U.S. patent application number 10/335690 was filed with the patent office on 2004-04-01 for single-tube, ready-to-use assay kits, and methods using same.
This patent application is currently assigned to Applera Corporation. Invention is credited to Eddins, Susan K., Gilbert, Dennis A., Hunkapiller, Michael W., Livak, Kenneth J., Lucero, Michael, Stevens, Junko.
Application Number | 20040063109 10/335690 |
Document ID | / |
Family ID | 27671405 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040063109 |
Kind Code |
A2 |
Hunkapiller, Michael W. ; et
al. |
April 1, 2004 |
SINGLE-TUBE, READY-TO-USE ASSAY KITS, AND METHODS USING SAME
Abstract
An assay kit is provided that includes assay reagents stored in
a single-tube container, and a data storage medium containing
information about the contents of the container. Methods are
provided for using the data provided with the kit to direct
instruments and/or processes, for example, to control an instrument
to perform amplification and/or sequencing reactions.
Inventors: |
Hunkapiller, Michael W.;
(San Carlos, CA) ; Gilbert, Dennis A.; (San
Francisco, CA) ; Livak, Kenneth J.; (San Jose,
CA) ; Stevens, Junko; (Menlo Park, CA) ;
Eddins, Susan K.; (San Diego, CA) ; Lucero,
Michael; (South San Francisco, CA) |
Correspondence
Address: |
KILYK & BOWERSOX, P.L.L.C.
3603 CHAIN BRIDGE ROAD
SUITE #
FAIRFAX
VA
22030
US
|
Assignee: |
Applera Corporation
850 Lincoln Centre Drive
Foster City
CA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0175774 A1 |
September 18, 2003 |
|
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Family ID: |
27671405 |
Appl. No.: |
10/335690 |
Filed: |
January 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60/352,039 |
Jan 25, 2002 |
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60/352,356 |
Jan 28, 2002 |
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60/369,127 |
Apr 1, 2002 |
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60/369,657 |
Apr 3, 2002 |
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60/370,921 |
Apr 9, 2002 |
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60/376,171 |
Apr 26, 2002 |
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60/380,057 |
May 6, 2002 |
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60/383,627 |
May 28, 2002 |
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60/390,708 |
Jun 21, 2002 |
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60/394,115 |
Jul 5, 2002 |
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60/399,860 |
Jul 31, 2002 |
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Current U.S.
Class: |
435/6.11 ;
702/20 |
Current CPC
Class: |
C12Q 1/6858 20130101;
G16B 30/00 20190201; G16B 50/00 20190201; G06Q 30/0633 20130101;
B01L 3/5453 20130101; G16C 20/60 20190201; G06Q 30/0621 20130101;
G06Q 50/22 20130101; G16B 30/20 20190201; G16H 10/40 20180101; G16B
35/00 20190201; G16B 25/10 20190201; G16B 50/10 20190201; B01L
2300/021 20130101; G16B 25/00 20190201; G01N 35/00663 20130101;
G16B 30/10 20190201; G16B 45/00 20190201; G06Q 30/0641 20130101;
G16B 35/20 20190201; C12Q 2600/156 20130101; B01L 3/545 20130101;
C12Q 1/6858 20130101; C12Q 2547/101 20130101 |
Class at
Publication: |
435/006 ;
702/020 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50 |
Claims
What is Claimed is:
1. An assay kit comprising: a container, the container containing a
mixture having reagents adapted to perform an allelic
discrimination or expression analysis reaction when admixed with at
least one target polynucleotide sequence, and a data storage medium
containing data about the contents of the first container.
2. The assay kit of claim 1, further comprising a second container
that contains assay reagents.
3. The assay kit of claim 1, wherein the container has a
machine-readable label that provides information about the contents
of the container.
4. The assay kit of claim 1, further comprising at least one first
probe, wherein the at least one first probe has a first marker
dye.
5. The assay kit of claim 1, wherein the container is sealed.
6. The assay kit of claim 1, wherein the kit further comprises a
target polynucleotide.
7. The assay kit of claim 1, wherein the reagents utilize
fluorogenic 5' nuclease chemistry during the performance of the
reaction.
8. The assay kit of claim 1, wherein the mixture further comprises
a DNA polymerase, one or more polynucleotide monomers, at least one
salt, and a buffer.
9. The assay kit of claim 8, wherein the polynucleotide monomers
are peptide nucleic acids.
10. The assay kit of claim 1, wherein the container is a tube.
11. The assay kit of claim 5, wherein the sealed container is
sealed with a removable cap.
12. The assay kit of claim 1, wherein the data storage medium is a
compact disk (CD) and the data is electronically formatted.
13. The assay kit of claim 1, wherein the data includes sequence
information about at least one of at least one first probe and at
least one primer.
14. The assay kit of claim 4, wherein the data includes information
about the first marker dye.
15. The assay kit of claim 4, wherein the data includes information
that is capable of controlling at least one scientific laboratory
instrument.
16. The assay kit of claim 15, wherein the at least one instrument
is an instrument designed to perform at least one of a polymerase
chain reaction and a polynucleotide sequencing reaction.
17. The assay kit of claim 15, wherein the data storage medium is a
compact disk (CD) and the data is electronically formatted.
18. The assay kit of claim 1, wherein the data storage medium
contains a software program that can be utilized with data
contained on the data storage medium to control at least one
scientific or laboratory instrument.
19. The assay kit of claim 18, wherein the software program is
adapted to receive output data from at least one scientific or
laboratory instrument.
20. The assay kit of claim 1, further comprising a second data
storage medium, and the second data storage medium contains a
software program that can be utilized to control at least one
scientific or laboratory instrument.
21. The assay kit of claim 20, wherein the software program is
adapted to receive output data from at least one instrument.
22. The assay kit of claim 1, wherein the data storage medium is
separate from the container.
23. The assay kit of claim 1, wherein the data storage medium is
affixed to the container.
24. The assay kit of claim 23, wherein the data storage medium
includes an optically detectable code.
25. The assay kit of claim 23, wherein the data storage medium is
the form of a 2-dimensional barcode.
26. The assay kit of claim 1, wherein the data storage medium is
etched in the container.
27. A method of performing an assay, comprising:providing an assay
kit according to claim 1, using the data to control an instrument
to process the assay reagents, and performing an assay.
28. The method of claim 27, further comprising processing the data
with a software program to control the instrument.
29. A method comprising:providing the assay kit of claim 1;
andshipping the assay kit to a user.
30. The method of claim 29, wherein the assay kit comprises
reagents for conducting a homogeneous assay.
31. A genetic-analysis kit, comprising: a first container; a
reaction mixture held in the first container, the reaction mixture
being adapted for allelic discrimination or expression analysis; a
data storage medium including information in electronic form
thereon, the information pertaining at least in part to the
reaction mixture; and a second container holding the first
container and the data storage medium for transport as a unit.
32. The kit of claim 31, wherein the first container comprises a
tube.
33. The kit of claim 31, wherein the second container comprises a
package.
34. The kit of claim 31, wherein the data storage medium comprises
a disk.
35. The kit of claim 34, wherein the disk comprises a removable
disk.
36. The kit of claim 31, wherein the reaction mixture comprises a
homogeneous reaction mixture.
37. The kit of claim 36, wherein the homogeneous reaction mixture
comprises components for conducting a fluorogenic 5' nuclease
assay.
38. The kit of claim 31, wherein the information is comprised, at
least in part, of American Standard Code for Information
Interchange (ascii)-formatted text.
39. The kit of claim 31, wherein the data storage medium further
includes machine-readable code thereon.
40. A method for facilitating genetic analysis: receiving from a
user a request for one or more genetic-analysis assays; and
providing to the user (i) the one or more assays, with each assay
being provided in a single-tube format, and (ii) information
relating to the one or more assays, with the information being in
electronic form.
41. The method of claim 40, wherein the one or more assays each
comprises a homogeneous reaction mixture.
42. The method of claim 41, wherein the homogeneous reaction
mixture comprises components for conducting a fluorogenic 5'
nuclease assay.
43. The method of claim 40, wherein the information is provided on
a disk.
44. The method of claim 43, wherein the disk is a removable
disk.
45. The method of claim 40, wherein the information is provided via
a computer network.
46. The method of claim 45, wherein the information is transmitted
to the user via electronic mail.
47. The method of claim 45, wherein the information is provided via
a file transfer protocol (FTP).
48. The method of claim 40, wherein the information is comprised,
at least in part, of ascii-formatted text.
49. The method of claim 40, wherein at least one of the one or more
assays and the information are shipped to the user together in a
single package.
50. A method for genetic analysis, comprising:requesting a
plurality of assay mixtures for genetic analysis from a supplier;
receiving from the supplier (i) the plurality of assay mixtures,
and (ii) information, in electronic form, pertaining to each assay
mixture; transferring at least a portion of the electronic
information to a computer system adapted for electronic
communication with a thermal cycler; and employing the computer
system and at least a portion of the information, instructing the
operation of the instrument, whereby each of the assay mixtures is
analyzed.
51. The method of claim 50, wherein each of the plurality of assays
comprises a homogeneous reaction mixture.
52. The method of claim 51, wherein the homogeneous reaction
mixture comprises components for conducting a fluorogenic 5'
nuclease assay.
53. The method of claim 50, wherein the information is provided on
a removable disk.
54. The method of claim 50, wherein the thermal cycler is
configured to perform a real-time polymerase chain reaction.
55. The method of claim 50, wherein the information is provided via
a computer network.
56. The method of claim 55, wherein the information is transmitted
by the supplier via electronic mail.
57. The method of claim 55, wherein the information is downloaded
by the user from a server.
58. The method of claim 50, wherein the information is comprised,
at least in part, of American Standard Code for Information
Interchange (ASCII)-formatted text.
59. The method of claim 50, wherein at least one of the plurality
of assays and the information are shipped to the user together in a
single package.
60. The method of claim 50, wherein each of the plurality of assay
mixtures is contained in a single tube.
61. The method of claim 50, wherein the requesting step is carried
out via the Internet.
62. A method for facilitating genetic analysis, comprising:
receiving a request for a plurality of assay mixtures for genetic
analysis from a user; and supplying to the user (i) the plurality
of assay mixtures, (ii) information, in electronic form, pertaining
to each assay mixture, and (iii) a program storage device readable
by a computer system, embodying a program of instructions
executable by the computer system to perform method steps for
genetic analysis, the method steps comprising (a) transferring at
least a portion of the electronic information to a memory of the
computer system, (b) employing at least a portion of the
information, generating instructions for the operation of a thermal
cycler adapted to perform real-time polymerase chain reaction, and
(c) sending the instructions to the thermal cycler whereby the
thermal cycler acts upon the assay mixtures.
63. The method of claim 62, wherein the plurality of assay mixtures
and the information is supplied to the user together, at the same
time.
64. The method of claim 62, wherein the information is provided on
a removable disk.
65. The method of claim 64, wherein the removable disk comprises a
compact disk (CD).
66. The method of claim 62, wherein the information is provided via
a computer network.
67. The method of claim 62, wherein each of the plurality of assay
mixtures comprises a homogeneous reaction mixture.
68. The method of claim 67, wherein each homogeneous reaction
mixture comprises a fluorogenic 5' nuclease assay.
69. The method of claim 62, wherein each of the plurality of assay
mixtures is contained in a single tube.
70. The method of claim 62, wherein the program storage device
comprises a removable disk.
71. The method of claim 62, further comprising providing a web site
configured for receiving requests from users for assay mixtures for
genetic analysis.
72. A program storage device readable by a computer system,
embodying a program of instructions executable by the computer
system for genetic analysis, the method steps comprising: (i)
uploading electronic information relating to a plurality of assay
mixtures from a data storage device to the computer system, (ii)
employing the information, generating a file containing
instructions for operation of a thermal cycler; and (iii)
tranferring the file to the thermal cycler for implementation.
73. The device of claim 72, wherein the method steps further
comprise: monitoring for electronic information ready for
uploading.
74. The device of claim 72, wherein the method steps further
comprise: receiving a results file from the thermal cycler, and
causing the results file to be saved.
75. The device of claim 72, wherein the method steps further
comprise: building an assay information database.
76. The device of claim 72, wherein the method steps further
comprise: generating plate documents and providing the plate
documents in the file for transfer to the thermal cycler.
77. The device of claim 72, wherein the method steps further
comprise: generating or updating an activity log.
78. The device of claim 72, wherein the method steps further
comprise: outputting the instructions to a printer for
printing.
79. The device of claim 72, wherein the method steps further
comprise: determining errors in the information or instructions
that could adversely impact analysis of the assay mixtures.
80. The device of claim 72, wherein the data storage device is a
removable disk.
81. The device of claim 72, wherein the thermal cycler is adapted
to perform a real-time polymerase chain reaction.
Description
Detailed Description of the Invention
Cross Reference to Related Applications
[0001] This application claims the benefit under 35 U.S.C. Section
119(e) of prior U.S. Provisional Patent Applications Nos.
60/352,039, filed January 25, 2002, 60/352,356, filed January 28,
2002, 60/369,127, filed April 1, 2002, 60/369,657, filed April 3,
2002, 60/370,921, filed April 9, 2002, 60/376,171, filed April 26,
2002, 60/380,057, filed May 6, 2002, 60/383,627, filed May 28,
2002, 60/390,708, filed June 21, 2002, 60/394,115, filed July 5,
2002, and 60/399,860, filed July 31, 2002, all of which are
incorporated herein in their entireties by reference. Cross
reference is made to U.S. Provisional Patent Application No.
60/383,954, filed May 29, 2002, and concurrently-filed U.S. Patent
Application Nos. 10/334,793 and 10/335,707, all of which are
incorporated herein in their entireties by reference.
Background
[0002] Polymerase chain reaction (PCR) is a common scientific
technique for amplification of polynucleotides and deoxyribonucleic
acid (DNA) complexes. PCR requires the use of several assay
reagents as well as a target analyte, such as a target
polynucleotide. The target analyte can be contained in a sample and
amplified during a PCR process. During the course of the reaction,
the target analyte can be amplified many times. Currently, the
assay reagents must be gathered and mixed, typically immediately
prior to subjecting the sample to PCR. The many assay reagents are
typically obtained from multiple sources and typically mixed
together on a laboratory bench top in a physical location that
commonly houses laboratory equipment necessary to perform PCR.
Summary
[0003] According to various embodiments, an assay kit is provided
that includes: a container containing assay reagents; and a
separate data storage medium that contains data about the assay
reagents. The assay reagents can be adapted to perform an allelic
discrimination or expression analysis reaction when admixed with at
least one target polynucleotide. The other reagents can be, for
example, components conventionally used for polymerase chain
reactions (PCR), and can include non-reactive components. The
container can be sealed and can be packaged with the separate data
storage medium in a package, for example, in a box. The container
can have a machine-readable label that provides information about
the contents of the container.
[0004] According to various embodiments, the data stored on the
data storage medium can include computer-readable code that can be
used to adjust, calibrate, direct, set, run, or otherwise control
an apparatus, for example, a scientific or laboratory instrument.
According to various embodiments, methods are provided wherein the
data is used to cause an apparatus to automatically perform a
polymerase chain reaction of a target analyte that is mixed with
the assay reagents. Methods are also provided whereby the kit is
shipped to a customer.
Brief Description of Drawings
[0005] Fig. 1 is a top plan segmented view of a plurality of
containers in a shipping and storage tray that can be included in a
kit, according to various embodiments;
[0006] Fig. 2 is a side view of a container of one of the
containers shown in Fig. 1;
[0007] Fig. 3 is a compact disk that can be included in a kit
according to various embodiments;
[0008] Fig. 4 is a flowchart outlining a method of use of a
container and a data storage medium according to an embodiment;
and
[0009] Fig. 5 is a schematic diagram of a quenchable dye that can
be part of a mixture of reagents according to various
embodiments.
[0010] It is intended that the specification and examples be
considered as exemplary only. The true scope and spirit of the
present teachings includes various embodiments.
Detailed Description
[0011] According to various embodiments, an assay kit is provided
that can include, for example: a container containing assay
reagents; and a separate data storage medium that contains data
about the assay reagents. The assay reagents can include reagents
adapted to perform an allelic discrimination or expression analysis
reaction when admixed with at least one target polynucleotide
sequence. Reagents can be, for example, reagents used for
polymerase chain reaction (PCR) amplification, ligase chain
reaction (LCR), oligonucleotide reaction assays (OLA),
self-sustaining sequence replication, enzyme kinetic studies,
homogeneous ligand binding assays, deoxyribonucleic acid or amino
acid sequencing, and/or other chemical or biochemical reaction, and
can include non-reactive components. The container can be sealed
and can be packaged with the separate data storage medium in a
package, for example, in a box. The container can have a
machine-readable label that provides information about the contents
of the container.
[0012] According to various embodiments, the terms "polynucleotide"
and "DNA," as used herein, can include nucleic acid analogs that
can be used in addition to or instead of nucleic acids. Examples of
nucleic acid analogs includes the family of peptide nucleic acids
(PNA), wherein the sugar/phosphate backbone of DNA or RNA has been
replaced with acyclic, achiral, and neutral polyamide linkages. For
example, a probe or primer can have a PNA polymer instead of a DNA
polymer. The 2-aminoethylglycine polyamide linkage with nucleobases
attached to the linkage through an amide bond has been well-studied
as an embodiment of PNA and shown to possess exceptional
hybridization specificity and affinity. An example of a PNA is as
shown below in a partial structure with a carboxyl-terminal amide:
1
[0013] "Nucleobase" as used herein means any nitrogen-containing
heterocyclic moiety capable of forming Watson-Crick hydrogen bonds
in pairing with a complementary nucleobase or nucleobase analog,
e.g. a purine, a 7-deazapurine, or a pyrimidine. Typical
nucleobases are the naturally occurring nucleobases such as, for
example, adenine, guanine, cytosine, uracil, thymine, and analogs
of the naturally occurring nucleobases, e.g. 7-deazaadenine,
7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine,
inosine, nebularine, nitropyrrole, nitroindole, 2-aminopurine,
2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,
pseudouridine, pseudocytosine, pseudoisocytosine,
5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine,
2-azapurine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine,
4-thiouracil, O.sup.6-methylguanine, N.sup.6-methyladenine,
O.sup.4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil,
4-methylindole, pyrazolo[3,4-D]pyrimidines, "PPG", and
ethenoadenine.
[0014] "Nucleoside" as used herein refers to a compound consisting
of a nucleobase linked to the C-1' carbon of a sugar, such as, for
example, ribose, arabinose, xylose, and pyranose, in the natural or
the anomeric configuration. The sugar can be substituted or
unsubstituted. Substituted ribose sugars can include, but are not
limited to, those riboses having one or more of the carbon atoms,
for example, the 2'-carbon atom, substituted with one or more of
the same or different Cl, F, -R, -OR, -NR.sub.2 or halogen groups,
where each R is independently H, C.sub.1-C.sub.6 alkyl or
C.sub.5-C.sub.14 aryl. Ribose examples can include ribose,
2'-deoxyribose, 2',3'-dideoxyribose, 2'-haloribose,
2'-fluororibose, 2'-chlororibose, and 2'-alkylribose, e.g.
2'-O-methyl, 4'--anomeric nucleotides, 1'--anomeric nucleotides,
2'-4'- and 3'-4'-linked and other "locked" or "LNA", bicyclic sugar
modifications. Exemplary LNA sugar analogs within a polynucleotide
can include the following structures: 2where B is any
nucleobase.
[0015] Sugars can have modifications at the 2'- or 3'-position such
as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy,
methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro,
chloro and bromo. Nucleosides and nucleotides can have the natural
D configurational isomer (D-form) or the L configurational isomer
(L-form). When the nucleobase is a purine, e.g. adenine or guanine,
the ribose sugar is attached to the N.sup.9-position of the
nucleobase. When the nucleobase is a pyrimidine, e.g. cytosine,
uracil, or thymine, the pentose sugar is attached to the
N.sup.1-position of the nucleobase.
[0016] "Nucleotide" as used herein refers to a phosphate ester of a
nucleoside and can be in the form of a monomer unit or within a
nucleic acid. "Nucleotide 5'-triphosphate" as used herein refers to
a nucleotide with a triphosphate ester group at the 5' position,
and can be denoted as "NTP", or "dNTP" and "ddNTP" to particularly
point out the structural features of the ribose sugar. The
triphosphate ester group can include sulfur substitutions for the
various oxygens, e.g. -thio-nucleotide 5'-triphosphates.
[0017] As used herein, the terms "polynucleotide" and
"oligonucleotide" mean single-stranded and double-stranded polymers
of, for example, nucleotide monomers, including
2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by
internucleotide phosphodiester bond linkages, e.g. 3'-5' and 2'-5',
inverted linkages, e.g. 3'-3' and 5'-5', branched structures, or
internucleotide analogs. Polynucleotides can have associated
counter ions, such as H.sup.+, NH.sub.4 .sup.+, trialkylammonium,
Mg.sup.2+, Na.sup.+ and the like. A polynucleotide can be composed
entirely of deoxyribonucleotides, entirely of ribonucleotides, or
chimeric mixtures thereof. Polynucleotides can be comprised of
internucleotide, nucleobase and sugar analogs. For example, a
polynucleotide or oligonucleotide can be a PNA polymer.
Polynucleotides can range in size from a few monomeric units, e.g.
5-40 when they are more commonly frequently referred to in the art
as oligonucleotides, to several thousands of monomeric nucleotide
units. Unless otherwise denoted, whenever a polynucleotide sequence
is represented, it will be understood that the nucleotides are in
5' to 3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytidine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise
noted.
[0018] "Internucleotide analog" as used herein means a phosphate
ester analog or a non-phosphate analog of a polynucleotide.
Phosphate ester analogs can include: (i) C.sub.1-C.sub.4
alkylphosphonate, e.g. methylphosphonate; (ii) phosphoramidate;
(iii) C.sub.1-C.sub.6 alkyl-phosphotriester; (iv) phosphorothioate;
and (v) phosphorodithioate. Non-phosphate analogs can include
compounds wherein the sugar/phosphate moieties are replaced by an
amide linkage, such as a 2-aminoethylglycine unit, commonly
referred to as PNA.
[0019] "Heterozygous" as used herein means both members of a pair
of alleles of a gene are present in a sample obtained from a single
source, wherein a gene can have two alleles due to, for example,
the fusion of two dissimilar gametes with respect to the gene.
[0020] "Heterozygous assay" as used herein means an assay adapted
to identify the allelic state of a gene having one or both members
of a pair of alleles.
[0021] "Homozygous" as used herein means one member of a pair of
alleles is present in a sample obtained from a single source,
wherein a gene can have one allele due to, for example, the fusion
of two identical gametes with respect to the gene.
[0022] "Homozygous assay" as used herein means an assay adapted to
identify only one of two possible allelic states of a gene having
one or both members of a pair of alleles.
[0023] As used herein, the terms "customer" and "user" can be
interchangeable.
[0024] According to various embodiments, the container can contain
all assay reagents and components necessary to conduct PCR, with
the exception of a target polynucleotide, also referred to as a
target nucleic acid sequence or a target DNA. The target
polynucleotide can be provided by a user and mixed with the assay
reagents or the target polynucleotide can be provided to the user
in a second container to be mixed with the assay reagents. For
example, the kit can include a container that contains a target
polynucleotide and a container that does not contain a target
polynucleotide.
[0025] According to various embodiments, the container can be a
tube, vial, jar, capsule, ampule, or like vessel. The tube can have
a removable cap and/or a replaceable cap. The cap can maintain the
container sealed such that the container is water-tight and
air-tight. The container can be hermetically sealed. The container
can be open at a first end and closed at a second end. According to
various embodiments, the first end can be tapered, for example,
along the length of the container. According to various
embodiments, the container can hold a mixture including assay
reagents and have a volume of at least about 5L. The container can
have a volume of about 10L or less. The maximum volume of the
container can be about 25L or less, and according to other
embodiments, the volume can be greater than about 25L.
[0026] According to various embodiments, standardized assay designs
are provided for custom assays and/or stock assays, including
either universal concentration or uniform thermal cycling
parameters, or both, allowing results to be more easily compared
with and/or transferred to other researchers and labs. Also, in
some embodiments, assays are formulated in a single-tube 20X mix
format that is convenient and easy to use, requiring no preparation
or clean-up and providing faster time to results.
[0027] According to various embodiments, the assay reagents in the
container can contain a volume of assay reagents for more than one
respective assay. For example, the assay reagents in the container
can be divided and transferred into five respective reaction wells,
for example, to conduct five identical and/or different assays. For
another example, the assay reagents in the container can be removed
from the container and aliquoted into ten respective reaction
wells. According to various embodiments, the container can contain
a sufficient volume of assay reagents to complete at least 1, at
least 5, at least 10, or at least 25 assays.
[0028] According to various embodiments, the container can have a
label that provides information about the contents of the
container. For embodiments including a cap, the label can be
secured to the container, or to the cap of the container, if
desired. The label can comprise a barcode and can comprise a
2-dimensional barcode that can be provided on the container. In
addition to or instead of a barcode, the label can include a serial
number, a lot number, a date, and/or other identifying or
descriptive indicia. The label can identify the reporter dye or
dyes in the container. The label can provide sequence information
regarding polynucleotide or peptide reagents provided in the
container. The label can contain information about the target
polynucleotide sequence, including a common or scientific name or
gene name for the target polynucleotide or sequence information
about a target analyte. The assay kit can be packaged in, for
example, a box. Packaging such as a carton or box can instead or
additionally be labeled with identifying and/or descriptive indicia
and/or coating as described above.
[0029] In addition to a human-readable label, for example, an
English-language label with the assay name can be located on each
tube. In some embodiments, a 2-D barcode can be laser-etched on the
bottom of each assay tube and a 1-D barcode can be laser-etched on
each 96-tube rack of assays, thereby making the assay tubes and
racks machine-identifiable so that the assays are compatible with
automation for high throughput applications.
[0030] According to various embodiments, the container can contain
at least one probe reactive with a target polynucleotide, wherein
the probe can include a polynucleotide, a marker compound, for
example, a marker dye, a quenchable dye, or a fluorescent reporter
dye, a non-fluorescent quencher, a minor groove binder, or a
combination thereof.
[0031] The probe can include a reporter dye such as VIC or 6-FAM
linked to the 5' end of the polynucleotide. VIC and 6-FAM
dye-labeled probes are available from Applied Biosystems, Foster
City, CA. The minor groove binder can increase the melting
temperature T.sub.m without increasing the length of the
polynucleotide. This can result in greater differences in T.sub.m
values between matched and mismatched probes that therefore enables
more accurate allelic discrimination. The probe can include a
quencher (e.g., a non-fluorescent quencher) linked to the 3' end of
the polynucleotide. The quencher can inhibit fluorescence that can
facilitate greater discrimination of reporter dye fluorescence.
[0032] According to various embodiments, the container can contain
two different types of probes, wherein the polynucleotide and the
reporter dyes differ. For example, the first type of probe can have
a first polynucleotide with a VIC reporter dye attached to the 5'
end of the first polynucleotide and the second type of probe can
have a second polynucleotide with a 6-FAM reporter dye attached to
the 5' end of the second polynucleotide and the first and second
polynucleotides differ by at least one monomeric unit at the same
location in the polynucleotide when the polynucleotides are aligned
5' to 3'. The dye-labeled probes can be adapted to perform a
heterozygous assay or a homozygous assay.
[0033] The probe can anneal to a complementary sequence between the
forward and reverse primer sites. At the time of annealing, the
probe is intact and the proximity of the reporter dye to the
quencher can result in suppression of fluorescence of the reporter
dye. A polymerase can cleave a reporter dye only when the probe has
completely, mostly, or substantially hybridized to the target
polynucleotide sequence. When the reporter dye is cleaved from the
probe, the relative fluorescence of the reporter dye increases. The
increase in relative fluorescence can only occur if the amplified
target polynucleotide sequence is complementary, mostly
complementary, or substantially complementary to the probe.
Therefore, the fluorescent signal generated by PCR amplification
can indicate which alleles are present in a sample. Mismatches
between a probe and a target polynucleotide sequence can reduce
efficiency of probe hybridization and/or a polymerase can be more
likely to displace a mismatched probe without cleaving it and
therefore not produce a fluorescent signal. For example, if one of
two possible reporter dyes fluoresce during an assay, then the
presence of a homozygous gene is indicated. For further example, if
both possible reporter dyes fluoresce during an assay, then the
presence of a heterozygous gene is indicated.
[0034] According to various embodiments, the container can contain
at least one primer, wherein the primer can comprise a sequence
that is shorter than the target polynucleotide. The primer can
comprise a polynucleotide and/or a minor groove binder. The primer
can comprise a sequence that is complimentary to, or mostly
complimentary to, the target polynucleotide. For example, the
primer can be at least 90% homologous to a corresponding length of
the target polynucleotide, at least 80% homologous to a
corresponding length of the target polynucleotide, at least 70%
homologous to a corresponding length of the target polynucleotide,
or at least 50% homologous to a corresponding length of the target
polynucleotide.
[0035] According to various embodiments, the container can contain
a thermostable DNA polymerase, such as, for example, thermus
aquaticus (Taq), and at least 4 embodiments of a deoxyribonucleic
acid (e.g., adenosine, tyrosine, cytosine, and guanine). The
polymerase can be, for example, AMPLITAQ GOLD, available from
Applied Biosystems, Foster City, CA. According to various
embodiments, the container can container components of a
fluorogenic 5' nuclease assay or other assay reagents that utilize
5' nuclease chemistry, for example, TAQMAN minor groove binder
probes, available from Applied Biosystems, Foster City, CA. Some or
all of the above-listed components can be replaced by or used with
commercially-available products, for example, buffers or AMPLITAQ
GOLD PCR MASTER MIX (Applied Biosystems, Foster City, CA).
[0036] According to various embodiments, the assay kit can further
include a target polynucleotide in the first or another container,
for example, as can be used to prepare a positive control. The
assay kit can include more than one container and an Assay
Information File (AIF) and/or Electronic Data Sheet (EDS) can be
provided on the data storage medium and can contain information
about the containers. For example, an AIF or EDS can contain
information about 96 containers or 384 containers.
[0037] According to various embodiments, a multi-well plate can
also be provided in the kit and can include, for example, 96 or 384
positions for container placement. A plate can be substantially
rectangular with an optional integrated structural feature for
plate orientation. A plate can have a plurality of wells. The assay
kit can include a plate adapted to hold a plurality of containers
or tubes. The plate can be of unitary construction. One or more
containers can be integrated into a single plate, and the plate can
have a plurality of containers in physical contact with each other.
For example, the plate can be of unitary construction and have 96
containers in the form of receptacles. For further example, the
plate can be of unitary construction and have 384 containers.
[0038] Assays may be delivered with certain sequence information.
For example, some sequence context information (e.g., forward
primer location in the RefSeq sequence) can denote which exon-exon
junction the assay covers so that users can get a sense of where
the assay is positioned in the transcript. More information can be
provided, as desired.
[0039] For example, according to various embodiments, data can have
the following columns (non-limiting examples are listed in
paratheticalls following the item): customer name (assigned by the
supplier); order number (assigned by the supplier, in some
configurations, and can correspond to a number on a 1-D bar code on
the plate); ship date (date shipped by the supplier); set ID (an
assay name created from record information in the requestor's
submission file, including record name and target site name from a
target site coordinate; if the sequence record submitted contained
multiple target sites, the value of the Set ID can be used to
determine which site was used to create the assay); set No. (may be
used for internal quality control by the supplier); plate ID
(assigned by the supplier, can include the order number value, and
can appear on the plate rack as the 1-D bar code); vial ID (a 2-D
bar code number can be attached to the bottom of each tube; entry
in the datasheet may have leading zeros dropped in some
configurations); well location (location of assay tube in the plate
rack); line item (may be used for internal quality control by the
supplier); VIC probe name (may be used for internal quality control
by the supplier); VIC probe sequence (5' to 3' sequence of the
probe labeled with VIC dye; in some configurations, the 3'
non-fluorescent quencher-minor groove binder (NFQ-MGB) may not be
listed but is present on the probe); VIC (M) concentration (probe
concentration); line item (may be used for internal quality control
by the supplier); 6-FAM probe name (may be used for internal
quality control by the supplier); 6-FAM probe sequence (5' to 3'
sequence of the probe labeled with 6-FAM dye; in some
configurations, the 3' NFQ-MGB may not be listed but is present on
the probe); 6-FAM (M) concentration (probe concentration); line
item (may be used for internal quality control by the supplier);
forward primer name (may be used for internal quality control by
the supplier); forward primer sequence; forward (M) primer
concentration; line item (may be used for internal quality control
by the supplier); reverse primer name (may be used for internal
quality control by the supplier); reverse primer sequence; reverse
(M) primer concentration; and/or part number (the part number
ordered by the requestor).
[0040] The shipped worksheet can be provided to enable a user of
the assays to determine that the tubes are in the same positions in
the plate rack as when the assays were shipped. For example,
according to various embodiments, the following columns can appear
in the shipped worksheet: position (position in the plate); and/or
vial ID (a 2-D bar code number that can be attached to the bottom
of the tube; in some configurations, leading zeros are
dropped).
[0041] According to various embodiments, the assay kit can be
shipped to a customer. The data storage medium can be shipped to
the customer along with, concurrent to, separately, previously, or
subsequently to shipment of the container and the contents of the
container. Alternatively, or additionally, data can be transferred
electronically to the customer. The data can be sent to the
customer by electronic mail. The customer can retrieve or download
the information over a computer network, such as, for example, the
Internet, a Wide Area Network, a Local Area Network, or a Virtual
Private Network. The customer can retrieve the data using a file
transfer protocol or by a hypertext transfer protocol. The protocol
can be secured using, for example, 128 bit encryption.
[0042] According to various embodiments, the manufactured assays
are shipped as homogenous assays in a single tube format. For
example, in at least some embodiments, a single-tube, ready to use
format is provided that is suitable for immediate use on an ABI
PRISM.sup..RTM. Sequence Detection System (SDS) instrument for one
or more applications.
[0043] Data stored on the data storage medium can include
information about a variety of items, for example, a stock number,
an assay ID number, a plate number, a well location, a gene symbol
or name, a category ID or name, a group ID or name, a chromosome
number, a cytogenetic band identification, an NCBI gene reference,
an NCBI SNP reference, a minor allele frequency, a minor allele
frequency of a particular population, an SNP type, a context
sequence, a reporter dye identification, barcode information, or a
combination thereof. The context sequence can include, for example,
up to 20 bases, more than 20 bases, more than 30 bases, or more
than 40 bases. The data stored on the data storage medium can
include information about some of the previously-mentioned items,
all of the previously-mentioned items, or none of the
previously-mentioned items. Furthermore, the information can
include more information than that information listed above and/or
other identifying or descriptive indicia.
[0044] According to various embodiments, the data storage medium
can be separate from the container or can be affixed to the
container. The data storage medium can be a label attached to the
container, for example, with a pressure sensitive adhesive or other
glue. The container or cap can serve as the data storage medium and
the data can be printed, etched, inscribed, or otherwise encoded on
a surface of the container or cap. The data storage medium can
include an optically detectable code and/or the data storage medium
can be a 2-dimensional barcode.
[0045] According to various embodiments, the data can be stored on
the data storage medium in electronic format. The data storage
medium can be a compact disk (CD). The information can be contained
in an Assay Information File (AIF) and/or the Assay Information
File can be in the form of an ascii-compatible text file. The data
can be contained in an Electronic Data Sheet (EDS) and/or the EDS
can be in the form of an ascii-compatible text file. The EDS can
contain information that links container identification
information, such as, for example, the information contained on a
2-dimensional barcode on the container, to assay identification
information. The link can be to assay information contained in, for
example, an Assay Information File. The AIF, the EDS, or other
computer-readable data or code contained or stored on the data
storage medium can be adapted to control an apparatus such as a
scientific or laboratory instrument, for example, a thermal cycler,
or sequence detection system.
[0046] According to various embodiments, the data storage medium
can, in addition or in the alternative, contain executable code.
The executable code can be in the form of stand-alone software,
updates to stand-alone software, or modules to third-party
software. The software can be adapted to run on an operating system
that controls an SDS instrument, such as, for example, UNIX. The
software can include computer code written in assembly language or
machine language. The software can be transferred to the SDS
instrument by computer and can be saved onto a storage device in
the SDS instrument, loaded into a memory device of the SDS
instrument, incorporated into previously-loaded software on the SDS
instrument, or can be overwritten onto previously-loaded software
on the SDS instrument. The storage device can be a hard drive or
optical drive. The memory device can be random access memory (RAM)
or an erasable, programmable read only memory (EPROM) chip. The
software can be provided to a customer on a data storage medium or
can be transferred to the customer over a computer network.
[0047] According to various embodiments, an Assay Information File
(AIF) or Electronic Data Sheet (EDS) can be provided with an assay
or assays. The AIF and/or EDS can be, in some embodiments,
electronic files or data electronically stored on a data storage
medium. The files or data can contain, for example, information on
one or more assays, information on one or more polynucleotide
sequences, an alphanumeric sequence representing a polynucleotide
sequence, or the like. Alternatively, or in addition, a print copy
or a printout of the AIF, EDS, and/or information in the AIF and/or
EDS can be provided.
[0048] According to various embodiments, a printed copy of the AIF
and/or EDS can also be provided and can contain information about
each assay. This information may include, among other things, the
position of each assay in the plate rack. Some embodiments provide,
either in place of, or in addition to the printed copy of the AIF
and/or EDS, a CD-ROM with one or more data files recorded thereon.
The data may include any or all of the following files, and may
include other files as well: an electronic assay workbook,
including data sheet(s) and shipped worksheet(s); an electronically
readable and/or printable copy of instructions for SNP assay
protocol for ordering by design; an electronically readable and/or
printable copy of protocols for submitting requests; and/or an
electronically readable copy of a product.
[0049] According to various embodiments, a data sheet and/or an
electronic assay workbook is provided with custom assays. In some
embodiments, an electronic assay workbook is included with each
order of up to 92 assays. The workbook file name can include the
number on a bar code for easy correlation. The workbook can contain
two worksheets, namely, a "data sheet" worksheet and a "shipped"
worksheet. The workbook can be a spreadsheet file, such as a
MICROSOFT EXCEL spreadsheet software file, that may contain macros
and/or be password protected. Cells of the workbook can be copied
and pasted into a new worksheet and modified in the new worksheet.
A printed copy of the data from the electronic file may be included
with a shipment of assays ordered, for example, by design. The data
can include a correlation of the 2-D barcodes on the tubes to the
corresponding assay names and primer and probe specific
information.
[0050] According to various embodiments, data included with an
order can include at least some of the following information: an
identification of the assay in each tube; assay names; which target
site was used, if the requestor submitted a sequence record that
included more than one target site; locations of each tube in the
assay rack; sequences of the primers and probes; and concentrations
(M) of primers and probes. Other configurations can necessarily
include all of this information and may include more
information.
[0051] According to various embodiments, a computer program,
comprised of lines of machine-readable and/or executable computer
code, can obtain data contained in the AIF, EDS, or in another data
file, and use the data to control a scientific or laboratory
instrument. For example, a microcomputer-based software program can
be provided that can load computer-readable data from an AIF or
EDS. The AIF or EDS can be stored, for example, on a compact disk
that can be shipped to a user along with, concurrent to, previous
to, or subsequent to shipping and providing the at least one
container of the assay kit. According to various embodiments, the
software program can configure, direct, or operate an instrument
that can perform PCR, sequencing, and/or sequence detection, for
example, an Applied Biosystems 7900HT Sequence Detection System
(SDS). The software program can control, for example, an
instrument, to perform PCR, sequencing, and/or sequence detection,
without human intervention. The SDS can directly operate and run
without human interaction The same or a different software program
can perform basecalling of detected sequence data.
[0052] According to various embodiments, the kit can include a
software program stored on the data storage medium or stored
separately on a second data storage medium, for example, a CD. The
software program can build an internal assay information database
from AIF or EDS files and/or generate plate information adapted to
control, at least in part, an SDS instrument. The software can
deliver the plate information to the SDS. The software program can
flag or note problems with the plate information or plate setup
data. The software program can generate an activity log file. The
software can detect new plate information setup files, new AIF
files, new EDS files, or a combination thereof. The software can
import data generated by the customer. The software program can add
to, modify, or delete data from an AIF or EDS. The software program
can control or direct an SDS instrument to perform, for example,
PCR, sequencing, sequence detection, or a combination thereof. The
software program can control an SDS instrument to perform PCR,
sequencing, or sequence detection using a protocol obtained from an
AIF or EDS.
[0053] According to various embodiments, the software can receive
data from an instrument, for example, from a real-time PCR or SDS
instrument, wherein the data is generated during the course of, for
example, PCR, sequencing, or sequence detection. An SDS data file
can be generated by an SDS instrument and can be transmitted to the
software program. The SDS data file can contain information
generated by an SDS instrument, or information from an AIF or EDS
stored on the data storage medium. The SDS data file can contain
error codes or error information generated by the SDS instrument as
a result of problems with the SDS instrument, the AIF, and/or the
EDS. The data file can include error codes related to, for example,
failure to detect at least one fluorescent probe or failure of a
component of the SDS instrument, such as a heating element. The SDS
instrument can send a log file containing, for example, information
about AIF or EDS files transmitted from the software program. A
detector list can be generated from an AIF or an EDS and saved in a
format suitable for input to a detector manager, such as, for
example, an ascii-compatible format.
[0054] According to various embodiments, the software can save an
AIF, EDS, and/or SDS data file, or other file in separate,
respective folders on a data storage device, for example, on a
microcomputer. The microcomputer can include, for example, a hard
drive, an optical drive, or both. The software program can remain
in the memory of a microcomputer before, during, or after
transmission of a data file to or from the software program. The
software program can continuously monitor data on a microcomputer
for new or modified AIF, EDS, and/or SDS data files.
[0055] According to various embodiments, a manual method can be
used by a customer or user of the assays to validate each tube
position in the rack plate. The rack plate position and assay name
on the tube label can be compared with the values in the well
location and set ID columns of the data. This "validation" is
different from the validation of assays, in that validation of each
tube position in a plate rack is performed by the user, and merely
confirms that the tubes are in positions matching the "shipped"
worksheet. If the tubes are not in the correction position, they
may be rearranged to match the worksheet. The operational quality
of the assays contained within the tubes is validated at the
supplier's factory.
[0056] According to various embodiments, an automated method can be
used by a customer to validate each tube position in the rack
plate. This method can include scanning the plate and tubes using a
2-D bar code reader, and executing a plate validation spreadsheet
macro (for example, a MICROSOFT EXCEL spreadsheet software macro).
To scan the plate and tubes, the plate rack can be placed on the
2-D bar code reader in a standard orientation. For example, tube
position "A1" is placed in the top left corner of the reader. The
1-D bar code on the plate rack can then be scanned. The bar code
reader can be configured, if necessary, to read positions in one
column and to read bar codes in a column next to the positions
column. Next, the plate rack is scanned and the results are saved
to a director that can be accessed from the computer containing the
electronic file. In some configurations, the scanning results are
saved as a tab-delimited file.
[0057] According to various embodiments, to validate, the "shipped"
worksheet can be opened in the spreadsheet and, with macros
enabled, the validation macro can be run. In some embodiments
utilizing MICROSOFT EXCEL spreadsheet software, the validation is
performed by opening the electronic workbook, clicking a mouse on a
"shipped" tab to view the worksheet containing the validation
macro, clicking on the "validate" button to start the plate
validation macro, and, when an "import plate scan" dialog box is
presented, selecting "browse" to locate the file from the 2-D bar
code scan. After "browse" is selected, the file that resulted from
the 2-D bar code scan is selected and imported into a new
worksheet, which, in some embodiments, is called "received". The
macro can then compare each bar code and its position in the plate
rack with the corresponding bar code in the "shipped" worksheet
(for example, a value in the "Vial ID" column). The macro then
enters the result in a "validation" column in the "shipped"
worksheet. According to various embodiments, the results for each
entry may either be "OK" (or any entry understood as indicating a
match) or "ERROR" (or any other entry understood as indicating a
non-match). A "shipment validation" dialog box can then alert that
the validation is complete, and the user clicks "OK" to dismiss the
dialog box.
[0058] Plate validation errors indicate that the tubes are not in
the same position as they were shipped by the supplier to the
requestor. The user can resolve plate validation errors by
rearranging the tubes to match the "shipped" worksheet. The user
can then rescan the plate and execute the validation macro again to
validate the plate.
[0059] According to various embodiments, an assay kit can be
provided that includes at least one assay for allelic
discrimination or expression analysis of genomic material. An
information source can be provided that has at least one member of
the group consisting of an electronic data sheet, an assay
information file, and at least one printed datasheet and
combinations thereof. The assay can be a SNP assay or a gene
expression assay. The assay can be provided in a single tube.
[0060] According to various embodiments, the assay can comprise at
least one probe and two primers. The assay can be a SNP assay
comprising one probe for each of two alleles and two primers.
According to various embodiments, the probe can have at least one
fluorophore and at least one fluorescence quencher. The
fluorescence quencher can be non-fluorescent fluorescence quencher.
The probe can have at least one minor groove binder. The assay can
have PCR reagents or RT-PCR reagents. The assay can have universal
master mix, where the universal master mix has at least one salt, a
buffer, and a DNA polymerase.
[0061] According to various embodiments, the single tube can have a
bar code label. The bar code label can be a two-dimensional bar
code label. The single tube can have a human-readable assay number.
The kit can be comprised of a plurality of assays, each of which is
in a single tube, thereby constituting a plurality of tubes. The
plurality of tubes can be contained in a rack and the rack can have
a bar-code identification. The kit can also have at least one
datasheet containing information on the assay. The kit can have at
least one machine-readable medium containing information on the
assay. The at least one machine-readable medium can be at least one
datasheet containing information on the assay. The machine-readable
medium can be a compact disk.
[0062] Referring to the drawing figures, Fig. 1 is a plan sectional
view of an embodiment of an assay kit having several containers 10
that are held in place for shipping in a tray 12. A 2-dimensional
barcode 14 is located on the top side of a container cap 16. The
2-dimensional barcode 14 is adapted to be machine readable. A
serial number 18 is located next to the 2-dimensional barcode 14.
The serial number 18 is printed in Arabic numerals.
[0063] Fig. 2 is a side view of one of the containers 10 shown in
Fig. 1. The container 10 has a 2-dimensional barcode 14 and a
serial number 18 printed on a cap 16 for the container 10. The
cylindrical body 20 of the container 10 is adapted to hold a
mixture of assay reagents and is adapted to fit into a tray 12 for
shipping and storage.
[0064] Fig. 3 is a compact disk 22 that can be included in an assay
kit according to various embodiments, and can be provided to a user
along with a container (not shown in Fig. 3) containing a mixture
including assay reagents.
[0065] Fig. 4 is a flowchart outlining a method of use of a
container and a data storage medium according to an embodiment. A
customer can request one or more assays from a supplier. The
customer receives from the supplier a CD containing an electronic
data file and an assay container or containers, and loads the CD
into a CD reader of a computer (e.g., a microcomputer) having
software that can communicate with an SDS instrument, for example,
an ABI 7900HT system. The software on the microcomputer detects the
new electronic data file on the CD and loads the electronic data
file into memory. The software can read the contents of the
electronic data file and can: build an internal assay information
database from the electronic data file; flag problems with any file
necessary to run or operate the SDS instrument; generate or update
an activity log; and monitor for any new electronic data files. The
software can then generate a file or files necessary to program and
operate the SDS instrument. The software can then export the file
to the SDS instrument. Alternately or additionally, the software
can output a printed copy of the protocols and/or parameters used
to program the SDS instrument. The SDS instrument can then receive
and implement the file and perform the assay or assays using the
contents of the assay containers. Upon completion of the assay or
assays, the SDS instrument can export to a results file containing
an electronic copy of data generated by the assay or assays to the
software. The software can then save the results file to a disk,
such as, for example, a hard drive or CD, for future use. If
desired, the customer can paste the printed copy of the protocols
and/or parameters in a lab notebook, for example, or can use the
information on the printed copy to manually program the SDS
instrument.
[0066] Fig. 5a-5e are schematic diagrams showing the interaction of
components that can comprise, at least in part, a geneous
homogeneous reaction mixture, according to various embodiments. In
Fig. 5a, primer 52 has annealed to template strand 54. Replication
of the template strand from primer 52 will occur in the 5' to 3'
direction. Probe 50, including a generic reporter dye R, quencher
Q, and minor groove binder MGB, has annealed to the template strand
54. Arrow 53 shows that as the complementary strand (not shown) is
produced from the template strand 54 starting at the forward primer
52, the complementary strand will meet probe 50. Fig. 5b shows the
complementary strand 55 as it meets probe 50a. Polymerase 60
cleaves VIC reporter dye V during the production of complementary
strand 55 given that probe 50a has annealed to the target strand 54
because the target strand 54 and the probe 50a are completely
complementary. Fig. 5c shows the complementary strand 55 as it
meets probe 50b. Polymerase 60 does not cleave FAM reporter dye F
during the production of complementary strand 55 given that probe
50b has not hybridized with the target strand 54 because of a
mismatched base pair at location 64. Fig. 5d shows the
complementary strand 55 as it meets probe 50b. Polymerase 60
cleaves FAM reporter dye F during the production of complementary
strand 55 given that probe 50b has annealed to the target strand 54
because the target strand 54 and the probe 50b are completely
complementary. Fig. 5e shows the complementary strand 55 as it
meets probe 50a. Polymerase 60 does not cleave VIC reporter dye V
during the production of complementary strand 55 given that the
probe 50a has not hybridized with the target strand 54 because of a
mismatched base pair at location 66.
[0067] Those skilled in the art can appreciate from the foregoing
description that the broad teachings herein can be implemented in a
variety of forms. Therefore, while the present teachings have been
described in connection with various embodiments and examples, the
scope of the present teachings should not be so limited
* * * * *