U.S. patent application number 14/060422 was filed with the patent office on 2014-05-08 for allelic discrimination analysis using an efficiency related value (efr).
This patent application is currently assigned to ABBOTT LABORATORIES. The applicant listed for this patent is ABBOTT LABORATORIES. Invention is credited to Eric B. Shain.
Application Number | 20140127793 14/060422 |
Document ID | / |
Family ID | 40824719 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140127793 |
Kind Code |
A1 |
Shain; Eric B. |
May 8, 2014 |
ALLELIC DISCRIMINATION ANALYSIS USING AN EFFICIENCY RELATED VALUE
(EFR)
Abstract
In various embodiments this invention provides novel methods for
discriminating two or more different target nucleic acids. In
certain embodiments the methods comprise providing data
amplification reactions comprising reagents to amplify two or more
different target nucleic acids where the data comprise signals
comprising an amplitude measurement representing the degree of
amplification of each target nucleic acid in the amplification
reaction and the time point in the amplification reaction at which
the amplitude is measured; determining an efficiency related
transform of the data, determining an efficiency related value for
each target nucleic acid that is the maximum magnitude of the
efficiency related transform; and outputting the efficiency related
values in the amplification reaction for each target nucleic acid,
where the relative amplitudes of the efficiency related values for
each target nucleic acid is an indicator of the presence of each of
said nucleic acids in said sample.
Inventors: |
Shain; Eric B.; (Glencoe,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBOTT LABORATORIES |
ABBOTT PARK |
IL |
US |
|
|
Assignee: |
ABBOTT LABORATORIES
Abbott Park
IL
|
Family ID: |
40824719 |
Appl. No.: |
14/060422 |
Filed: |
October 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13315133 |
Dec 8, 2011 |
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14060422 |
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12343233 |
Dec 23, 2008 |
8093020 |
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13315133 |
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61060742 |
Jun 11, 2008 |
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61017531 |
Dec 28, 2007 |
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Current U.S.
Class: |
435/287.2 ;
702/19 |
Current CPC
Class: |
G16B 30/00 20190201;
G16B 40/00 20190201; C12Q 1/6858 20130101; C12Q 1/6858 20130101;
C12Q 2537/165 20130101 |
Class at
Publication: |
435/287.2 ;
702/19 |
International
Class: |
G06F 19/22 20060101
G06F019/22 |
Claims
1.-31. (canceled)
32. A machine-readable medium that provides instructions that, if
executed by a machine, will cause the machine to perform operations
comprising: receiving signals from one or more amplification
reactions comprising reagents to amplify two or more different
target nucleic acids from a single sample where the signals provide
data comprising an amplitude measurement representing the degree of
amplification of each target nucleic acid in the amplification
reaction and the time point in the amplification reaction at which
the amplitude is measured, and where the signal provides such data
for a multiplicity of time points in the amplification reaction(s);
determining an efficiency related transform of said data where said
efficiency related transform provides an amplitude measure that is
related to the efficiency of amplification in said reaction;
determining an efficiency related value for each target nucleic
acid that is the maximum magnitude of the efficiency related
transform determined for that target nucleic acid; and outputting
to a display, printer, or storage medium the efficiency related
values and corresponding points in the amplification reaction for
each target nucleic acid, where the relative amplitudes of the
efficiency related values for each target nucleic acid is an
indicator of the presence of each of said nucleic acids in said
sample.
33. The medium of claim 32, wherein said data provide amplitude
measurements for two or more target nucleic acids are in a single
amplification reaction.
34. The medium of claim 32, wherein said data provide amplitude
measurements from a separate amplification reaction each reaction
amplifying different target nucleic acids.
35. The medium of claim 32, wherein said data provide amplitude
measurements from a separate amplification reaction each reaction
amplifying a single target nucleic acid.
36. The medium of claim 32, wherein the time points in the
amplification reaction are measured in cycle number.
37. The medium of claim 32, wherein the points in the amplification
reaction are measured in reaction time.
38. The medium of claim 32, wherein said amplifying comprises
amplifying at least three different target nucleic acids.
39. The medium of claim 32, wherein said amplifying comprises
amplifying at least five different target nucleic acids.
40. The medium of claim 32, wherein said target nucleic acids
comprise a nucleic acids derived from a first allele of a gene and
a nucleic acid derived from a second allele of a said gene.
41. The medium of claim 40, wherein outputting comprises outputting
information indicating whether said sample is homozygous for said
first allele, homozygous for said second allele or heterozygous for
both alleles.
42. The medium of claim 32, wherein the efficiency related
transform is selected from the group consisting of the ratio
transform of the signals, the shifted ratio transform of the
signals, the first derivative of the signals, the differences
between sequential signals, and the slope or gradient of the log of
the signals.
43. The medium of claim 32, wherein the efficiency related
transform (ERT) is calculated as:
ERT=(Signal.sub.n+1/Signal.sub.n)-1 (a) or
ERT=(Signal.sub.n/Signal.sub.n-1)-1 (b) where Signal.sub.n is the
signal produced at cycle number n, Signal.sub.n+1 is the signal
produced at the subsequent cycle number, Signaln-1 is the signal
produced at the previous cycle number, and n ranges from 1 up to
the number of amplification cycles analyzed in the reaction for
formula (a) and n ranges from 2 up to the number of amplification
cycles -1 analyzed in the reaction for formula (b).
44. The medium of claim 32, wherein the efficiency related value is
the maximum of the efficiency related transform.
45. The medium of claim 32, wherein the efficiency related value is
the maximum gradient of the log of the amplification response.
46. The medium of claim 32, wherein the efficiency related value is
the maximum ratio of the amplification response.
47. The medium of claim 32, wherein the efficiency related value is
the maximum first derivative of the amplification response.
48. The medium of claim 32, wherein additional signal values are
generated by interpolating points between the measured signal
values.
49. The medium of claim 48, wherein said additional signal values
are generated by interpolating points between the measured signal
values using cubic splines.
50. The medium of claim 32, wherein said efficiency related
transform additionally provides a measure of the time or cycle
number in said amplification reaction(s).
51.-66. (canceled)
67. A system comprising: a device for performing a nucleic acid
amplification and providing output signals that comprises a measure
of the time point of the reaction, and the magnitude of the
amplification of a target nucleic acid; a processor operably
coupled to said device; and a machine-readable medium according to
claim 32.
68.-69. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S. Ser.
No. 61/060,742, filed on Jun. 11, 2008 and U.S. Ser. No.
61/017,531, filed on Dec. 28, 2007, both of which are incorporated
herein by reference in their entirety for all purposes.
COPYRIGHT NOTICE
[0002] Pursuant to 37 C.F.R. 1.71(e), applicants note that this
disclosure contains material that is subject to and for which is
claimed copyright protection, such as, but not limited to, source
code listings, screen shots, user interfaces, user instructions,
and any other aspects of this submission for which copyright
protection is or may be available in any jurisdiction. The
copyright owner has no objection to the facsimile reproduction by
anyone of the patent document or patent disclosure, as it appears
in the records of the Patent and Trademark Office. All other rights
are reserved, and all other reproduction, distribution, creation of
derivative works based on the contents, public display, and public
performance of the application or any part thereof are prohibited
by applicable copyright law.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0003] [Not Applicable]
FIELD OF THE INVENTION
[0004] The present invention relates to analysis of data of nucleic
acid amplification reactions. More specifically, in certain
embodiments the invention relates to an information system and
methods for making performing allelic discrimination and/or the
detection/discrimination of other nucleic acids using real-time
nucleic acid amplification including, but not limited to, PCR
analysis.
BACKGROUND OF THE INVENTION
[0005] Nucleic acid sequence analysis is becoming increasingly
important in many research, medical, and industrial fields (see,
e.g., Caskey (1987) Science 236: 1223-1228; Landegren et al. (1988)
Science, 242: 229-237; Arnheim et al. (1992) Ann. Rev. Biochem.,
61: 131-156, etc.). In particular, more than 2,000 conditions have
been identified as single-gene defects for which the risk of
producing affected offspring can be mathematically predicted. Among
these conditions in man include Huntington's chorea, cystic
fibrosis, .alpha..sub.1 antitrypsin deficiency, muscular dystrophy,
Hunter's syndrome, Lesch-Nyhan syndrome, Down's syndrome, Tay-Sachs
disease, hemophilias, phenylketonuria, thalasemias, and sickle-cell
anemia. In addition to various genetic diseases can be diagnosed
utilizing nucleic acid sequence analysis, various infectious
diseases can be diagnosed by the presence in a clinical sample of a
specific DNA sequence characteristic of the causative
microorganism. These include, but are not limited to bacteria,
viruses, and parasites. In addition, particular pathogen strains
(e.g., drug resistant pathogens) can be identified by nucleic acid
analysis. Also the identification of various nucleic acid
polymorphisms has utility for basic research, genotyping, and
forensics.
[0006] Current diagnostic techniques for the detection of known
nucleotide differences include: hybridization with allele-specific
oligonucleotides (ASO) (Ikuta, et al., Nucleic Acids Research 15:
797-811 (1987); Nickerson, et al., PNAS (USA) 87: 8923-8927 (1990);
Saiki, et al., PNAS (USA) 86: 6230-6234 (1989); Verlaan-de Vries,
et al., Gene 50: 313-320 (1980); Wallace, et al., Nucleic Acids
Research 9:879-894 (1981); Zhang, Nucleic Acids Research 19:
3929-3933 (1991)); allele-specific PCR (Gibbs, et al., Nucleic
Acids Research 17: 2437-2448 (1989); Newton, et al., Nucleic Acids
Research 17: 2503-2516 (1989)); solid-phase minisequencing
(Syvanen, et al., American Journal of Human Genetics 1993; 52:
46-59 (1993)); oligonucleotide ligation assay (OLA) (Grossman, et
al., Nucleic Acids Research 22: 4527-4534 (1994); Landegren, et
al., Science 241: 1077-1080 (1988)); and allele-specific ligase
chain reaction (LCR) (Abravaya, et al. (1995) Nucleic Acids Res.
23: 675-682; Barany, et al. (1991) Proc. Natl. Acad. Sci., USA, 88:
189-193; Wu, et al., (1989) Genomics 4: 560-569). Genomic DNA is
analyzed with these methods by the amplification of a specific DNA
segment followed by detection analysis to determine which allele is
present.
[0007] The routine use of nucleic acid amplification reactions for
allelic detection/discrimination, particularly in clinical
settings, has been hampered because the quantification of nucleic
acids is made more difficult or less accurate or both because data
captured during amplification reactions are often significantly
obscured by signals that are not generated in response to the
target nucleic acid (i.e., noise). Furthermore, the data captured
by many monitoring methods can be subject to variations and lack of
reproducibility due to conditions that can change during a reaction
or change between different instances of a reaction.
SUMMARY OF THE INVENTION
[0008] In certain embodiments this invention pertains to the
discovery that the use of the maximum value of an efficiency
related transform of amplification data provides an effective
analytical tool for distinguishing different nucleic acid targets
in such an amplification.
[0009] Accordingly in certain embodiments methods are provided for
discriminating two or more different target nucleic acids. These
methods typically involve providing data from one or more
amplification reactions comprising reagents to amplify two or more
different target nucleic acids from a single sample where the data
comprise signals comprising an amplitude measurement representing
the degree of amplification of each target nucleic acid in the
amplification reaction and the time point in the amplification
reaction at which the amplitude is measured where the signal
provides such data for a multiplicity of time points in the
amplification reaction(s); determining an efficiency related
transform of the data where the efficiency related transform
provides an amplitude measure that is related to the efficiency of
amplification in the reaction; determining an efficiency related
value for each target nucleic acid that is the maximum magnitude of
the efficiency related transform determined for that target nucleic
acid; and outputting to a display, printer, or storage medium the
efficiency related values for each target nucleic acid, where the
relative amplitudes of the efficiency related values for each
target nucleic acid is an indicator of the presence of each of the
nucleic acids in the sample. In certain embodiments the reagents to
amplify two or more target nucleic acids are in a single
amplification reaction. In certain embodiments the reagents to
amplify two or more target nucleic acids distributed/segregated so
that each amplification reaction comprises reagents to amplify
different target nucleic acids. In certain embodiments reactions
are run in one combined reaction mix and in other segregated
reaction mixes. In certain embodiments the providing comprises
reading a data file from a PCR reaction, or real-time monitoring of
a PCR reaction, or receiving such values through a network
connection. In various embodiments the time points in the
amplification reaction are measured in cycle number or in reaction
time. In certain embodiments the methods involve discriminating at
least 3, or at least 4 or at least 5 or at least 6 different target
nucleic acids. In certain embodiments the target nucleic acids
comprise a first nucleic acid derived from a first allele of a gene
and a second nucleic acid derived from a second allele of the gene.
In certain embodiments the outputting comprises outputting
information indicating whether the sample is homozygous for the
first allele, homozygous for the second allele or heterozygous for
both alleles. In various embodiments the efficiency related
transform is selected from the group consisting of the ratio
transform of the signals, the shifted ratio transform of the
signals, the first derivative of the signals, the differences
between sequential signals, and the slope or gradient of the log of
the signals. In certain embodiments the efficiency related
transform (ERT) is calculated as:
ERT=(Signal.sub.n+1/Signal.sub.n)-1 (a)
or
ERT=(Signal.sub.n/Signal.sub.n-1)-1 (b)
where Signal.sub.n is the signal produced at cycle number n,
Signal.sub.n+1 is the signal produced at the subsequent cycle
number, Signaln-1 is the signal produced at the previous cycle
number, and n ranges from 1 up to the number of amplification
cycles analyzed in the reaction for formula (a) and n ranges from 2
up to the number of amplification cycles -1 analyzed in the
reaction for formula (b). In certain embodiments the efficiency
related value is the maximum of the efficiency related transform,
or the maximum gradient of the log of the amplification response,
or the maximum ratio of the amplification response, or the maximum
first derivative of the amplification response. In certain
embodiments additional signal values are generated by interpolating
points between the measured signal values (e.g., using cubic
splines). In certain embodiments the efficiency related transform
additionally provides a measure of the time or cycle number in the
amplification reaction(s). In certain embodiments the method
further comprises calculating a reaction point that is the
fractional cycle number or time at which the maximum magnitude of
the efficiency related transform occurs. In certain embodiments the
method further comprises calculating an adjusted reaction point
(e.g., an adjusted reaction point equal to the reaction point minus
the log base 2 of the efficiency related value). In certain
embodiments the adjusted reaction point is equal to the reaction
point minus the log base 2 of the signal intensity above
background. In certain embodiments the determining an efficiency
related value for each target nucleic acid that is the maximum
magnitude comprises identifying a peak in the efficiency related
transform as a function of time or cycle number. The method can
then further comprise determining the width of the peak; comparing
the width of the peak to a selected range of acceptable peak
widths; and outputting to a display, printer, or storage medium and
indicator identifying the nucleic acid amplification reaction as
possibly abnormal if the peak width determined is greater than or
less than a selected range of acceptable peak widths. In certain
embodiments the peak width is calculated using only efficiency
related transforms that occur at or before the reaction point value
of the efficiency related value. In various embodiments the
amplification reaction is performed with a set of probes that
comprises a FRET probe that is complementary to all or a portion of
one of the amplified target nucleic acids. In certain embodiments
the amplification reaction is performed with a set of probes that
comprise a molecular beacon that is complementary to all or a
portion of one of the amplified target nucleic acids. In certain
embodiments the providing data comprises a modality selected from
the group consisting of reading a data file containing the data,
receiving the data from a network connection or feed, and receiving
the data from an amplification reaction in realtime.
[0010] In various embodiments this invention also provides a
machine-readable medium that provides instructions that, if
executed by a machine, will cause the machine to perform operations
comprising the analyses as described herein.
[0011] Also provided is a system comprising a device for performing
a nucleic acid amplification and providing output signals that
comprises a measure of the time point of the reaction, and the
magnitude of the amplification of a target nucleic acid; a
processor operably coupled to said device; and a machine-readable
medium as described herein.
DEFINITIONS
[0012] The term "target nucleic acid" refers to a nucleic acid
(often derived from a biological sample), that the amplification
reaction is designed to amplify and or detect and/or quantify. It
is either the presence or absence of the target nucleic acid that
is to be detected, or the amount of the target nucleic acid that is
to be quantified. In various embodiments, the term "target nucleic
acid" refers to a nucleic acid all or a portion of which is to be
amplified. Thus the target nucleic acid can comprise the template
for an amplification reaction or a nucleic acid derived
therefrom.
[0013] As used herein, the term "derived from a nucleic acid"
refers to a nucleic acid nucleic acid for whose synthesis the
referenced nucleic acid or a subsequence thereof has ultimately
served as a template. Thus, for example, a DNA reverse transcribed
or RT-PCR'd from an mRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the mRNA. A DNA amplified from a
template comprising a gene, a DNA reverse transcribed from the
transcript of that gene, a DNA amplified from the reverse
transcript are all derived from that gene (nucleic acid).
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 illustrates a ratio transform showing MR, FCN and
width definition.
[0015] FIGS. 2A and 2B show the results of a real time PCR allelic
discrimination analysis using a conventional Ct analysis (FIG. 2A)
and a MaxRatio analysis (FIG. 2B).
[0016] FIG. 3 shows the results of a real time PCR allelic
discrimination analysis using a conventional Ct analysis (upper
panels) and a MaxRatio analysis (lower panels).
[0017] FIG. 4 shows a plot illustrating ratio transform of reaction
target and control data according to embodiments of this
invention.
[0018] FIG. 5 is a plot illustrating shifted ratio transform of
reaction target and control data according to embodiments of this
invention.
[0019] FIG. 6 shows the analysis by maxRatio of RealTime HIV-1
assay amplification plots. HIV-1 RNA ranging from 7.44 log 10
copies/mL to 1.56 log 10 copies/mL were tested in replicates of
four using the m2000sp and m2000rt instruments. (A) Amplification
plots of the HIV-1 normalized FAM fluorescence versus cycle number.
(B) Corresponding plots after applying the ratio transformation.
(C) Plot of MR versus FCN values derived from the peaks of the
ratio responses.
[0020] FIG. 7 shows reaction data for a number of target nucleic
acids plotted as a function of cycle number (top panel) and ratio
transforms of these data (bottom panel).
[0021] FIG. 8 from parent is a plot illustrating reaction data
showing target and control data that have been scaled according to
certain embodiments of this invention.
[0022] FIG. 9 illustrates an example of a user interface displaying
an FCN-MR plot according to embodiments of this invention.
[0023] FIG. 10 illustrates an example of a user interface
displaying a shifted ratio plot according to embodiments of this
invention.
[0024] FIG. 11 is a block diagram showing a representative example
of a logic device in which various aspects of the present invention
may be embodied.
[0025] FIG. 12 shows a flowchart for an illustrative embodiment of
the methods of this invention.
DETAILED DESCRIPTION
[0026] This invention pertains to improved methods of detecting and
discriminating closely related nucleic acid in a nucleic acid
amplification reaction. The methods are easily implemented using
conventional technology and are effectively detect and discriminate
even single nucleotide differences thereby provide powerful methods
for allelic discrimination, the detection of single nucleotide
polymorphisms, and the like.
[0027] The method are applicable to the analysis of multiple target
nucleic acids (e.g., different alleles of a gene) in a single
amplification reaction. Typical allelic discrimination assays are
multiplexed amplification assays comprising where at least two
different target nucleic acids are amplified in the same reaction
mixture. In various embodiments the multiplexed reaction mixture
contain reagents to amplify at least 3, at least four, or at least
5 different target nucleic acids.
[0028] Conventional" allelic discrimination analysis is performed
using an "end-point" assay system which attempts to determine the
"amount" of amplification by measuring the amount of fluorescence
(signal) generated for each target nucleic acid (e.g., allele) in
the reaction, which should relate to whether that target nucleic
acid is present. Total fluorescence generated in a PCR reaction,
however, is not necessarily well related to efficiency of
amplification. A higher concentration but less efficient
amplification can generate more fluorescence than a higher
efficiency but lower concentration amplification In addition, final
fluorescence is generally determined after the PCR reaction has
gone beyond the exponential amplification region where other
aspects of the reaction can significantly affect performance. For
this reason, final fluorescence levels are variable indicators of
amplification. In addition in order to get adequate fluorescence
measurements, a series of pre and post PCR fluorescence reads are
required which increases the processing time.
[0029] More particularly, previous analysis methods primarily
concentrate on quantitative responses that involve cycle number
determination. These approaches provide a quantitative assessment
by focusing on one portion of the amplification growth curve,
namely the region of observed exponential growth. The cycle
threshold or Ct-method (Heid et al. (1996) Genome Res., 6: 986-989)
determines a cycle number based on the point where the fluorescence
response grows above the background level to cross a predetermined
fluorescence threshold value. The critical steps involved in Ct
determination include defining the baseline and establishing a
suitable threshold for quantification of the target for use with
either an external calibration curve or an internal quantitation
standard. However, these methods are challenged when the growth
curve signal exhibits anomalous features. In such cases, analysis
often requires some measure of interpretation on the part of the
data reviewer to assess whether a particular response is truly an
amplification or not.
[0030] In contrast, the present invention utilizes an efficiency
related transform (ERT) of amplification signals where the
efficiency related transform provides a measure of the time or
cycle number in the amplification reaction and an amplitude measure
that is related to the efficiency of amplification in said
reaction. It was a surprising discovery that the use of such
efficiency related transforms in the analysis/discrimination of
related nucleic acid targets provides improved sensitivity and
discrimination of the targets.
[0031] In various embodiments the efficiency related transform
involves the calculation of a ratio between sequential
amplification measurements thereby yielding a series of ratios,
each of which can be indexed to a time value or cycle number. In
various embodiments amplification efficiency related values (MR
values) are determined in the early cycles as the amplification
rises above the background. Because these MR values are determined
while the reaction is still near exponential, they are more
directly related to amplification efficiency and provide better
discrimination between target nucleic acids than conventional Ct
analyses (see, e.g., FIGS. 2A, 2B, and 3) and are more useful for
determining AD or SNP calls than total fluorescence. MaxRatio
analysis uses most of the measurements from a real-time PCR
reaction. For this reason, there is the ability to make
measurements of the quality and validity of the PCR amplification
not available in the total fluorescence method. In addition, using
MR values only requires the PCR cycling protocol and eliminates the
need the pre and post reads significantly reducing processing
time.
I. Amplification Methods
[0032] The methods described herein are useful in discriminating
related target nucleic acid sis any of a number of amplification
methods.
[0033] Many systems have been developed that are capable of
amplifying and detecting nucleic acids. Similarly, many systems
employ signal amplification to allow the determination of
quantities of nucleic acids that would otherwise be below the
limits of detection. The present invention can utilize any of these
systems, provided that a signal indicative of the presence of a
nucleic acid or of the amplification of copies of the nucleic acid
can be measured in a time-dependent or cycle-dependent manner. Some
preferred nucleic acid detection systems that are useful in the
context of the present invention include, but are not limited to,
PCR, LCR, 3SR, NASBA, TMA, and SDA.
[0034] Polymerase Chain Reaction (PCR) is well-known in the art and
is essentially described in Saiki et al. (1985) Science 230:
1350-1354; Saiki et al. (1988) Science 239: 487-491; and in U.S.
Pat. Nos. 5,538,848; 5,723,591; and 5,876,930, and other
references. PCR can also be used in conjunction with reverse
transcriptase (RT) and/or certain multifunctional DNA polymerases
to transform an RNA molecule into a DNA copy, thereby allowing the
use of RNA molecules as substrates for PCR amplification by DNA
polymerase (see, e.g., Myers et al. (1991) Biochem. 30:
7661-7666).
[0035] Ligation chain reactions (LCR) are similar to PCR with the
major distinguishing feature that, in LCR, ligation instead of
polymerization is used to amplify target sequences. LCR is
described inter alia in European Patent 320 308; and by Landegren
et al. (1988) Science 241(4869): 1077-1080; by Wu et al. (1989)
Genomics 4(4): 560-569, and the like. In some advanced forms of
LCR, specificity can be increased by providing a gap between the
oligonucleotides, which gaps must be filled in by
template-dependent polymerization. This can be especially
advantageous if all four dNTPs are not needed to fill the gaps
between the oligonucleotide probes and all four dNTPS are not
supplied in the amplification reagents. Similarly, rolling circle
amplification (RCA) is described by Lisby (19999) Mol. Biotechnol,
12(1): 75-99), Hatch et al. (19999) Genet. Anal. 15(2): 35-40, and
others, and is useful in the context of the present invention.
[0036] Isothermal amplification reactions are also known in the art
and useful in the context of the present invention. Examples of
isothermal amplification reactions include 3SR as described by Kwoh
et al. (1989) Proc. Natl. Acad. Sci., USA, 86: 1173-1177 and
further developed in the art; NASBA as described by Kievits et al.
(1991) J. Virol. Meth. 35: 273-286, and further developed in the
art; and Strand Displacement Amplification (SDA) method as
initially described by Walker et al. (1992) Proc. Natl. Acad. Sci.,
USA, 89: 392-396 and U.S. Pat. No. 5,270,184, and further developed
in the art.
[0037] Thus, many amplification or detection systems requiring only
that signal gains indicative of the quantity of a target nucleic
acid can be measured in a time-dependent or cycle-dependent manner
are useful in the context of the present invention. Other systems
having these characteristics are known to the skilled artisan, and
even though not discussed above, are useful in the context of the
present invention.
[0038] For clarity, the invention will be illustrated with
reference to real-time PCR reactions, however, it will be
recognized that the methods are equally applicable to other
amplification systems including, but not limited to the other
amplification systems describe herein.
[0039] Real-time PCR combines amplification of nucleic acid (NA)
sequence targets with substantially simultaneous detection of the
amplification product. Optionally, detection can be based on
fluorescent probes or primers that are quenched or are activated
depending on the presence of a target nucleic acid. The intensity
of the fluorescence is dependent on the concentration or amount of
the target sequence in a sample (assuming, of course, that the
quantity of the target is above a minimal detectable limit and is
less than any saturation limit). This quench/fluoresce capability
of the probe allows for homogeneous assay conditions, i.e., all the
reagents for both amplification and detection are added together in
a reaction container, e.g., a single well in a multi-well reaction
plate. Electronic detection systems, target-capture based systems,
and aliquot-analysis systems and techniques are other forms of
detection systems useful in the context of the present invention so
long as a given system accumulates data indicative of the quantity
of target present in a sample during various time points of a
target amplification reaction.
[0040] In allelic discrimination systems, the amplification is
multiplexed. That is, each reaction typically comprises primers
that specifically amplify at least two different target nucleic
acids. In addition, the systems typically include probes for the
detection of the amplification products.
[0041] In PCR reactions, the quantity of target nucleic acid
doubles at each cycle until reagents become limiting or are
exhausted, there is significant competition, an inadequate supply
of reactants, or other factors that accumulate over the course of a
reaction. At times during which a PCR reaction causes doubling
(exactly) of the target in a particular cycle, the reaction is said
to have an efficiency (e) of 1 (e.g., e=1). After numerous cycles,
detectable quantities of the target can be created from very small
and initially undetectable quantity of target. Typically, PCR
cycling protocols consist of between around 30-50 cycles of
amplification, but PCR reactions employing more or fewer cycles are
known in the art and useful in the context of the present
invention.
[0042] In the real-time PCR reactions described below to illustrate
the present invention, the reaction mixture includes an appropriate
reagent cocktail of oligonucleotide primers, fluorescent
dye-labeled oligonucleotide probes capable of being quenched (or
de-quenched) when not bound to a complementary target nucleic acid,
or intercalating dyes, amplification enzymes, deoxynucleotide
triphosphates (dNTPs), and additional support reagents. Also, a
second fluorescent dye-labeled oligonucleotide probe for detection
of an amplifiable "control sequence" or "internal control" and a
"reference dye", which optionally may be attached to an
oligonucleotide that remains unamplified throughout a reaction
series, can optionally be added to the mixture for a real-time PCR
reaction. Thus, some real-time PCR systems use a minimum of three
fluorescent dyes in each sample or reaction container (e.g., a
well).
[0043] In various amplification systems, particularly where
multiple target nucleic acids are amplified (e.g., in allelic
discrimination), it is often desirable to multiplex amplification
reactions. Thus a single amplification reaction can include primers
to amplify and probes to detect two or more, in certain
embodiments, three or more, four or more, five or more different
target nucleic acids. In such systems probes and/or labels are
selected to provide a different an distinguishable signal for the
amplification produce of each target nucleic acid.
[0044] While allelic discrimination reactions (e.g., reactions to
determine the presence of two or more closely related nucleic
acids) are often performed in multiplexed amplification reactions
such multiplexing is not required. Thus for example different
target nucleic acids can be detected in different reaction mixes
(e.g., in different wells on a PCR plate). Also combinations of
multiplexed and individual target amplification reactions can be
utilized. Thus for example, three alleles can be detected using one
reaction mix for all three targets, using a different reaction mix
for each target nucleic acid, or using one reaction for two target
nucleic acids and a second amplification reaction for the third
target nucleic acid. In the various multi-reaction analyses, it is
desirable that the target nucleic acids be derived from the same
sample.
[0045] Systems that plot and display data for each of one, or
possibly more, reactions (e.g., each well in a multi-well plate)
are also useful in the context of the present inventions. These
systems optionally calculate values representing the fluorescence
intensity of the probe as a function of time or cycle number
(C.sub.N) or both as a two-dimensional plot (y versus x). Thus, the
plotted fluorescence intensity can optionally represent a
calculation from multiple dyes (e.g., different probe dyes, and/or
optional control dyes and/or optional reference dyes) and can
include subtraction of a calculated background signal. In PCR
systems, such a plot is generally referred to as a PCR
amplification curve and the data plotted can be referred to as the
PCR amplification data.
[0046] In PCR, data analysis can be made difficult by a number of
factors. Accordingly, various steps can be performed to account for
these factors. For example, captured light signals can be analyzed
to account for imprecision in the light detection itself. Such
imprecision can be caused by errors or difficulties in resolving
the fluorescence of an individual dye among a plurality of dyes in
mixture of dyes (described below as "bleedover"). Similarly, some
amount of signal can be present (e.g., "background signal") and can
increase even when no target is present (e.g., "baseline drift").
Thus, a number of techniques for removing the background signal,
preferably including the baseline drift, trend analysis, and
normalization are described herein and/or are known in the art.
These techniques are useful but are not required in the context of
the present invention. (Baseline drift or trending can be caused by
many sources, such as, for example, dye instability, lamp
instability, temperature fluctuations, optical alignment, sensor
stability, or combinations of the foregoing. Because of these
factors and other noise factors, automated methods of identifying
and correcting the baseline region are prone to errors).
[0047] As used herein, nucleic acid amplification reaction can
refer both to amplification of a portion of the sequence of a
target nucleic acid and to amplification and accumulation of a
signal indicative of the presence of a target nucleic acid, with
the former often being preferred to the latter.
II. Analytic Methods
[0048] The real time PCR (or other amplification) curve is a
fluorescence response with a roughly sigmoidal shape that
correlates to the growth of amplified product during the PCR
amplification process. The shape of the PCR amplification curve
reflects the dynamics of the PCR reaction for an individual sample
which is uniquely controlled by the assay design which includes
reactive components (primer and probe designs and concentrations,
concentrations for enzymes, activators, buffers, dNTPs, etc.) and
cycling conditions for the reaction. Traditional real time PCR data
reduction methods utilize the Ct method. The Ct method utilizes a
threshold, which is chosen to be fairly close to the baseline
signal level that corresponds to the exponential growth region of
the PCR curve. The interpolated cycle at which the signal rises
above the threshold is the Ct value for the curve. The Ct method is
an excellent method for providing quantitative PCR analysis because
of the consistency in signal intensity during the exponential
growth phase of the PCR. However, it is susceptible to error when
challenged with signal anomalies such as spectral crosstalk or
discontinuities due to bubbles or noise. In order to detect in the
exponential growth region of the PCR curve, a low threshold is
required. With a low threshold, it is difficult to discriminate
between a false threshold crossing due to an anomalous signal,
e.g., spectral crosstalk, which results in a Ct error and a true
signal Ct value. Even small errors in the baselining process can
cause even negative reactions to cross the threshold or for
reactive signals to cross early or late.
[0049] Accordingly in various embodiments, the methods of this
invention utilize a MaxRatio method that involves the ratio between
sequential measurement in the amplification reaction. In this
method, the ratio between sequential measurements is calculated,
thereby yielding a series of ratios, each of which can be indexed
to a time value or cycle number. Many suitable means of calculating
these ratios exist, and any suitable means can be used. The
simplest way of performing this ratio calculation utilizes the
following function:
Ratio ( n ) = s ( n + 1 ) s ( n ) I ##EQU00001##
where n represents the cycle number and s(n) represents the signal
at cycle n. This calculation provides a curve that starts at
approximately 1 in the baseline region of the response, increases
to a maximum during the growth region, and returns to approximately
1 in the plateau region. A MATLAB expression that performs this
calculation efficiently is the following:
Ratio=s(2:end,:)./s(1:end-1,:),
where "s" represents the signal response matrix, with each column
representing a separate response.
[0050] FIG. 4 shows an example of this ratio transform. Because of
the intrinsic background fluorescence, the ratio does not reach 2
as would be expected of a PCR reaction if the signal were doubling.
Regardless, the magnitude of the peak is independent of
multiplicative intensity variations and is proportional to the rate
of growth or efficiency at that point. The method of calculating
ratios is simple and efficiently calculated. Other equivalent
calculations could be made. An example would involve calculating
the forward and reverse ratios and then averaging them. On can use
the inverse of the ratio, in which case the curve will begin at a
value of approximately 1 in the baseline region, decrease in the
growth region, and return to a value of approximately 1 in the
plateau region. One would then use the magnitude and location of
the trough instead of a peak for analysis. This transform can be
implemented in a manner essentially equivalent to the ratio
method.
[0051] Although the MaxRatio algorithm is usable as described, it
is convenient to shift the curve by subtracting a constant, e.g.,
about one (1), from each point. This operation provides a
transformation of the original response, which starts near zero in
the baseline region, rises to a peak in the growth region of the
curve, and returns near zero in the plateau region (see, e.g., FIG.
1). This shifted ratio calculation is described by the following
function:
Ratio n = Signal n Signal n - 1 - 1 II ##EQU00002##
where Signal.sub.n is the measured real-time PCR fluorescence
response for the target of interest at cycle n. The ratio
calculation transforms the roughly sigmoidal shaped amplification
curve to a ratio curve with a well-defined peak. FIG. 1 illustrates
this transformation. The ratio curve exhibits several well-defined
features.
[0052] The maximum value of the ratio curve defines two values. The
cycle number at which the maximum occurs is defined as the FCN
value or fractional cycle number. The magnitude of the ratio curve
at the maximum is defined as the MR (maxRatio) value. The ratio
curve has a characteristic width, measured as the half width at
half maximum, referred to as the width parameter.
[0053] The ratio curve is a relative measure of the fluorescence
signal growth throughout the PCR reaction. The early cycle ratio
curve near zero represents the baseline region of the PCR curve and
the late cycle region corresponds to the plateau phase. The
ascending part of the ratio curve corresponds to the exponential
growth phase; the descending part of the ratio curve is the
transition from the exponential to the plateau phase in the PCR
curve. The ratio equation is similar to the equation for the PCR
reaction efficiency (Peirson et al. (2003) Nucleic Acids Res.,
31(14 e73)) at cycle n.
Efficiency n = .DELTA. R n .DELTA. R n - 1 - 1 III ##EQU00003##
Where .DELTA.R.sub.n is the baselined PCR signal intensity at cycle
n. In practice, applying equation III to real amplification
responses is problematic. Because the baselined PCR signal is
approximately zero in the baseline portion of the curve, equation
III suffers from division by zero problems. In addition, even
trivial background slope variations cause significant changes in
efficiency measurement in the exponential region. The signal value,
Signal.sub.n, in the ratio equation II includes the PCR signal
intensity and the inherent background fluorescence level. As such,
MR values are a relative measure of reaction efficiency. The
magnitude of the MR value even for a perfectly efficient reaction
is always less than one because of the inherent level of background
fluorescence incorporated in the ratio equation. By including the
background fluorescence in the ratio equation, the resulting ratio
curve avoids division by zero problems and is highly insensitive to
even moderate baseline slope variation.
[0054] FIG. 1 illustrates this the relationship of this calculation
to simple Ct analysis, while FIG. 5 shows real output of this
shifted ratio calculation. The reaction point and magnitude of the
peak of the shifted ratio curve is then determined. The reaction
point (i.e., distance along the x-axis) specifies the FCN value of
the MR and the magnitude specifies the efficiency related value MR
(Maximum of the Ratio).
[0055] FIG. 6 illustrates an example dilution series of Abbott
RealTime.TM. HIV-1 normalized FAM fluorescence processed with the
maxRatio algorithm. Plotting the MR versus FCN values generates the
characteristic MR-FCN plot for this data.
[0056] FIG. 6 represents amplification plots for a run of reactive
samples. The only negative response is from the negative control,
which is identified in the MR versus FCN plot with an MR value near
zero. Since there is no signal growth in reactions without target,
the ratio curve is nearly equal to zero throughout the
amplification process. The MR value of approximately zero easily
distinguishes the negative response from all the reactive samples.
In practice a line can easily be established to separate these two
populations of responses. This line is called the Ratio
Threshold.
[0057] It will be appreciated that there are equivalent ratio
calculations that provide a similar or essentially identical
result. For example, a ratio calculation essentially equivalent to
Formula II is:
Ratio n = Signal n + 1 Signal n - 1 ##EQU00004##
[0058] This is meant to be illustrative and not limiting. Using the
teachings provided herein, other efficiency related transforms, in
particular ratio calculations will be available to one of skill in
the art.
[0059] In one illustrative, but not limiting embodiment, the
maxRatio method is implemented as part of the Abbott m2000 system.
Because the m2000 system has an effective automatic baselining
algorithm, baseline slope correction (but not offset) is applied.
Although normalization and baseline slope correction are not
required by the maxRatio method, a small but significant
improvement in performance is achieved using them. In addition, the
signal has a smoothing filter applied. It is a feature of the
maxRatio method that a much more aggressive noise filter can be
applied without significantly affecting the cycle number compared
to the Ct method. The m2000rt instrument implements a fourth order,
zero-phase noise filter. In order to obtain 0.01 cycle resolution,
a cubic spline interpolation can be applied to the ratio curve.
[0060] It has been found that for assay responses with suppressed
signal levels, the FCN value can shift slightly early. In order to
provide more linear results, the adjusted FCN (FCNA) value can,
optionally, be calculated using formula IV.
FCNA=FCN-Log.sub.2(MR) IV
[0061] Because the ratio transformation is inherently
self-compensating for reaction signal intensity, it can be applied
to a reaction's raw fluorescence signal. When a reference dye is
available, the normalized fluorescence signal can be analyzed. It
should be noted that the fluorescence signal naturally has a
background level of unquenched fluorescence. Because of the
division in the ratio transformation, it is necessary to maintain
this background fluorescence level to avoid division by zero. As an
alternative to utilizing the raw or normalized fluorescence
response directly, the response may be shifted to fixed positive
background fluorescence level. The advantage of this response
shifting is to eliminate sensitivity to factors that can change the
level of background fluorescence such as variability in probe
manufacture or fluorescence contamination in the thermal cycler
block. The disadvantage to shifting the response is that it removes
the inherent insensitivity to signal intensity and can introduce
some instrument-to-instrument variability. For this reason, if
shifting is implemented, using a shift value near the natural level
of background fluorescence is recommended. It should be noted that
shifting will directly affect the magnitude of the MR value.
Shifting to a low value will increase both the MR value of positive
reactions as well as the mean and standard deviation of the MR for
negative reactions. In terms of statistical separation of
populations, this rarely makes significant difference. However
shifting to a low level can reduce robustness to spectral
crosstalk, initial signal transients and other anomalies in the
baseline portion of the amplification response. It is important
therefore when developing the assay, to focus on separation of
reactive from non-reactive populations by MR, not on maximizing the
MR value.
III. Discriminating Alleles or Other "Related" Nucleic Acids
[0062] As indicated above, the above-described analytic methods are
particular valuable in detecting/discriminating related nucleic
acids (e.g., different alleles of a gene, strain variants of a
pathogen, single nucleotide polymorphisms, and the like). In such
assays, a nucleic acid sample is derived (obtained) from a
biological sample. The term "biological sample" refers to sample
that comprises a biological tissue, cell, fluid, pathogen, and the
like that contains a nucleic acid that is to be detected/screened
according to the assays described herein. Such samples include, but
are not limited to, cultured cells, primary cell preparations,
sputum, amniotic fluid, blood, tissue or fine needle biopsy
samples, urine, peritoneal fluid, and pleural fluid, or cells
therefrom. Biological samples can also include samples of pathogens
(e.g., bacteria, viruses, parasites, etc.) that are either in
primary samples (e.g., taken from an organism) or in samples that
have been cultures. Biological samples may also include sections of
tissues (e.g, frozen sections taken for histological purposes), and
the like. The sample may be pretreated as necessary by dilution in
an appropriate buffer solution or concentrated, if desired. Any of
a number of standard aqueous buffer solutions, employing one of a
variety of buffers, such as phosphate, Tris, or the like, at
physiological pH can be used.
[0063] In various embodiments, the sample used for amplification
can comprise genomic DNA and/or a nucleic acid derived from such.
Thus, for example in certain embodiments, the sample can comprise
an RNA, a DNA reverse transcribed from the RNA, and the like.
[0064] Amplification reactions are run according to standard
methods well known to those of skill in the art. Typically the
amplification reactions will be run with reagents (e.g., primers
and probes) to specifically detect the target nucleic acids of
interest. Thus, for example, where it is desired to detect
different alleles (SNPs, etc.) primers and probes will be selected
to amplify and detect all or part of the target nucleic acid. Where
only a fragment of the target nucleic acid is to be detected,
probes and primers are selected to amplify and detect that fragment
of the nucleic acid that is expected to differ between the
alleles.
[0065] The assays can be "multiplexed", or segregated, or both. In
a multiplexed assay, a single amplification reaction (reaction mix)
will contain primers and probes to amplify and detect at least two
(in certain embodiments, at least 3, at least 4, at least 5, at
least 6, etc.) different target nucleic acids. In segregated
assays, a separate amplification reaction (reaction mix) will be
used to amplify and detect each different target nucleic acid. In
certain embodiments certain amplification reaction(s) (reaction
mixes) can be used to each amplify and detect a single target
nucleic acid while simultaneously other amplification reaction(s)
(reaction mixes) each contain primers and probes to amplify and
detect at least two different target nucleic acids. In "segregated"
and "combined" assays it is desirable that the different
amplification reactions are performed on a nucleic acid derived
from the same sample.
[0066] Amplification data from the amplification reaction(s) can be
acquired (e.g., using a computer system) and analyzed (e.g., as
described above) to provide a measure of the presence and/or
quantity of each target nucleic acids. In allelic discrimination
analysis it is sometimes desirable to provide the analyzed
information as a scatter plot showing the amplified values of each
target nucleic acid (see, e.g., FIG. 2). In certain embodiments,
the resulting data can be statistically analyzed (e.g., using
cluster analysis, discriminant function analysis, and the like) to
optimize the separation and detection of each target nucleic
acid.
IV. Captured/Received Data
[0067] By way of example, a typical real-time PCR reaction
detection system generates a data file that stores the signal
generated from one or more detection dyes. These dyes can
represent, for example, amplification data for two or more
different target nucleic acids, and optionally, internal control
data, and optionally reference data. FIG. 7, top panel, illustrates
a plot of received/captured reaction data for a plurality of target
nucleic acids that can be used in an analytical method according to
the present invention. In this plot, the x-axis provides an
indication of cycle number (e.g., 1 to 40) and the y-axis indicates
dye intensity detected, in relative fluorescence units. In this
figure, the different data sets are illustrated as continuous
curves. However, the actual captured data values are generally
discrete signal values captured at each cycle number.
[0068] As shown in FIG. 7, bottom panel, the data can be
transformed (e.g., as described herein) using a ratio
transformation which can provide a maximum ratio value, and
optionally, a point at which the maximum ratio occurs, and
optionally a peak width (e.g., full width at half max).
V. Optional Error Correction
[0069] a) Normalization
[0070] Although optional, normalization can be performed on the
captured data in several different ways. One method involves
dividing the target and control values at each cycle reading by the
corresponding reference dye signal. Alternatively, the divisor can
be the average reference value over all cycles or an average over
certain cycles. In another alternative embodiment, the divisor can
be the average of the target dye or the control dye or the target
dye and the control dye over one or more earlier (baseline) cycles,
when no amplification signal is detected. Any known normalization
method can be employed in a data analysis. The invention can be
used with data that has already been normalized by a PCR
system.
[0071] Because normalization is optional, the present invention can
be used to analyze reaction data without the use of a normalization
or reference dye. Alternatively, the target signal or the control
signal or both can be used for normalization.
[0072] b) Scaling
[0073] Scaling is optional but can be performed to make it easier
for a human operator to visualize the data. Scaling does not affect
analytical results. Scaling can be carried out in addition to
normalization, in the absence of normalization, or before or after
normalization.
[0074] One method of scaling involves dividing each data set value
by the average of the values during some early cycles, generally in
the baseline region before any positive data signal is detected. In
this example, readings 4 through 8 were averaged and normalization
was performed first. FIG. 8 is a plot of reaction data showing
target and control data that have been scaled. In this example,
scaling forces the early values of the target and control to one,
and because the early values are less than one, the division forces
the later values to slightly larger pure numbers.
[0075] c) Digital Filtering
[0076] One or more digital filtering methods can be applied to the
captured data to "clean up" the signal data sets and to improve the
signal to noise ratio. Many different filtering algorithms are
known. The present invention can employ a four-pole filter with no
zeros. This eliminates the potential for overshoot of the filtered
signal. As an example, this can be implemented with the MATLAB
function "filtfilt" provided with the MATLAB Signal Processing
Toolbox, which both forward and backward filters to eliminate any
phase lag (time delays). An example of parameters and MATLAB
function call is as follows:
[0077] b=0.3164;
[0078] a=[1.0000 -1.0000 0.3750 -0.0625 0.0039];
[0079] data(:,:,assay)=filtfilt(b,a,data(:,:,assay));
[0080] data(:,:,ic)=filtfilt(b,a,data(:,:,ic));
[0081] In this example, "b" and "a" contain the filter
coefficients. "data(:,:,assay)" and "data(:,:,ic)" contain the
captured data that may or may not have been normalized, scaled, or
both. In this case, the filtered data is both normalized and
scaled.
[0082] d) Slope Removal/Baselining
[0083] An optional slope removal method can be used to remove any
residual slope that is present in the early baseline signal before
any detectable actual signal is produced. This procedure may also
be referred to as baselining, but in some embodiments, the offset
is not removed, only the slope. In certain embodiments, for slope
removal, both the target (DYE1) and, when present, control (DYE2)
signals are examined simultaneously. Whichever signal (when
present) comes up first defines the forward regression point, and
the method generally goes back 10 cycles. If 10 cycles back is
before cycle 5, then cycle 5 is used as the initial regression
point to avoid any earlier signal transients. A linear regression
line can be calculated using the signal data between these points
and the slope of the regression for each dye is subtracted from
that dye's signal. In this case, the slope removal is applied to
the normalized, scaled, and filtered data discussed above.
VI. Systems, Devices, and Software
[0084] The methods of this invention can be incorporated into a
multiplicity of suitable systems, computer products, and/or
information instruments. Some details of a MR software
implementation are provided below. Specific user interface
descriptions and illustrations are taken to illustrate specific
embodiments only and any number of different user interface methods
known in the information processing art can be used in systems
embodying this invention. The invention can also be used in systems
where virtually all of the options described below are preset,
calculated, or provided by an information system, and,
consequently, provide little or no user interface options. In some
cases, details and/or options of a prototype system are described
for illustrative purposes; many of these options and/or details may
not be relevant or available for a production system.
[0085] Furthermore, software embodiments can include various
functionalities, such as, for example, processing reactions with
two, three, four, or five or more target reactions, and,
optionally, or one or more internal control reactions, or reference
data, or combinations of the foregoing. A software system suitable
for use in this invention can provide any number of standard file
handling functions such as open, close, printing, saving, etc.
[0086] A) Illustrative User Interface.
[0087] FIG. 9 illustrates a user interface for processing PCR
allelic discrimination data according to this invention. In this
interface, the selection of appropriate dye(s) corresponding to the
various targets (e.g., target 1 (allele 1), target 2 (allele 2),
and the like), and optional internal control, and reference
responses are selected from popup lists as shown in the window.
Tabs for selecting different viewing options (e.g., MR-FCN plot,
shifted ratio curve, scatter plot of target signal as a function of
target, etc.) are positioned in the middle of the window and are
arranged horizontally. FIG. 9 shows that the tab displaying the
MR-FCN plot has been selected. FIG. 10 illustrates a user interface
showing the same data for well 1, but displaying the shifted ratio
curve. Other tabs allow viewing of the raw fluorescence data,
normalized fluorescence, baselined data, and the like for all the
responses. Drop-down selectors are provided to permit selection of
each dye (target). In addition, a tab allows inspection of each
response individually. Fields to the right of the plot show
calculated response values such as MR, FCN, C.sub.t, and standard
deviation in the baseline region. Below these calculated values are
radio buttons allowing the user to display either the assay data,
internal control data, and the like.
[0088] B) Embodiment in a Programmed Information Appliance/Device
and/or System.
[0089] FIG. 11 is a block diagram schematically illustrating one
example of a logic device and/or system in which various aspects of
the present invention may be embodied. As will be understood from
the teachings provided herein, the invention can be implemented in
hardware or software or both. In some embodiments, different
aspects of the invention can be implemented in either hardware or
software and in either client-side logic or server-side logic.
Moreover, the invention or components thereof can be embodied in a
fixed media (e.g., a computer accessible/computer readable) program
component containing logic instructions or data, or both, that when
loaded into an appropriately configured computing device can cause
that device to perform operations to the invention. In various
embodiments a fixed media component containing logic instructions
can be delivered to a viewer on a fixed medium for physically
loading into a viewer's computer or a fixed medium containing logic
instructions can reside on a remote server that a viewer can access
through a communication medium in order to download a program
component.
[0090] As illustrated in FIG. 11, the system comprises an
information instrument or digital device 700 that can be used as a
logical apparatus for performing logical operations regarding image
display or analysis, or both, as described herein. Such a device
can be embodied as a general-purpose computer system or workstation
running logical instructions to perform according to various
embodiments of the present invention. Such a device can also be
customized and/or specialized laboratory or scientific hardware
that integrates logic processing into a machine for performing
various sample handling operations. In general, the logic
processing components of a device according to the present
invention are able to read instructions from media 717 or network
port 719, or both. The central processing unit can optionally be
connected to server 720 having fixed media 722. Apparatus 700 can
thereafter use those instructions to direct actions or perform
analysis as described herein. One type of logical apparatus that
can embody the invention is a computer system as illustrated in
700, containing CPU 707, optional input devices 709 and 711,
storage media 715, e.g., disk drives, and optional monitor 705.
Fixed media 717, or fixed media 722 over port 719, can be used to
program such a system and can represent disk-type optical or
magnetic media, magnetic tape, solid state dynamic or static
memory, etc. The invention can also be embodied in whole or in part
as software recorded on this fixed media. Communication port 719
can also be used to initially receive instructions that are used to
program such a system and represents any type of communication
connection.
[0091] FIG. 11 shows that the system can comprise a diagnostic
system or an amplification system. Thus, for example the system can
include an amplification device such as a thermocycler 785 and
optional sample handler 790 for loading and unloading the
thermocycler. These additional components can be components of a
single system that includes logic analysis and/or control. These
devices may also be essentially stand-alone devices that are in
digital communication with an information instrument such as 700
via a network, bus, wireless communication, etc., as will be
understood in the art. Components of such a system can have any
convenient physical configuration and/or appearance and can be
combined into a single integrated system. Thus, the individual
components shown in FIG. 11 represent just one example system.
[0092] C) Embodiment in a Computer-Accessible/Readable Medium.
[0093] As indicated above, in certain embodiments, this invention
contemplates a computer (machine) accessible/computer (machine)
readable medium that provides an instruction set that, if executed
by a machine (e.g., a computer processor), will cause the machine
to perform the various analytical operations described herein.
Thus, in certain embodiments, the machine-readable medium provides
instructions that, if executed by a machine, will cause the machine
to perform operations comprising: receiving signals from one or
more amplification reactions comprising reagents to amplify two or
more different target nucleic acids from a single sample where the
signals provide data comprising an amplitude measurement
representing the degree of amplification of each target nucleic
acid in the amplification reaction and the time point in the
amplification reaction at which the amplitude is measured, and
where the signal provides such data for a multiplicity of time
points in the amplification reaction(s); determining an efficiency
related transform of said data where said efficiency related
transform provides an amplitude measure that is related to the
efficiency of amplification in said reaction; determining an
efficiency related value for each target nucleic acid that is the
maximum magnitude of the efficiency related transform determined
for that target nucleic acid; and outputting to a display, printer,
or storage medium the efficiency related values and corresponding
points in the amplification reaction for each target nucleic acid,
where the relative amplitudes of the efficiency related values for
each target nucleic acid is an indicator of the presence of each of
said nucleic acids in said sample. One illustrative embodiment of
such instructions is shown in FIG. 12.
[0094] In various embodiments the machine readable medium comprises
any tangible medium capable of holding/storing an instruction set.
Such media include, but are not limited to a magnetic medium, a
flash memory, an optical memory, a DRAM, an SRAM, and the like.
[0095] D) Embodiment in Circuitry.
[0096] In various embodiments the invention can also be embodied in
whole or in part within the circuitry of an application specific
integrated circuit (ASIC) or a programmable logic device (PLD). In
such a case, the invention can be embodied in a computer
understandable descriptor language, which may be used to create an
ASIC, or PLD, that operates as described herein.
VII. Other Embodiments
[0097] The invention has been described with reference to specific
embodiments. Other embodiments will be apparent to those of skill
in the art. In particular, a viewer digital information appliance
has generally been illustrated as a computer workstation such as a
personal computer. However, the digital computing device is meant
to be any information appliance suitable for performing the logic
methods of the invention, and could include such devices as a
digitally enabled laboratory systems or equipment, digitally
enabled television, cell phone, personal digital assistant, etc.
Modification within the spirit of the invention will be apparent to
those skilled in the art. In addition, various different actions
can be used to effect interactions with a system according to
specific embodiments of the present invention. For example, a voice
command may be spoken by an operator, a key may be depressed by an
operator, a button on a client-side scientific device may be
depressed by an operator, or selection using any pointing device
may be effected by the user.
EXAMPLES
[0098] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0099] The Applied Biosystems SDS system performs allelic
discrimination using an end-point assay system which attempts to
determine the "amount" of amplification by measuring the amount of
fluorescence generated which should relate to whether that allele
is present. Total fluorescence generated in a PCR reaction is not
necessarily well related to efficiency of amplification. A higher
concentration but less efficient amplification can generate more
fluorescence than a higher efficiency but lower concentration
amplification In addition, final fluorescence is generally
determined after the PCR reaction has gone beyond the exponential
amplification region where other aspects of the reaction can
significantly affect performance. For this reason, final
fluorescence levels are variable indicators of amplification. In
addition in order to get adequate fluorescence measurements, the
SDS system makes a series of pre and post PCR fluorescence reads
which increases the processing time.
[0100] MaxRatio generated MR values are determined in the early
cycles as the amplification rises above the background Because
these MR values are determined while the reaction is still near
exponential, they are more directly related to amplification
efficiency and should be more useful for determining AD or SNP
calls than total fluorescence MaxRatio analysis uses most of the
measurements from a real-time PCR reaction. For this reason, there
is the ability to make measurements of the quality and validity of
the PCR amplification not available in the total fluorescence
method. In addition, using MR values would only require the PCR
cycling protocol and would eliminate the need the pre and post
reads significantly reducing processing time.
[0101] Assay runs from DVT SNP reactions were utilized to test the
methods described herein. The deep vein thromobosis prototype assay
consisted of identification of SNPs (Single Nucleotide
Polymorphisms) within 3 genes: Factor V (G1691A) ("Factor V
Leiden"), Factor II (G20210A) and MTHFR (C677T). The Factor V
Leiden mutation is the most common genetic risk for venous
thrombosis and pulmonary embolism, present in 5% of the Caucasian
population and in 20-40% of individuals with a history of venous
thromboembolism. Factor V Leiden heterozygotes are at a 7-fold
increased risk for venous thromboembolism. The Factor V Leiden
mutation is responsible for 85-95% of APC resistance. APC is a
natural anticoagulant that inactivates factors Va and VIIIa. The
Factor II (Prothrombin) mutation is associated with elevated
circulating levels of prothrombin. Greater availability of
prothrombin is believed to lead to greater conversion to thrombin
and an increased chance of thrombosis. The MTHFR (methylene
tetrahydrofolate reductase) C677T mutation is tentatively
associated with increased risk of venous thrombosis.
[0102] These files were processed in SDS for the allelic
discrimination results. Because these are known samples, calls were
predetermined. An SDS results report was generated. Component
fluorescence files were exported from SDS and run in MultiAnalyze
3.0 to generate MR values. SDS generated total fluorescence values
and MR values were imported into Excel for plot generation. Results
are shown in FIGS. 2A and 2B. FIG. 2A shows the results generated
by SDS. FIG. 2B shows the results generated using MaxRatio.
[0103] The MTHFR Major cluster is much more clearly separated from
the no template control (NTC) using MR values. In general, clusters
are at least as well separated using the MR method as with SDS.
[0104] A second set of comparisons is provided in FIG. 3. MaxRatio
clearly reduces the variability and provides a cleaner signal
(tighter clustering) which facilitates discrimination of the
alleles. It is noted that the assay conditions were optimized for
SDS and not for a maxratio analysis. It is believed that
optimization of assays for maxratio can provide even cleaner
results.
[0105] It is understood that the examples and embodiments described
herein are for illustrative purposes and that various modifications
or changes in light thereof will be suggested by the teachings
herein to persons skilled in the art and are to be included within
the spirit and purview of this application and scope of the
claims.
[0106] All publications, patents, and patent applications cited
herein or filed with this application, including any references
filed as part of an Information Disclosure Statement, are
incorporated by reference in their entirety
[0107] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
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