the chamber 17.
[0171] Referring again to FIG. 20, each of the vessels 12 may be
inserted between the thermal plates of a respective heat-exchanging
module 60 either prior to filling and pressurizing the vessel or
after filling and pressurizing the vessel. In either case, as shown
in FIG. 5, the pressure in the chamber 17 forces the flexible major
walls 18 to contact and conform to the inner surfaces of the plates
50. Further, each of the vessels may be manually filled and
pressurized by a human operator or the vessels may be filled and
pressurized by an automated machine, e.g., a pick-and-place
machine. Various automated embodiments of the apparatus are
described in U.S. application Ser. No. 09/468,690 filed Dec. 21,
1999 the disclosure of which is incorporated by reference
herein.
[0172] Referring again to FIG. 20, the user then selects a desired
thermal profile to be executed at each reaction site at which one
of the vessels 12 is present. For example, for a PCR amplification,
the user may select the thermal profile to begin with a 30 second
induction hold at 95.degree. C., followed by 45 thermal cycles in
which the reaction mixture is cycled between higher and lower
temperatures for denaturization, annealing, and polymerization. For
example, each thermal cycle may include a first set point
temperature of 95.degree. C. which is held for 1 second to denature
double-stranded bNA, followed by a second set point temperature of
60.degree. C. which is held for 6 seconds for annealing of primers
and polymerization. The user also enters into the controller 112
specific values related to the calibration and test samples. In
particular, the user specifies in a setup table the specific site
at which each sample is located, the starting quantity of each
calibration nucleic acid sequence in each calibration sample, the
specific dye being used to label the calibration sequence (e.g.,
FAM, TET, TAMRA, or ROX), and the specific dye being used to label
each target nucleic acid sequence in the test sample(s).
[0173] The reaction mixtures contained in the vessels 12 are then
subjected to the thermal profile selected by the user. The
controller 112 preferably implements standard
proportional-integral-derivative (PID) control to execute the
selected thermal profile. Referring again to FIG. 23, for each
heat-exchanging module in use, the controller receives signals
indicating the temperatures of the plates 50A, 50B from the
temperature sensors 52. Polling of the plate temperatures
preferably occurs regularly throughout the running of the
temperature profile. After each polling, the controller averages
the temperatures of the two plates 50A, 50B to determine an average
plate temperature. The controller then determines the difference
(delta) between the profile target temperature, i.e. the set point
temperature defined by the user for the particular time in the
profile, and the average plate temperature. Based on the
relationship between the average plate temperature and the current
target temperature, the controller controls the amount of power
supplied to the heating elements on the plates 50A, 50B or to the
fan 66 as appropriate to reach or maintain the current set point
temperature. Standard PID control is well known in the art and need
not be described further herein.
[0174] The controller may optionally be programmed to implement a
modified version of PID control described in International
Publication Number WO 99/48608 published Sep. 30, 1999, the
disclosure of which is incorporated by reference herein. In this
modified version of PID control, the controller is programmed to
compensate for thermal lag between the plates 50A, 50B and a
reaction mixture contained in a reaction vessel inserted between
the plates. The thermal lag is caused by the need for heat to
transfer from the plates 50A, 50B through the flexible walls of the
vessel and into the reaction mixture during heating, or by the need
for heat to transfer from the reaction mixture through the walls of
the vessel to the plates 50A, 50B during cooling. In standard PID
control, the power supplied to a heating or cooling element is
dependent upon the difference (error) between the actual measured
temperature of the plates and the desired set point temperature.
The average power being supplied to either the heating or cooling
element therefore decreases as the actual temperature of the plates
approaches the set point temperature, so that the reaction mixture
does not reach the set point temperature as rapidly as possible.
The modified version of PID control overcomes this disadvantage of
standard PID control during rapid heating or cooling steps.
[0175] To compensate for the thermal lag during heating steps
(i.e., to raise the temperature of the reaction mixture to a
desired set point temperature that is higher than the previous set
point temperature), the controller sets a variable target
temperature that initially exceeds the desired set point
temperature. For example, if the set point temperature is
95.degree. C., the initial value of the variable target temperature
may be set 2 to 10.degree. C. higher. The controller next
determines a level of power to be supplied to the heating elements
to raise the temperature of the plates 50A, 50B to the variable
target temperature by inputting the variable target temperature and
the current average plate temperature to a standard PID control
algorithm. The level of power to be supplied to the heaters is
therefore determined in dependence upon the difference (error)
between the average plate temperature and a target temperature that
is higher than the desired set point temperature. The higher target
temperature ensures that a higher level of power is supplied to
heat the plates 50A, 50B, and therefore the reaction mixture, to
the set point temperature more rapidly. The controller then sends a
control signal to the power and source control circuit in the base
instrument to provide power to the heating elements at the level
determined.
[0176] When the temperature of the plates 50A, 50B is subsequently
polled, the controller determines if the actual measured
temperature of the plates is greater than or equal to a
predetermined cutoff value. Suitable cutoff values are: the desired
set point temperature itself; or 1 to 2.degree. C. below the set
point temperature, e.g., 93 to 94 .degree. C. for a set point
temperature of 95.degree. C. If the average plate temperature does
not exceed the predetermined value, then the controller again
determines a level of power to be supplied to the heating elements
in dependence upon the difference between the average plate
temperature and the target temperature and sends another control
signal to provide power to the heaters at the level determined.
This process is repeated until the average plate temperature is
greater than or equal to the cutoff value.
[0177] When the average plate temperature is greater than or equal
to the cutoff value, the controller decreases the variable target
temperature, preferably by exponentially decaying the amount by
which the variable target temperature exceeds the set point
temperature. For example, the amount by which the variable target
temperature exceeds the desired set point temperature may be
exponentially decayed as a function of time according to the
equation: .DELTA.=(.DELTA..sub.max)*e.sup.(-t/tau) where .DELTA. is
equal to the amount by which the variable target temperature
exceeds the desired set point temperature, .DELTA..sub.max is equal
to the difference between the initial value of the variable target
temperature and the desired set point temperature, t is equal to
the elapsed time in seconds from the start of decay, and tau is
equal to a decay time constant. In the system of the present
invention, tau preferably has a value in the range of 1 to 4
seconds. It is presently preferred to determine tau empirically for
the heat-exchanging module during testing and calibration of the
module and to store the value of tau in the memory 160 of the
module before shipping it to the end user. Although the exponential
equation given above is presently preferred, it is to be understood
that many other decay formulas may be employed and fall within the
scope of the invention. Moreover, the variable target temperature
may be decreased by other techniques, e.g., it may be decreased
linearly.
[0178] After decreasing the variable target temperature, the
controller determines a new level of power to be supplied to the
heating elements to raise the temperature of the plates 50A, 50B to
the decreased target temperature. The controller determines the
level of power by inputting the current plate temperature and
decreased target temperature to the PID control algorithm. The
controller then sends a control signal to provide power to the
heaters at the new level determined. As the time in the thermal
profile progresses, the controller continues to decrease the
variable target temperature until it is equal to the set point
temperature. When the variable target temperature is equal to the
set point temperature, standard PID control is resumed to maintain
the plates 50A, 50B at the set point temperature.
[0179] To compensate for the thermal lag during cooling steps
(i.e., to lower the temperature of the reaction mixture to a
desired set point temperature that is lower than the previous set
point temperature), the controller preferably activates the fan 66
just prior to the completion of the previous set point temperature
to allow the fan to achieve maximum speed for cooling (i.e., to
allow for spin-up time). The controller then sets a variable target
temperature that is initially lower than the desired set point
temperature. For example, if the set point temperature is
60.degree. C., the initial value of the variable target temperature
may be set 2 to 100.degree. C. lower, i.e., 50 to 580.degree. C.
The controller continues cooling with the fan 66 until the actual
measured temperature of the plates 50A, 50B is less than or equal
to a second cutoff value, preferably the variable target
temperature. When the average plate temperature is less than or
equal to the variable target temperature, the controller
deactivates the fan 66 and increases the target temperature,
preferably by exponentially decaying the amount by which the
variable target temperature differs from the set point temperature
using the exponential decay equation given above. For cooling, tau
is preferably in the range of 1 to 5 seconds with a preferred value
of about 3 seconds. As in the heating example given above, tau may
be determined empirically for the heat-exchanging module during
testing or calibration and stored in the memory 160.
[0180] The controller next determines a level of power to be
supplied to the heating elements to raise the temperature of the
plates 50A, 50B to the increased target temperature by inputting
the current average plate temperature and the increased target
temperature to the PID control algorithm. The controller then sends
a control signal to the power and source control circuit in the
base instrument to provide power to the heating elements at the
level determined. As time in the thermal profile continues, the
controller continues to increase the variable target temperature
and issue control signals in this manner until the variable target
temperature is equal to the set point temperature. When the
variable target temperature is equal to the set point temperature,
the controller resumes standard PID control to maintain the plates
50A, 50B at the set point temperature.
[0181] Referring again to FIG. 20, the reaction mixtures in the
vessels 12 are optically interrogated in real-time as they are
thermally processed. If the mixtures are being subjected to thermal
cycling, then each mixture is preferably optically interrogated
once per thermal cycle at the lowest temperature in the cycle. If
isothermal amplification is employed, then each mixture is
preferably optically interrogated at regular time intervals (e.g.,
every 10 seconds) during the amplification. Referring again to
FIGS. 16 and 18, optical interrogation of an individual mixture in
a reaction vessel 12 is accomplished by sequentially activating
LEDs 100A, 100B, 100C, and 100D to excite different
fluorescently-labeled nucleic acid sequences in the mixture and by
detecting fluorescent signals emitted from the chamber 17 using
detectors 102A, 102B, 102C, and 102D. In the following example of
operation, the fluorescent dyes FAM, TAMRA, TET, and ROX are used
to label the target nucleotide sequences in the reaction
mixture.
[0182] There are four pairs of LEDs 100A, 100B, 100C, and 100D and
four detectors 102A, 102B, 102C, and 102D for a total of sixteen
combinations of LED/detector pairs. It is theoretically possible to
collect output signals from the detectors for all sixteen
combinations. Of these sixteen combinations, however, there are
only four primary detection channels. Each primary detection
channel is formed by a pair of LEDs in the optics assembly 68 whose
excitation beams lie in the peak excitation wavelength range of a
particular dye and by one corresponding channel in the optics
assembly 70 designed to detect light emitted in the peak emission
wavelength range of the same dye. The first primary detection
channel is formed by the first pair of LEDs 100A and the fourth
detector 102D (the ROX channel). The second primary detection
channel is formed by the second pair of LEDs 100B and the third
detector 102C (the TAMRA channel). The third primary detection
channel is formed by the third pair of LEDs 100C and the first
detector 102A (the FAM channel). The fourth primary detection
channel is formed by the fourth pair of LEDs 100D and the second
detector 102B (the TET channel).
[0183] Prior to activating any of the LEDs 100A, 100B, 100C, 100D,
a "dark reading" is taken to determine the output signal of each of
the four detectors 102A, 102B, 102C, 102D when none of the LEDs are
lit. The "dark reading" signal output by each detector is
subsequently subtracted from the corresponding "light reading"
signal output by the detector to correct for any electronic offset
in the optical detection circuit. This procedure of obtaining "dark
reading" signals and subtracting the dark signals from the
corresponding "light reading" signals is preferably performed every
time that a reaction vessel is optically interrogated, including
those times the vessel is interrogated during the development of
calibration data (described in detail below). For clarity and
brevity of explanation, however, the steps of obtaining "dark
reading" signals and subtracting the dark signals from the
corresponding "light reading" signals will not be further repeated
in this description.
[0184] Following the dark reading, a "light reading" is taken in
each of the four primary optical detection channels as follows. The
first pair of LEDs 100A is activated and the LEDs generate an
excitation beam that passes through the pair of 593 nm low pass
filters 203, reflects off of the 593 nm low pass reflector 212,
passes through the 555 nm low pass reflector 211, reflects off of
the 527 nm high pass reflector 209, and passes through the lens 215
into the reaction chamber 17. The excitation beam from the LEDs
100A is thus filtered to a wavelength range of 555 to 593 nm
corresponding to the peak excitation range for ROX. As shown in
FIG. 18, emitted light (fluorescence emission radiation) from the
chamber 17 passes through the lens 232 of the detection assembly 70
and strikes the 565 nm low pass reflector 229. The portion of the
light having a wavelength over 605 nm (corresponding to the peak
emission wavelength range of ROX) passes through the 565 nm low
pass reflector 229, reflects from the 605 nm high pass reflector
227, reflects from the mirror 228, passes through the pair of 605
nm high pass filters 226, through the lens 242, through the 620 nm
Schott Glass.RTM. filter 222D, and is detected by the fourth
detector 102D. The fourth detector 102D outputs a corresponding
signal that is converted to a digital value and recorded.
[0185] Next, as shown in FIG. 16, the second pair of LEDs 100B is
activated and the LEDs generate an excitation beam that passes
through the pair of 555 nm low pass filters 204, reflects off of
the 555 nm low pass reflector 211, reflects off of the 527 nm high
pass reflector 209, and passes through the lens 215 into the
reaction chamber 17. The excitation beam from LEDs 100B is thus
filtered to a wavelength range of 527 to 555 nm corresponding to
the peak excitation range for TAMRA. As shown in FIG. 18, emitted
light from the chamber 17 then passes through the lens 232 of the
detection assembly 70 and strikes the 565 nm low pass reflector
229. The portion of the light having a wavelength in the range of
about 565 to 605 nm (corresponding to the peak emission wavelength
range of TAMRA) passes through the 565 nm low pass reflector 229,
through the 605 nm high pass reflector 227, through the pair of 565
nm high pass filters 225, through the lens 242, through the 570 nm
Schott Glass.RTM. filter 222C, and is detected by the third
detector 102C. The third detector 102C outputs a corresponding
signal that is converted to a digital value and recorded.
[0186] Next, as shown in FIG. 16, the pair of blue LEDs 100C is
activated and the LEDs generate an excitation beam that passes
through the pair of 495 nm low pass filters 205, through the 495 nm
high pass reflector 208, through the 527 nm high pass reflector
209, and through the lens 215 into the reaction chamber 17. The
excitation beam from LEDs 100C is thus filtered to a wavelength
range of about 450 to 495 nm corresponding to the peak excitation
range for FAM. As shown in FIG. 18, emitted light from the chamber
17 then passes through the lens 232 of the detection assembly 70
and strikes the 565 nm low pass reflector 229. The portion of the
light having a wavelength in the range of about 505 to 537 nm
(corresponding to the peak emission wavelength range of FAM)
reflects from the 565 nm low pass reflector 229, passes through the
537 nm high pass reflector 230, reflects from the 505 nm high pass
reflector 231, passes through the pair of 505 nm high pass filters
223, through the lens 242, through the 515 nm Schott Glass.RTM.
filter 222A, and is detected by the first detector 102A. The first
detector 102A outputs a corresponding signal that is converted to a
digital value and recorded.
[0187] Next, as shown in FIG. 16, the fourth pair of LEDs 100D is
activated and the LEDs generate an excitation beam that passes
through the pair of 527 nm low pass filters 206, reflects off of
the mirror 210, reflects off of the 495 nm high pass reflector 208,
passes through the 527 nm high pass reflector 209, and passes
through the lens 215 into the reaction chamber 17. The excitation
beam from LEDs 100D is thus filtered to a wavelength range of 495
to 527 nm corresponding to the peak excitation range for TET. As
shown in FIG. 18, emitted light from the chamber 17 then passes
through the lens 232 of the detection assembly 70 and strikes the
565 nm low pass reflector 229. The portion of the light having a
wavelength in the range of about 537 to 565 nm (corresponding to
the peak emission wavelength range of TET) reflects from the 565 nm
low pass reflector 229, reflects from the 537 nm high pass
reflector 230, passes through the pair of 537 nm high pass filters
224, through the lens 242, through the 550 nm Schott Glass.RTM.
filter 222B, and is detected by the second detector 102B. The
second detector 102B outputs a corresponding signal that is
converted to a digital value and recorded. The total time required
to activate each of the four LEDs 100A, 100B, 100C, 100D in
sequence and to collect four corresponding measurements from the
detectors 102A, 102B, 102C, 102D is typically five seconds or
less.
[0188] The spectrum of the fluorescence that is emitted by the dyes
used for detection is usually broad. As a result, when an
individual dye (e.g., FAM, TAMRA, TET, or ROX) emits fluorescence
from the reaction vessel 12, the fluorescence can be detected in
several of the primary detection channels, i.e. several of the
detectors 102A, 102B, 102C, and 102D detect the fluorescence.
However, each dye has its own `signature`, i.e., the ratios of the
optical signals in each detection channel are unique to each dye.
It is also a reasonable assumption that the fluorescent emission
from a mixture of dyes are simply additive in each of the detection
channels, so that the individual dye signals of a dye mixture can
be extracted from the mixed signals using linear algebra.
[0189] In the preferred embodiment, the controller is programmed to
convert the output signals of the detectors to values indicating
the true signal from each dye in a reaction mixture using linear
algebra and a calibration matrix. A preferred method for developing
the calibration matrix will now be described using the four-channel
optical system of the preferred embodiment as an example. First, a
reaction vessel containing only reaction buffer is optically read
using optics assemblies 68, 70. The reaction buffer should be a
fluid similar or nearly identical to the reaction mixtures that
will be optically read by the optics assemblies during production
use of the system to test samples. The reaction buffer should
contain no dyes, so that the concentrations of all dyes are zero.
The optical reading of the reaction buffer in the four primary
detection channels produces four output signals that are converted
to corresponding digital values. These four numbers are called
Buffer(I), where `I` is 1, 2, 3 or 4 depending upon which detection
channel is read. The buffer values are a measure of the background
signal or scattered light detected in each primary detection
channel without any added fluorescent signal from dyes.
[0190] Next, a reaction mixture containing a known concentration
(e.g., 100 nM) of dye #1 is placed into the vessel and again the
four channels are read. The four numbers produced are called
Rawdye(I, 1). Similar sets of four numbers are obtained for the
other three dyes to obtain Rawdye(I, 2), Rawdye(I, 3), and
Rawdye(I, 4). The buffer values are then subtracted from the raw
dye values to obtain net dye values as follows: Netdye(I,
J)=Rawdye(I, J)-Buffer (I); where I indicates the detection
channel, and J indicates the dye number.
[0191] The matrix Netdye(I, J) is then inverted using standard
numerical methods (such as Gaussian elimination) to obtain a new
matrix called the calibration matrix Cal(I,J). Note that the matrix
product of Netdye(I, J)*Cal (I,J) is the unity matrix. Now, any
reaction mixture can be read and the raw mixed fluorescent signals
detected and measured by the four detectors may be converted to
values representative of the individual signal emitted by each dye.
The optical reading of the mixture produces four numbers called
RawMix(I). The reaction buffer values are then subtracted from the
raw mix values to obtain four numbers called Mix(I) as follows:
Mix(I)=RawMix(I)-Buffer(I) Next, the true dye signals are obtained
by matrix multiplication as follows: Truedye(I)=100 nM*Cal(I,
J)*Mix(I) In the above equation, the factor of 100 comes from the
fact that a concentration of 100 nM was used for the initial
calibration measurements. The concentration of 100 nM is used for
purposes of example only and is not intended to limit the scope of
the invention. In general, the dye concentrations for calibration
measurements should be somewhere in the range of 25 to 2,000 nM
depending upon the fluorescent efficiency (strength) of the dyes
and their use in a particular assay. When displayed to a user, the
fluorescent signal values may be normalized to an arbitrary scale
having arbitrary units of fluorescent intensity (e.g., a scale
ranging from 0 to 1000 arbitrary units).
[0192] Referring again to FIGS. 22-23, the matrices Cal(I, J) and
Buffer(I) are preferably produced during the manufacture of each
heat-exchanging module 60 and stored in the memory 160. When the
module 60 is plugged into the base instrument 110, the controller
reads the matrices into memory and uses the matrices to deconvolve
the raw fluorescent signals. Because the calibration matrices
Cal(I, J) and Buffer(I) are dependent upon the particular set of
dyes calibrated and the volume of the reaction vessel, it is also
preferred to produce and store multiple sets of the matrices for
various combinations of dye sets and reaction vessel volumes. This
gives the end user greater flexibility in using the system.
[0193] As one example, calibration matrices could be stored for
three different dye sets to be used with three different sizes of
reaction vessels (e.g., 25 .mu.l, 50 .mu.l, 100 .mu.l) for a total
of nine different sets of calibration matrices. Of course, this is
just one example, and many other combinations will be apparent to
one skilled in the art upon reading this description. Further, in
alternative embodiments, the control software may include
functionality to guide the end user through the calibration
procedure to enable the user to store and use calibration data for
his or her own desired combination of dyes and reaction vessel
size.
[0194] In one possible implementation of the four-channel system,
three of the optical channels are used to detect amplified nucleic
acid sequences while the fourth channel is used to monitor an
internal control to check the performance of the system. For
example, beta actin is often used as an internal control in nucleic
acid amplification reactions because it has a predictable
amplification response and can be easily labeled and monitored to
verify that the amplification is occurring properly. In another
possible implementation of the four-channel system, two of the
optical channels are utilized to detect target nucleic acid
sequences, one of the channels is used to monitor an internal
control, and the fourth channel is used to monitor a passive
normalizer. The passive normalizer is a dye that is placed in a
reaction mixture in a known concentration and in a free form so
that it will not label any target nucleic acid sequence. For
example, ROX in a concentration of 100 to 500 nM makes a suitable
passive normalizer. Because the passive normalizer is placed in a
reaction mixture in a free form, the intensity of the fluorescent
signal output by the passive normalizer is substantially unaffected
by the presence or absence of a target nucleic acid sequence in the
reaction mixture. The intensity of the signal does vary, however,
due to such effects as evaporation of the mixture, variances in
reaction vessel shapes, or air bubbles in the vessel. The intensity
of the signal from the passive normalizer is monitored throughout
the reaction and used to normalize the optical signals collected
from the other three detection channels. If the signal from the
passive normalizer changes due to evaporation, variances in
reaction vessel shapes, or air bubbles in the vessel, the signals
received in the other three detection channels are normalized for
these variances.
[0195] Referring again to FIG. 20, the controller 112 stores in
memory the deconvolved fluorescent signal values determined for
each primary detection channel at each reaction site in use. The
signal values are preferably stored in an array indexed by reaction
site, detection channel, and cycle number (or time value for
isothermal amplification). The signal values stored for a
particular detection channel at a particular site define a growth
curve for a target nucleic acid sequence being amplified at that
site and detected in that channel.
[0196] FIG. 24A shows a typical growth curve for a nucleic acid
sequence being amplified in a thermal cycling reaction (e.g., PCR).
The growth curve shows fluorescent intensity (and hence the
relative quantity or concentration of the nucleic acid sequence) as
a function of cycle number in the reaction. As the reaction
proceeds, the concentration of detectable fluorescent dye
increases. In a typical reaction, every cycle of PCR results in a
doubling of product. As the reactants start to become depleted, the
reaction shifts from two-fold logarithmic growth to linear growth,
and eventually with additional cycles, a plateau is reached. The
plateau region can vary greatly from reaction to reaction, and
conventional endpoint measurements used for quantitative analysis
have very poor reproducibility. However, product accumulation in
the log phase is typically uniform. In order to perform
quantitative PCR, a threshold cycle value is determined for each
target nucleic acid sequence being amplified in the test and
calibration samples. It is important that the method used to
determine threshold values give reproducible values. By locating
the threshold value in the log phase of the growth curve, such
reproducibility is achievable.
[0197] In particular, it is presently preferred to calculate a
second derivative (with respect to cycle number) of the growth
curve and to calculate the threshold cycle number as the location,
in cycles, of the positive peak of the second derivative. For
example, FIG. 24A shows a threshold cycle number of 30.93 at the
positive peak of the second derivative. The method of the present
invention may also be applied to isothermal nucleic acid
amplification reactions. FIG. 24B shows a typical growth curve for
a nucleic acid sequence being amplified in an isothermal reaction
(e.g., Rolling Circle Amplification). The growth curve shows
fluorescent intensity (and hence the relative quantity of the
nucleic acid sequence) as a function of amplification time. A
second derivative (with respect to time) of the growth curve is
calculated, and a threshold time value of 15.47 minutes has been
calculated at the positive peak of the second derivative. In
alternative embodiments, characteristics other than the positive
peak of the second derivative may be used to determine threshold
values. For example, the threshold value may be calculated from the
location of the negative peak of the second derivative, the
zero-crossing of the second derivative, or the positive peak of the
first derivative. These embodiments are described in greater detail
below.
[0198] FIG. 25 is a flow chart showing the preferred method steps
executed by the controller to determine a threshold value (e.g., a
threshold cycle number or threshold time value) for a nucleic acid
sequence in a test or calibration sample. The threshold value is
determined from the deconvolved fluorescent signal values
calculated for the specific reaction site and detection channel at
which the growth of the nucleic acid sequence was measured. In
optional steps 302 and 304, the signal values are preprocessed
prior to threshold calculation. In optional step 302, boxcar
averaging is performed on the signal values as follows. The average
of the set of values {n, n-1, . . . , n+1-k} is used as the value
for cycle n. For example, if k=2, then the data from cycles 4 and 5
are averaged and used as the data for cycle 5. In optional step
304, background subtraction is performed on the signal values.
[0199] FIGS. 26-27 illustrate background subtraction. Using a least
squares algorithm, a line y=mX+b (where y is the signal value, m is
the slope, X is the cycle number and b is the intercept) is fit to
the signal values recorded for cycles M to N (preferably cycles 3
to 8). M and N are integers selected by the user. The equation of
the line is used to decrease each signal value recorded for a cycle
number by the value of the fitted line corresponding to the cycle
number, according to the equation: Optic(X)=Optic(X)-[mX+b] where X
is equal to the cycle number, Optic(X) is equal to the signal value
at cycle number X, and m and b are fitted parameters of the line.
The effect of the background subtraction is to subtract the
baseline signal and its linear drift from the signal values.
[0200] In step 306, second derivative data points are calculated
from the signal values. Preferred methods for calculating the
second derivative data points will now be described with reference
to FIGS. 28A-28C. FIG. 28A shows a segment of a growth curve
defined by five consecutive signal values {Optic.sub.(X-4),
Optic.sub.(X-3), . . . , Optic.sub.(X)} where x is equal to the
cycle number (or measurement time point in isothermal
amplification) at which the signal was measured. Thus,
Optic.sub.(X-2) is equal to the signal value two cycles prior to
cycle number x. The controller preferably calculates the second
derivative (with respect to x) of the growth curve at point
Optic.sub.(X-2) using equation (1):
2ndDeriv.sub.(X-2)=[Optic.sub.(X)-2*Optic.sub.(X-2)+Optic.sub.(X-4)]*k;
(1) where k is equal to a constant multiplier (e.g., 5). The
purpose of the constant multiplier is to make the second derivative
curve (FIG. 24A) appear taller when displayed to the user. Neither
the constant multiplier nor the displaying of the primary or second
derivative curves are necessary to practice the invention and may
be omitted in alternative embodiments.
[0201] The derivation of equation (1) will now be explained with
reference to FIG. 28A. The second derivative of the growth curve at
point Optic.sub.(X-2) is given by equation (2):
2ndDeriv.sub.(X-2)=[1stDeriv.sub.(X-1)-1stDeriv.sub.(X-3)]/2; (2)
The first derivative of the growth curve at point Optic.sub.(X-1)
is given by equation (3):
1stDeriv.sub.(X-1)=[Optic.sub.(X)-Optics.sub.(X-2)]/2; (3) In
addition, the first derivative of the growth curve at point
Optic.sub.(X-3) is given by equation (4):
1stDeriv.sub.(X-3)=[Optic.sub.(X-2)-Optic.sub.(X-4)]/2; (4)
Combining equations (2), (3), and (4) and multiplying by the
constant multiplier k yields equation (1).
[0202] Equation (1) may be used to calculate the second derivative
of the growth curve at any point on the curve for which the two
prior and two subsequent signal values are known. This is not
possible, however, for the last two signal values on the growth
curve. Therefore, different equations are necessary for second
derivative calculations for these points.
[0203] Referring to FIG. 28B, the second derivative of the growth
curve at point Optic.sub.(X-1) is given by equation (5):
2ndDeriv.sub.(X-1)=[Optic(x)-Optic.sub.(X-1)-1stDeriv.sub.(X-2)]/2;
(5)
[0204] The first derivative of the growth curve at point
Optic.sub.(X-2) is given by equation (6):
1stDeriv.sub.(X-2)=[Optic.sub.(X-1)-Optics.sub.(X-3)]/2; (6)
Combining equations (5) and (6) and multiplying by the constant
multiplier k yields equation (7):
2ndDeriv.sub.(X-1)=[2*Optic(x)-3*Optic.sub.(X-1)+Optic.sub.(X-3)]*k;
(7)
[0205] Equation (7) may be used to calculate the second derivative
of the growth curve at any point for which at least two previous
and one subsequent signal values are known. If no subsequent signal
value is known, then the second derivative may be calculated at a
point Optic(x) using another equation which will now be described
with reference to FIG. 28C. Specifically, the second derivative of
the growth curve at point Optic(x) is given by equation (8):
2ndDeriv.sub.(X)=[Optic.sub.(X)-Optic.sub.(X-1)]-1stDeriv.sub.(X-1);
(8)
[0206] The first derivative of the growth curve at point
Optic.sub.(X-1) is given by equation (3):
1stDeriv.sub.(X-1)=[Optic.sub.(X)-Optic.sub.(X-2)]/2; (3) Combining
equations (3) and (8) and multiplying by the constant multiplier k
yields equation (9):
2ndDeriv.sub.(X)=[Optic.sub.(X)-2*Optic.sub.(X-1)+Optic.sub.(X-2)]*2*k;
(9)
[0207] In the preferred embodiment, the controller displays the
growth curve and the second derivative of the growth curve to the
user in real-time on a graphical user interface. When a new
fluorescent signal value Optic(x) is received, the controller
calculates a second derivative of the growth curve at Optic(x)
using equation (9). When a subsequent signal value Optic.sub.(X+1)
is received, the controller recalculates the second derivative of
the growth curve at Optic(x) using equation (7). When another
signal value Optic.sub.(X+2) is received, the controller
recalculates the second derivative of the growth curve at Optic(x)
using equation (1). Thus, previously calculated second derivative
values are updated as new signals are measured. Although this
dynamic updating of second derivative values is useful for
real-time display, dynamic updating is not necessary to practice
the invention. For example, all signal values for an amplification
reaction may be recorded before calculating second derivative
values, and the second derivative values may be calculated using
just one equation rather than three.
[0208] In step 308, the controller calculates a noise-based
threshold level for the positive peak of the second derivative to
exceed. FIG. 29 is a flow chart showing the steps executed by the
controller to calculate the threshold level. For each primary
detection channel at each reaction site in use, the controller
calculates the standard deviation of the second derivative values
calculated for the detection channel for cycles M to N (preferably
cycles 3 to 8). The controller next sets the threshold level for
each detection channel equal to R times the maximum standard
deviation calculated for the channel. For example, assume that 8
sites are in use (labeled A1-A8) and the FAM channel is used to
detect and measure the growth of a target nucleic acid sequence at
each of the sites. For each site, based on the deconvolved signal
values calculated for the FAM channel for cycles M to N, the
controller calculates second derivative data points for cycles M to
N. The controller also calculates the standard deviation of the
second derivative data points and sets the threshold level for each
FAM channel equal to R times the largest standard deviation found.
Thus, the reaction site whose FAM channel has the largest standard
deviation in the values of its second derivative data points for
cycles M to N is used to set the threshold level for all FAM
channels in the batch. M, N, and R are preferably user-defined
integers. Reasonable values for M and N are 3 and 8, respectively.
The value of R is preferably in the range of 3 to 10, with a
preferred value of 5. Although it is presently preferred to
calculate an automatic, noise-based threshold level in this manner,
the threshold level may also be set manually by the user.
[0209] Referring again to FIG. 25, in step 310, the controller
detects a positive peak of the second derivative. The positive peak
is preferably detected using at least three second derivative data
points calculated at cycles X, X-1, an X-2. A positive peak is
detected if the second derivative value at cycle X is less than the
second derivative value at cycle X-1 and if the second derivative
value at cycle X-1 is greater than the second derivative value at
cycle X-2. After a peak is detected, a second order curve is fit to
the three second derivative data points, step 312. In decision step
314, it is determined if the height of the peak of the second order
curve exceeds the threshold level calculated in step 308. If the
peak of the second order curve does not exceed the threshold level,
the controller returns to step 310 and looks for the next positive
peak in the second derivative. If the peak of the second order
curve does exceed the threshold level, the controller proceeds to
step 316. In step 316, the controller calculates the threshold
value (e.g., the threshold cycle number in thermal cycling
amplification or time value in isothermal amplification) as the
location of the peak of the second order curve.
[0210] FIG. 30 illustrates the fitting of a second order curve to
the three second derivative data points used to detect a peak in
the second derivative. To locate the true maximum of the second
derivative peak and the cycle number (or time value) at which it
occurs, the controller executes a peak finding algorithm. Let (X1,
Y1), (X2, Y2), and (X3, Y3) be the three second derivative data
points for the analysis. Y2 is the value of the highest point, and
X2 is the cycle (or time of amplification) where that point has
occurred. In addition, X1=X2-1 and X3=X2+1. FIG. 31 illustrates the
steps executed to calculate the height of the peak of the second
order curve and the cycle number of the peak. In steps 400-406,
four determinants are calculated using the x-y values of the three
second derivative data points. In steps 408-412, three ratios R1,
R2, R3 are calculated from the determinants. In step 414, the
threshold value (which may be fractional) is calculated from the
ratios. In step 416, the height of the peak of the second order
curve is calculated using the formula shown.
[0211] FIGS. 32A-32B illustrate another embodiment of the invention
in which the zero-crossing of the second derivative of the growth
curve is used to calculate the threshold value (e.g., cycle number
or time value). FIG. 33 is a flow chart illustrating the steps for
calculating the threshold value for a target nucleic acid sequence
according to the second embodiment. In step 308, the controller
calculates a minimum, noise-based threshold level for the growth
curve to exceed. FIG. 34 is a flow chart showing the steps executed
by the controller to calculate the threshold level. For each
primary detection channel at each reaction site in use, the
controller calculates the standard deviation of the deconvolved
signal values calculated for the detection channel for cycles M to
N (preferably cycles 3 to 8). The controller next sets the
threshold level for each detection channel equal to R times the
maximum standard deviation calculated for the channel. For example,
assume that 8 sites are in use (labeled A1-A8) and the FAM channel
is used to detect and measure the growth of a target nucleic acid
sequence at each of the sites. For each site, the controller
calculates the standard deviation of the FAM channel signal values
for cycles M to N and sets the threshold level for each FAM channel
equal to R times the largest standard deviation found. M, N, and R
are preferably user-defined integers. Reasonable values for M and N
are 3 and 8, respectively. The value of R is preferably in the
range of 3 to 10, with a preferred value of 5. Although this
automatic, noise-based threshold level is presently preferred, the
threshold level may also be set manually by a user.
[0212] Referring again to FIG. 33, in step 454, the controller
calculates a second derivative of the growth curve. Preferably, the
controller calculates a plurality of second derivative data points
as previously described with reference to FIGS. 28A-28C. In step
456, the controller identifies a zero-crossing of the second
derivative. A zero-crossing is detected if the second derivative
value at cycle X is less than zero and the second derivative value
at cycle X-1 is greater than zero. In decision step 458, it is
determined if the deconvolved signal value at cycle X exceeds the
threshold level calculated in step 452. If the signal value does
not exceed the threshold level, the controller returns to step 456
and looks for the next zero-crossing of the second derivative. If
the signal value does exceed the threshold level, the controller
proceeds to step 460. In step 460, the controller calculates the
threshold value (e.g., the threshold cycle number in thermal
cycling amplification or time value in isothermal amplification) as
the location of the zero-crossing of the second derivative curve.
The threshold value may be calculated by linear interpolation
between the second derivative data points at cycle X and X-1.
[0213] FIGS. 35A-35B illustrate a third embodiment of the invention
in which a negative peak of the second derivative of the growth
curve is used to calculate the threshold value (e.g., cycle number
or time value). FIG. 36 is a flow chart illustrating the steps for
calculating the threshold value for a target nucleic acid sequence
according to the third embodiment. In step 502, the controller
calculates a minimum, noise-based threshold level for the growth
curve to exceed, as previously described with reference to FIG. 34.
In step 504, the controller calculates a second derivative of the
growth curve. Preferably, the controller calculates a plurality of
second derivative data points as previously described with
reference to FIGS. 28A-28C. In step 506, the controller identifies
a negative peak of the second derivative. A negative peak is
detected if the second derivative value at cycle X is less than
zero, the second derivative value at cycle X is greater than the
second derivative value at cycle X-1, and the second derivative
value at cycle X-1 is less than the second derivative value at
cycle X-2. In decision step 508, it is determined if the
deconvolved signal value at cycle X exceeds the threshold level
calculated in step 502. If the signal value does not exceed the
threshold level, the controller returns to step 506 and looks for
the next negative peak of the second derivative. If the signal
value does exceed the threshold level, the controller fits a second
order curve to the second derivative data points at cycles X, X-1,
and X-2, step 510. In step 512, the controller calculates the
threshold value (e.g., the threshold cycle number in thermal
cycling amplification or time value in isothermal amplification) as
the location of the negative peak (minimum value) of the fitted
second order curve.
[0214] FIGS. 37A-37B illustrate another embodiment of the invention
in which the threshold value is calculated as the cycle number or
time value associated with the positive peak of the first
derivative of the growth curve. It should be noted that the
positive peak of the first derivative is mathematically equivalent
to the zero-crossing of the second derivative in terms of the
x-location, although the y-location will vary between the two. FIG.
38 is a flow chart showing the preferred method steps executed by
the controller to determine a threshold value for a nucleic acid
sequence using the positive peak (maximum) of the first derivative
of the growth curve. In step 602, first derivative data points are
calculated from the deconvolved signal values calculated for the
nucleic acid sequence.
[0215] Preferred methods for calculating the first derivative data
points will now be described with reference to FIG. 28A. FIG. 28A
shows a segment of a growth curve defined by five consecutive
signal values {Optic.sub.(X-4), Optic.sub.(X-3), . . . ,
Optic.sub.(x)} where X is equal to the cycle number (or measurement
time point in isothermal amplification) at which the signal was
measured. Thus, Optic.sub.(X-2) is equal to the signal value two
cycles prior to cycle number X. The controller preferably
calculates the first derivative (with respect to x) of the growth
curve at point Optic.sub.(X-1) using equation (3):
1stDeriv.sub.(X-1)=[Optic.sub.(X)-Optics.sub.(X-2)]/2; (3)
[0216] Equation (3) may be used to calculate the first derivative
of the growth curve at any point on the curve for which at least
one prior and one subsequent signal value is known. This is not
possible, however, for the last signal value on the growth curve.
Therefore, a different equation is necessary to calculate a first
derivative value at the last point. Still referring to FIG. 28A,
the first derivative of the growth curve at point Optic(X) is
preferably calculated using equation (10):
1stDeriv.sub.(X)=Optic.sub.(X)-Optics.sub.(X-1); (10)
[0217] The controller preferably displays the growth curve and the
first derivative of the growth curve to the user in real-time on a
graphical user interface. When a new fluorescent signal value
Optic(x) is received, the controller calculates a first derivative
of the growth curve at Optic(x) using equation (10). When a
subsequent signal value Optic.sub.(X+1) is received, the controller
recalculates the first derivative of the growth curve at Optic(x)
using equation (3). Thus, previously calculated first derivative
values are updated as new signals are measured.
[0218] In step 604, the controller calculates a noise-based
threshold level for the positive peak of the first derivative to
exceed. FIG. 39 is a flow chart showing the steps executed by the
controller to calculate the threshold level. For each primary
detection channel at each reaction site in use, the controller
calculates the standard deviation of the first derivative values
calculated for the detection channel for cycles M to N (e.g.,
cycles 3 to 8). The controller next sets the threshold level for
each detection channel equal to R times the maximum standard
deviation calculated for the channel. For example, assume that 8
sites are in use (labeled A1-A8) and the FAM channel is used to
detect and measure the growth of a target nucleic acid sequence at
each of the sites. For each site, based on the deconvolved signal
values calculated for the FAM channel for cycles M to N, the
controller calculates first derivative data points for cycles M to
N. The controller also calculates the standard deviation of the
first derivative data points and sets the threshold level for each
FAM channel equal to R times the largest standard deviation found.
Thus, the reaction site whose FAM channel has the largest standard
deviation in the values of its first derivative data points for
cycles M to N is used to set the threshold level for all FAM
channels in the batch. M, N, and R are preferably user-defined
integers. Reasonable default values for M and N are 3 and 8,
respectively. A preferred default value for R is 5. Although it is
presently preferred to calculate an automatic, noise-based
threshold level in this manner, the threshold level may also be set
manually by the user.
[0219] Referring again to FIG. 38, in step 606, the controller
detects a positive peak of the first derivative. The positive peak
is preferably detected using at least three first derivative data
points calculated at X, X-1, an X-2, where X is equal to the cycle
number (or time point of measurement for isothermal amplification).
A positive peak is detected if the first derivative value at cycle
X is less than the first derivative value at cycle X-1 and if the
first derivative value at cycle X-1 is greater than the first
derivative value at cycle X-2. After a peak is detected, a second
order curve is fit to the three first derivative data points, step
608. In decision step 610, it is determined if the height of the
peak of the second order curve exceeds the threshold level
calculated in step 604. If the peak of the second order curve does
not exceed the threshold level, the controller returns to step 606
and looks for the next positive peak in the first derivative. If
the peak of the second order curve does exceed the threshold level,
the controller proceeds to step 612. In step 612, the controller
calculates the threshold value (e.g., the threshold cycle number in
thermal cycling amplification or time value in isothermal
amplification) as the location of the peak of the second order
curve. The location and height of the peak of the second order
curve may be calculated using the algorithm previously described
with reference to FIGS. 30-31.
[0220] Referring again to FIG. 20, the controller 112 is programmed
to calculate and store in memory a respective threshold value for
each target nucleic acid sequence that is amplified in each of the
reaction vessels 12. The threshold values may be calculated using
any of the four methods just described. Next, the controller
derives a calibration curve using the threshold values determined
for the known starting quantities of the calibration nucleic acid
sequence in the calibration samples. The calibration curve relates
the threshold value to the log of the starting quantity of the
nucleic acid sequence. To determine the unknown starting quantity
of the target nucleic acid sequence in the test sample, the
threshold value determined for the target sequence in the test
sample is entered into the equation of the calibration curve and
the equation returns a value that is the starting quantity of the
target nucleic acid sequence in the test sample.
[0221] The following three examples of operation demonstrate
various different methods for using threshold cycle values to
determine the unknown starting quantity of a target nucleic acid
sequence in a test sample according to the present invention.
EXAMPLE 1
External Standards
[0222] Referring to FIG. 20, test samples and calibration samples
are amplified in separate reaction vessels 12 at separate reaction
sites. In this example, there are sixteen heat-exchanging modules
60 (arranged in two rows of eight). Each heat-exchanging module
provides a reaction site for amplifying a sample contained in a
reaction vessel. The eight heat-exchanging modules in the first row
are designated reaction sites A1-A8 and the eight heat-exchanging
modules in the second row are designated reaction sites B1-B8.
Eight calibration samples (standards) are amplified at sites A1-A8
and eight test samples are amplified at sites B1-B8. Each test
sample is mixed with the necessary reagents and fluorescent probes
to amplify and detect up to three different target nucleic acid
sequences. Each calibration sample contains a known starting
quantity of three calibration nucleic acid sequences corresponding
to the three target nucleic acid sequences in the test samples.
Each calibration nucleic acid sequence is preferably the same or
similar to a respective one of the target nucleic acid sequences in
the test samples.
[0223] FIG. 40 shows a schematic representation of a setup table
that appears on a graphical user interface of the controller. Prior
to amplifying and detecting the nucleic acid sequences in the test
and calibration samples, the user enters in the setup table the
known starting quantity of each calibration nucleic acid sequence
in each calibration sample, as well as the specific dye (e.g., FAM,
TET, TAM, or ROX) used to label each nucleic acid sequence. For
example, at site A1, the user has specified 1,000 starting copies
of a first calibration nucleic acid sequence to be labeled with
FAM, 100 starting copies of a second nucleic acid sequence to be
labeled with TET, and 10 starting copies of a third nucleic acid
sequence to be labeled with TAM. The nucleic acid sequences in the
test and calibration samples are then amplified and a threshold
value (e.g., cycle number or time value) is determined for each
nucleic acid sequence using any of the four methods previously
described.
[0224] FIG. 41 is a table showing the threshold values computed by
the controller for each of the three nucleic acid sequences
(labeled with FAM, TET, and TAM, respectively) in each of the
calibration samples. From the data in the table, the controller
computes the average threshold value for each calibration nucleic
acid sequence at each starting quantity, as shown in FIG. 42. As
shown in FIG. 43, the controller next generates three calibration
curves, one for each calibration nucleic acid sequence. Each
calibration curve relates threshold value to the log of the
starting quantity of a nucleic acid sequence. Each calibration
curve is preferably generated using a least squares algorithm to
fit a line to the data points.
[0225] To determine the unknown starting quantity of each of the
three target nucleic acid sequences in a test sample, a respective
threshold value is determined for each target sequence. The
threshold value is then entered into the equation of the
corresponding calibration curve and the equation returns a value
that is the starting quantity of the target nucleic acid sequence
in the test sample. For example, FIG. 44 shows the results
determined for one of the test samples. The first target nucleic
acid sequence in the test sample (labeled with FAM) had a threshold
value of 29 corresponding to a starting quantity of 251 copies, the
second target nucleic acid sequence in the test sample (labeled
with TET) had a threshold value of 29 corresponding to a starting
quantity of 46 copies, and the third target nucleic acid sequence
in the test sample (labeled with TAMRA) had a threshold value of 24
corresponding to a starting quantity of 464 copies.
EXAMPLE 2
Quantitative Internal Controls
[0226] This example is similar to example 1, except that in example
2 each threshold value determined for a nucleic acid sequence is
normalized by the threshold value determined for a quantitative
internal control. Referring to FIG. 20, test samples and
calibration samples are amplified in separate reaction vessels 12
at separate reaction sites. Eight calibration samples (standards)
are amplified at sites A1-A8 and eight test samples are amplified
at sites B1-B8. Each test sample is mixed with the necessary
reagents and fluorescent probes to amplify and detect up to two
different target nucleic acid sequences. Each calibration sample
contains a known starting quantity of two different calibration
nucleic acid sequences corresponding to the two target nucleic acid
sequences in the test samples. In addition, a known quantity of a
quantitative internal control (QIC) is placed in each test and
calibration sample. The quantitative internal control is a nucleic
acid sequence different than the calibration and target nucleic
acid sequences in the samples and is used to normalize the
threshold values determined for the target and calibration
sequences. Suitable nucleic acid sequences to be used as a QIC
include, e.g., beta-actin, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), or any synthetic, amplifiable target. The same starting
quantity of the QIC is placed in each test and calibration sample.
The starting quantity of the QIC placed in each sample is
preferably in the range of about 10 to 1,000 copies, with a
preferred starting quantity of about 100 copies.
[0227] FIG. 45 shows a schematic representation of a setup table
that appears on a graphical user interface of the controller. Prior
to amplifying and detecting the nucleic acid sequences in the test
and calibration samples, the user enters in the setup table the
known starting quantity of each calibration nucleic acid sequence
in each calibration sample, as well as the specific dye (e.g., FAM,
TET, TAM, or ROX) used to label each nucleic acid sequence. For
example, at site A1, the user has specified 1,000 starting copies
of a first calibration nucleic acid sequence to be labeled with
FAM, and 100 starting copies of a second nucleic acid sequence to
be labeled with TET. In this example, TAM is the dye used to label
the QIC and the user is therefore prevented from entering values
for TAM in the standards column. The nucleic acid sequences in the
test and calibration samples (each containing the same starting
quantity of a QIC) are then amplified and a threshold value is
determined for each nucleic acid sequence, preferably using any of
the four methods previously described. Alternatively, threshold
values may be determined using any of the methods known in the
art.
[0228] FIG. 46 is a table showing the threshold values computed by
the controller for each of the two calibration nucleic acid
sequences (labeled with FAM and TET) and of the QIC (labeled with
TAM) in each of the calibration samples. As shown in the table of
FIG. 47, the threshold values for each calibration nucleic acid
sequence are normalized to the corresponding QIC by dividing the
threshold values of the calibration sequences by the threshold
values of the QIC. The controller next computes the average
normalized threshold value for each calibration nucleic acid
sequence at each starting quantity, as shown in FIG. 48. As shown
in FIG. 49, the controller derives two calibration curves, one for
each calibration nucleic acid sequence. Each calibration curve
relates normalized threshold value to the log of the starting
quantity of a nucleic acid sequence. Each calibration curve is
preferably generated using a least squares algorithm to fit a line
to the data points.
[0229] Referring to FIG. 50, to determine the unknown starting
quantity of each of the two target nucleic acid sequences in a test
sample, a respective threshold value is determined for each target
sequence and for the QIC amplified in the same reaction with the
target sequences. The threshold values determined for the target
sequences are then divided by the threshold value determined for
the QIC to normalize the threshold values to the QIC. Each
normalized threshold value is then entered into the equation of the
corresponding calibration curve and the equation returns a value
that is the starting quantity of the target nucleic acid sequence
in the test sample. For example, FIG. 50 shows the results
determined for one of the test samples. The first target nucleic
acid sequence in the test sample (labeled with FAM) has a
normalized threshold value of 1.006944 corresponding to a starting
quantity of 210 copies, and the second target nucleic acid sequence
in the test sample (labeled with TET) has a normalized threshold
value of 1.041667 corresponding to a starting quantity of 21
copies.
EXAMPLE 3
Internal Standards
[0230] In this example, the calibration nucleic acid sequences
(standards) are amplified together in the same reaction vessel with
the unknown quantity of a target nucleic acid sequence in a test
sample. Referring to FIG. 20, eight reaction vessels containing
reaction mixtures are placed at sites A1-A8. The reaction mixture
in each vessel comprises (1) a test sample mixed with the necessary
reagents and fluorescent probes to amplify and detect a target
nucleic acid sequence in the test sample; (2) a first internal
standard comprising a known quantity of a second nucleic acid
sequence different than the target sequence in the test sample, as
well as the necessary reagents and probes to amplify and detect the
second nucleic acid sequence; and (3) a second internal standard
comprising a known quantity of a third nucleic acid sequence
different than the target sequence in the test sample and the
second nucleic acid sequence, as well as the necessary reagents and
probes to amplify and detect the third nucleic acid sequence.
[0231] FIG. 51 shows a schematic representation of a setup table
that appears on a graphical user interface of the controller. Prior
to amplifying and detecting the nucleic acid sequences in the
reaction mixtures, the user enters in the setup table the known
starting quantity of the second and third nucleic acid sequences
(the internal standards) in each reaction mixture, as well as the
specific dye (e.g., FAM, TET, TAM, or ROX) used to label each
nucleic acid sequence. For example, at site Al, the user has
specified 100 starting copies of the first internal standard to be
labeled with TET, and 1000 starting copies of the second internal
standard to be labeled with TAM. In this example, FAM is the dye
used to label the target nucleic acid sequence in the test sample
and the user is therefore prevented from entering starting copy
numbers for FAM in the standards column. The nucleic acid sequences
in the reaction mixtures (each containing an unknown quantity of a
target sequence and known starting quantities of two internal
standards) are then amplified and a threshold value is determined
for each nucleic acid sequence, preferably using any of the four
methods previously described. Alternatively, threshold values may
be determined using any of the methods known in the art.
[0232] FIG. 52 is a table showing the threshold values computed by
the controller for the target sequence and first and second
standards at each reaction site. Next, a calibration curve is
generated for each individual site based on the threshold values
determined for the two internal standards. For example, FIG. 54
shows the calibration curve generated for site A2. The threshold
values and known starting quantities of the two internal standards
provide two data points to which a calibration line is fit. To
determine the unknown starting quantity of the target nucleic acid
sequence in the test sample amplified at site A2, the threshold
value determined for the target sequence is then entered into the
equation of the calibration curve and the equation returns a value
that is the starting quantity of the target nucleic acid sequence
in the test sample. For example, if the target sequence is
determined to have a threshold value of 29.9. then the starting
quantity is calculated as 124.81 copies.
[0233] One advantage to using internal standards is that a
calibration curve is developed based only on the reaction in which
the unknown quantity of the target nucleic acid sequence is being
amplified. Consequently, the method reduces problems arising from
the variability between reactions occurring in different reaction
vessels. Another advantage of the method is that it reduces the
number of reaction sites and the amount of expensive reagents
required to perform an assay.
Summary, Ramifications, and Scope
[0234] Although the above description contains many specificities,
it is to be understood that many different modifications or
substitutions may be made to the methods, apparatus, and computer
program products described without departing from the broad scope
of the invention. For example, the means for amplifying the test or
calibration samples need not be the specialized thermal cycler
described herein. The means for amplifying the test and calibration
samples may comprise a metal block having a plurality of wells for
receiving the samples. Alternatively, the means for amplifying the
test and calibration samples may comprise a forced air system for
heating and cooling samples contained in capillary tubes. These and
other apparatuses for amplifying and detecting nucleic acid are
known in the art.
[0235] Moreover, the controller for controlling the operation of
the apparatus may be a personal or network computer linked to the
heat-exchanger or may comprise a microprocessor and memory built
into the heat-exchanging instrument. The computer program product
(e.g., software) readable by the controller may comprise a storage
medium (e.g., a disk) embodying the program instructions.
Alternatively, the computer program product may be an electronic
file stored in the memory of the controller or downloadable to the
controller. Further, the specialized reaction vessels described
above are preferred, but the apparatus and methods of the present
invention are applicable to any type of vessel including plastic
reaction tubes, glass capillary tubes, microtiter plates,
cartridges or cuvettes, etc.
[0236] In addition, the threshold value (e.g., cycle number or time
value) determined using the methods of the present invention has
other uses besides quantitation of an unknown quantity of a nucleic
acid sequence. For example, the threshold value may be used to
determine an optimal termination point for a nucleic acid
amplification reaction so that the reaction may be terminated prior
to reaching the plateau phase to prevent degradation of amplicons
and/or accumulation of undesired products (e.g., primer
dimers).
[0237] Further, the mathematical methods described above for
calculating derivatives and threshold criteria are examples only
and other methods may be used to obtain similar data. For example,
one could fit a mathematical function as an approximation to an
entire growth curve and then calculate derivatives based on that
function. Moreover, the terminology in the claims related to the
steps of deriving growth curves, calculating derivatives, deriving
calibration curves, and/or fitting curves to data points is
intended to include the processing of data (e.g., x-y data) and
variables internal to a processing unit (e.g., a computer)
containing memory and is not limited to the physical acts of
printing, plotting, or displaying lines, curves, or graphs.
[0238] Therefore, the scope of the invention should be determined
by the following claims and their legal equivalents.
* * * * *