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`Ambry Exhibit 1002 - Page 1
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`Ambry Exhibit 1002 - Page 1
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`Ambry Exhibit 1002 - Page 2
`
`Ambry Exhibit 1002 - Page 2
`
`

`

`DIGITAL AMPLIFICATION
`
`This application is a continuation of US. Application Serial Number
`
`10/828,295 filed April 21, 2004, which is a divisional of US. Application
`
`Serial Number 09/981,356 filed October 12, 2001, now US Patent 6,753,147,
`
`which is a continuation of US. Application Serial Number 09/613,826 filed
`
`July 1 1, 2000, now US. Patent 6,440,706, which claims the benefit of
`
`provisional US. Application Serial Number 60/146,792, filed August 2, 1999.
`
`The disclosure of all priority applications is expressly incorporated herein.
`
`The US. government retains certain rights in this invention by virtue of
`
`its support of the underlying research, supported by grants CA 43460, CA
`
`57345, and CA 62924 from the National Institutes of Health.
`
`TECHNICAL FIELD OF THE INVENTION
`
`This invention is related to diagnostic genetic analyses.
`
`In particular it
`
`relates to detection of genetic changes and gene expression.
`
`BACKGROUND OF THE INVENTION
`
`In classical genetics, only mutations of the germ-line were considered
`
`importantfor understanding disease. With the realization that somatic
`
`mutations are the primary cause of cancer, and may also play a role in aging,
`
`new genetic principles have arisen. These discoveries have provided a wealth
`
`of new opportunities for patient management as well as for basic research into
`
`the pathogenesis of neoplasia. However, many of these opportunities hinge
`
`upon detection of a small number of mutant-containing cells among a large
`
`excess of normal cells. Examples include the detection of neoplastic cells in
`
`urine, stool, and sputum of patients with cancers of the bladder, colorectum,
`
`and lung, respectively. Such detection has been shown in some cases to be
`
`Ambry Exhibit 1002 - Page 3
`
`Ambry Exhibit 1002 - Page 3
`
`

`

`I)
`
`u
`
`possible at a stage when the primary tumors are still curable and the patients
`
`asymptomatic. Mutant sequences from the DNA of neoplastic cells have also
`
`been found in the blood of cancer patients. The detection of residual disease in
`
`lymph nodes or surgical margins may be useful in predicting which patients
`
`might benefit most from further therapy. From a basic research standpoint,
`
`analysis of the early effects of carcinogens is often dependent on the ability to
`
`detect small populations of mutant cells.
`
`Because of the importance of this issue in so many settings, many
`
`useful techniques have been developed for the detection of mutations. DNA
`
`sequencing is the gold standard for the detection of germ line mutations, but is
`
`useful only when the fraction of mutated alleles is greater than ~20%.
`
`Mutant-specific oligonucleotides can sometimes be used to detect mutations
`
`present in a minor proportion of the cells analyzed, but the signal to noise ratio
`
`distinguishing mutant and wild—type (WT) templates is variable. The use of
`
`mutant—specific primers or the digestion of polymerase chain reaction (PCR)
`
`products with specific restriction endonucleases are extremely sensitive
`
`methods for detecting such mutations, but it is difficult to quantitate the
`
`fraction of mutant molecules in the starting population with these techniques.
`
`Other innovative approaches for the detection of somatic mutations have been
`
`reviewed. A general problem with these methods is that it is difficult or
`
`impossible to independently confirm the existence of any mutations that are
`
`identified.
`
`Thus there is a need in the art for methods for accurately and
`
`quantitatively detecting genetic sequences in mixed populations of sequences.
`
`SUMMARY OF THE INVENTION
`
`It is an object of the present invention to provide methods for
`
`determining the presence of a selected genetic sequence in a population of
`
`genetic sequences.
`
`Ambry Exhibit 1002 - Page 4
`
`Ambry Exhibit 1002 - Page 4
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`

`

`I)
`
`It is another object of the present invention to provide molecular
`
`beacon probes useful in the method of the invention.
`
`These and other objects of the invention are achieved by providing a
`
`method for determining the presence of a selected genetic sequence in a
`
`population of genetic sequences. A biological sample comprising nucleic acid
`
`template molecules is diluted to form a set of assay samples. The template
`
`molecules within the assay samples are amplified to form a population of
`
`amplified molecules in the assay samples of the set. The amplified molecules
`
`in the assay samples of the set are then analyzed to determine a first number of
`
`assay samples which contain the selected genetic sequence and a second
`
`number of assay samples which contain a reference genetic sequence. The first
`
`number is then compared to the second number to ascertain a ratio which
`
`reflects the composition of the biological sample.
`
`Another embodiment of the invention is a method for determining the
`
`ratio of a selected genetic sequence in a population of genetic sequences.
`
`Template molecules within a set comprising a plurality of assay samples are
`
`amplified to form a population of amplified molecules in each of the assay
`
`samples of the set. The amplified molecules in the assay samples of the set are .
`
`analyzed to determine a first number of assay samples which contain the
`
`selected genetic sequence and a second number of assay samples which
`
`contain a reference genetic sequence. At least one-fiftieth of the assay samples
`
`in the set comprise a number (N) of moleculessuch that UN is larger than the
`
`ratio of selected genetic sequences to total genetic sequences required to
`
`determine the presence of the selected genetic sequence. The first number is
`
`compared to the second number to ascertain a ratio which reflects the
`
`composition of the biological sample.
`
`According to another embodiment of the invention, a molecular beacon
`
`probe is provided.
`
`It comprises an oligonucleotide with a stem-loop structure
`
`having a photoluminescent dye at one of the 5’ or 3’ ends and a quenching
`
`Ambry Exhibit 1002 - Page 5
`
`Ambry Exhibit 1002 - Page 5
`
`

`

`agent at the opposite 5’ or 3’ end. The loop consists of 16 base pairs which
`
`has a Tm of 50-51 DC. The stem consists of 4 base pairs having a sequence 5’-
`
`CACG-3’.
`
`A second type of molecular beacon probe is provided in another
`
`embodiment.
`
`It comprises an oligonucleotide with a stem-loop Structure
`
`having a photoluminescent dye at one of the 5’ or 3’ ends and a quenching
`
`agent at the opposite 5’ or 3’ end. The loop consists of 19—20 base pairs and
`
`has a Tm of 54-56DC. The stem consists of 4 base pairs having a sequence 5’-
`
`CACG-3’.
`
`Another embodiment provides the two types of molecular beacon
`
`probes, either mixed together or provided in a divided container as a kit.
`
`The invention thus provides the art with the means to obtain
`
`quantitative assessments of particular DNA or RNA sequences in mixed
`
`populations of sequences using digital (binary) signals.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1. Schematic of experimental design. (A) The basic two steps involved:
`
`PCR on diluted DNA samples is followed by addition of fluorescent probes
`
`which discriminate between WT and mutant alleles and subsequent
`
`fluorometry. (B) Principle of molecular beacon analysis. In the stem—loop
`
`configuration, fluorescence from a dye at the 5 ’ end of the oligonucleotide
`
`probe is quenched by a Dabcyl group at the 3’ end. Upon hybridization to a
`
`template, the dye is separated from the quencher, resulting in increased
`
`fluorescence. Modified from Marras et al.
`
`(C) Oligonucleotide design.
`
`Primers F1 and R1 are used to amplify the genomic region of interest. Primer
`
`INT is used to produce single stranded DNA from the original PCR products
`
`during a subsequent asymmetric PCR step (see Materials and Methods).
`
`MB-RED is a Molecular Beacon which detects any appropriate PCR product,
`
`Ambry Exhibit 1002 - Page 6
`
`Ambry Exhibit 1002 - Page 6
`
`

`

`II
`
`whether it is WT or mutant at the queried codons. MB—GREEN is a Molecular
`
`Beacon which preferentially detects the WT PCR product.
`
`FIG. 2. Discrimination between WT and mutant PCR products by Molecular
`
`Beacons. Ten separate PCR products, each generated from ~25 genome
`
`equivalents of genomic DNA of cells containing the indicated mutations of
`
`c-Ki-Ras, were analyzed with the Molecular Beacon probes described in the
`text. Representative examples of the PCR products used for Molecular
`
`I
`
`Beacon analysis were purified and directly sequenced.
`
`In the cases with
`
`Gly12Cys and GlylZArg mutations, contaminating non-neoplastic cells within
`
`the tumor presumably accounted for the relatively low ratios. In the cases with
`
`GlylZSer and Gly12Asp, there were apparently two or more alleles of mutant
`
`c-Ki-Ras for every WT allele; both these tumors were aneuploid.
`
`FIG. 3. Detecting Dig-PCR products with MB-RED. Specific Fluorescence
`
`Units of representative wells from an experiment employing colorectal cancer
`
`cells with Gly12Asp or Gly13Asp mutations of the c-Ki-Ras gene. Wells with
`
`values >10,000 are shaded yellow. Polyacrylamide gel electrophoretic
`analyses of the PCR products fromselected wells are shown. Wells with
`
`fluorescence values <3 500 had no PCR product of the correct size while wells
`
`with fluorescence values >10,000 SFU always contained PCR products of 129
`
`bp. Non-specific products generated during the large number of cycles
`
`required for Dig-PCR did not affect the fluorescence analysis. M1 and M2 are
`
`molecular weight markers used to determine the size of fragments indicated on
`
`the left (in base pairs).
`
`FIG. 4. Discriminating WT from mutant PCR products obtained in Dig-PCR.
`
`RED/GREEN ratios were determined from the fluorescence of MB-RED and
`
`MB-GREEN as described in Materials and Methods. The wells shown are the
`
`Ambry Exhibit 1002 - Page 7
`
`Ambry Exhibit 1002 - Page 7
`
`

`

`v)
`
`same as those illustrated in Fig. 3. The sequences of PCR products from the
`
`indicated wells were determined as described in Materials and Methods. The
`
`wells with RED/GREEN ratios >3.0 each contained mutant sequences while
`
`those with RED/GREEN ratios of ~1 .0 contained WT sequences.
`
`FIG. 5. Dig-PCR of DNA from a stool sample. The 384 wells used in the
`
`experiment are displayed. Those colored blue contained 25 genome
`
`equivalents of DNA from normal cells. Each of these registered positive with
`
`MB-RED and the RED/GREEN ratios were 1.0 +/- 0.1 (mean +/- 1 standard
`
`deviation). The wells colored yellow contained no template DNA and each
`
`was negative with MB-RED (i.e., fluorescence <3 500 fluorescence units.).
`
`The other wells contained diluted DNA from the stool sample. Those
`
`registering as positive with MB-RED were colored either red or green,
`
`depending on their RED/GREEN ratios. Those registering negative with
`
`MB-RED were colored white. PCR products from the indicated wells were
`
`used for automated sequence analysis.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The method devised by the present inventors involves separately
`
`amplifying small numbers of template molecules so that the resultant products
`
`have a proportion of the analyte sequence which is detectable by the detection
`
`means chosen. At its limit, single template molecules can be amplified so that
`
`the products are completely mutant or completely wild-type (WT). The
`
`homogeneity of these amplification products makes them trivial to distinguish
`
`through existing techniques.
`
`The method requires analyzing a large number of amplified products
`
`simply and reliably. Techniques for such assessments were developed, with
`
`the output providing a digital readout of the fraction of mutant alleles in the
`
`analyzed population.
`
`Ambry Exhibit 1002 - Page 8
`
`Ambry Exhibit 1002 - Page 8
`
`

`

`I)
`
`The biological sample is diluted to a point at which a practically usable
`
`number of the diluted samples contain a proportion of the selected genetic
`
`sequence (analyte) relative to total template molecules such that the analyzing
`
`technique being used can detect the analyte. A practically usable number of
`
`diluted samples will depend on cost of the analysis method. Typically it would
`be desirable that at least 1/50 of the diluted samples have a detectable
`
`proportion of analyte. At least 1/10, 1/5, 3/10, 2/5, 1/2, 3/5, 7/10, 4/5, or 9/10
`
`of the diluted samples may have a detectable proportion of analyte. The higher
`
`the fraction of samples which will provide useful information, the more
`
`economical will be the overall assay. Over-dilution will also lead to a loss of
`
`economy, as many samples will be analyzed and provide no signal. A
`
`particularly preferred degree of dilution is to a point where each of the assay
`
`samples has on average one-half of a template. The dilution can be performed
`
`from more concentrated samples. Alternatively, dilute sources of template
`nucleic acids can be used. All of the samples may contain amplifable template
`
`molecules. Desirably each assay sample prior to amplification will contain
`
`less than a hundred or less than ten template molecules.
`
`Digital amplification can be used to detect mutations present at
`
`relatively low levels in the samples to be analyzed. The limit of detection is
`defined by the number of wells that can be analyzed and the intrinsic mutation
`
`rate of the polymerase used for amplification. 384 well PCR plates are
`
`commercially available and 1536 well plates are on the horizon, theoretically
`
`allowing sensitivities for mutation detection at the ~0.1% level. It is also
`
`possible that Digital Amplification can be performed in microarray format,
`
`potentially increasing the sensitivity by another order of magnitude. This
`
`sensitivity may ultimately be limited by polymerase errors. The effective error
`
`rate in PCR as performed under our conditions was 1.1%, i.e., four out of 351
`
`PCR products derived from WT DNA sequence appeared to contain a mutation
`
`by RED/GREEN ratio criteria. However, any individual mutation (such as a
`
`Ambry Exhibit 1002 - Page 9
`
`Ambry Exhibit 1002 - Page 9
`
`

`

`O!
`
`G to T transversion at the second position of codon 12 of c—Ki-Ras), are
`
`expected to occur in < l in 50 of these polymerase-generated mutants (there
`
`are at least 50 base substitutions within or surrounding codons 12 and 13 that
`
`should yield high RED/GREEN ratios). Determining the sequence ofthe
`putative mutants in the positive wells, by direct sequencing as performed here
`
`or by any of the other techniques, provides unequivocal validation of a
`
`prospective mutation: a significant fraction of the mutations found in
`
`individual wells should be identical if the mutation occurred in vivo.
`
`Significance can be established through rigorous statistical analysis, as positive
`
`signals should be distributed according to Poisson probabilities. Moreover, the
`
`error rate in particular Digital Amplification experiments canibe precisely
`
`determined through performance of Digital Amplification on DNA templates
`
`from normal cells.
`
`Digital Amplification is as easily applied to RT—PCR products
`
`generated from RNA templates as it is to genomic DNA. For example, the
`
`fraction of alternatively spliced or mutant transcripts from a gene can be easily
`
`determined using photoluminescent probes specific for each of the PCR
`
`products generated. Similarly, Digital Amplification can be used to quantitate
`
`relative levels of gene expression within an RNA population. For this
`
`amplification, each well would contain primers which are used to amplify a
`
`reference transcript expressed constitutively as well as primers specific for the
`
`experimental transcript. One photoluminescent probe would then be used to
`
`detect PCR products from the reference transcript and a second
`
`photoluminescent probe used for the test transcript. The number of wells in
`
`which the test transcript is amplified. divided by the number of wells in which
`
`the reference transcript is amplified provides a quantitative measure of gene
`
`expression. Another group of examples involves the investigations of allelic
`
`status when two mutations are observed upon sequence analysis of a standard
`
`DNA sample. To distinguish whether one variant is present in each allele (vs.
`
`Ambry Exhibit 1002 - Page 10
`
`Ambry Exhibit 1002 - Page 10
`
`

`

`(I
`
`both occurring in one allele), cloning ofPCR products is generally performed.
`
`The approach described here would simplify the analysis by eliminating the
`need for cloning. Other potential applications of Digital Amplification are
`listed in Table 1. When the goal is the quantitation of the proportion of two
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`relatively common alleles or transcripts rather than the detection of rare alleles,
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`techniques such as those employing TaqMan and real time PCR provide an
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`excellent alternative to use of molecular beacons. Advantages of real time
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`PCR methods include their simplicity and the ability to analyze multiple
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`samples simultaneously. However, Digital Amplification may prove useful for
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`these applications when the expected differences are small, (e.g., only ~2-fold,
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`such as occurs with allelic imbalances.)
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`The ultimate utility of Digital Amplification lies in its ability to convert
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`the intrinsically exponential nature of PCR to a linear one.
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`It should thereby
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`prove useful for experiments requiring the investigation of individual alleles,
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`rare variants/mutations, or quantitative analysis of PCR products.
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`In one preferred embodiment each diluted sample has on average one
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`half a template molecule. This is the same as one half of the diluted samples
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`having one template molecule. This can be empirically determined by
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`amplification. Either the analyte (selected genetic sequence) or the reference
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`genetic sequence can be used for this determination.
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`If the analysis method
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`being used can detect analyte when present at a level of 20%, then one must
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`dilute such that a significant number of diluted assay samples contain more
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`than 20% of analyte. If the analysis method being used requires 100% analyte
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`to detect, then dilution down to the single template molecule level will be
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`required.
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`To achieve a dilution to approximately a single template molecule
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`level, one can dilute such that between 0.1 and 0.9 of the assay samples yield
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`an amplification product. More preferably the dilution will be to between 0.1
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`and 0.6, more preferably to between 0.3 and 0.5 ofthe assay samples yielding
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`an amplification product.
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`The digital amplification method requires analysis ofa large number of
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`samples to get meaningful results. Preferably at least ten diluted assay samples
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`are amplified and analyzed. More preferably at least 15, 20, 25, 30, 40, 50, 75,
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`100, 500, or 1000 diluted assay samples are amplified and analyzed. As in any
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`method, the accuracy of the determination will improve as the number of
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`samples increases, up to a point. Because a large number of samples must be
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`analyzed, it is desirable to reduce the manipulative steps, especially sample
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`transfer steps. Thus it is preferred that the steps of amplifying and analyzing
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`are performed in the same receptacle. This makes the method an in situ, or
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`“one-pot” method.
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`The number of different situations in which the digital amplification
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`method will find application is large. Some of these are listed in Table 1. ‘As
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`shown in the examples, the method can be used to find a tumor mutation in a
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`population of cells which is not purely tumor cells. As described in the
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`examples, a probe for a particular mutation need not be used, but diminution in
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`binding to a wild-type probe can be used as an indicator of the presence of one
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`or more mutations. Chromosomal translocations which are characteristic of
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`leukemias or lymphomas can be detected as a measure of the efficacy of
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`therapy. Gene amplifications are characteristic of certain disease states. These
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`can be measured using digital amplification. Alternatively spliced forms of a
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`transcript can be detected and quantitated relative to other forms of the
`transcript using digital amplification on (DNA made from mRNA. Similarly,
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`using cDNA made from mRNA one can determine relative levels of
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`transcription of two different genes. One can use digital amplification to
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`distinguish between a situation where one allele carries two mutations ”and one
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`mutation is carried on each of two alleles in an individual. Allelic imbalances
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`fl
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`often result from a disease state. These can be detected using digital
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`amplification.
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`Biological samples which can be used as the starting material for the
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`analyses may be from any tissue or body sample from which DNA or mRNA
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`can be isolated. Preferred sources include stool, blood, and lymph nodes.
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`Preferably the biological sample is a cell-free lysate.
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`Molecular beacon probes according to the present invention can utilize
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`any photoluminescent moiety as a detectable moiety. Typically these are dyes.
`Often these are fluorescent dyes. Photoluminescence is any process in which a
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`material is excited by radiation such as light, is raised to an excited electronic
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`or vibronic state, and subsequently re-emits that excitation energy as a photon
`
`of light. Such processes include fluorescence, which denotes emission
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`accompanying descent from an excited state with paired electrons (a “singlet”
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`state) or unpaired electrons (a “triplet” state) to a lower state with the same
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`multiplicity, i.e., a quantum-mechanically “allowed” transition.
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`Photoluminescence also includes phosphorescence which denotes emission
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`accompanying descent from an excited triplet or singlet state to a lower state of
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`different multiplicity, i. e., a quantum mechanically “forbidden” transition.
`Compared to “allowed” transitions, “forbidden” transitions are associated with
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`relatively longer excited state lifetimes.
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`The quenching of photoluminescence may be analyzed by a variety of
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`methods which vary primarily in terms of signal transduction. Quenching may
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`be transduced as changes in the intensity of photoluminescence or as changes
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`in the ratio of photoluminescence intensities at two different wavelengths, or as
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`changes in photoluminescence lifetimes, or even as changes in the polarization
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`(anisotropy) of photoluminescence. Skilled practitioners will recognize that
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`instrumentation for the measurement of these varied photoluminescent
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`responses are known. The particular ratiometric methods for the analysis of
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`quenching in the instant examples should not be construed as limiting the
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`DI
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`invention to any particular form of signal transduction. Ratiometric
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`measurements of photoluminescence intensity can include the measurement of
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`changes in intensity, photoluminescence lifetimes, or even polarization
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`(anisotropy).
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`Although the working examples demonstrate the use of molecular
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`beacon probes as the means of analysis of the amplified dilution samples, other
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`techniques can be used as well. These include sequencing, gel electrophoresis,
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`hybridization with other types of probes, including TaqManTM (dual-labeled
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`fluorogenic) probes (Perkin Elmer Corp/Applied Biosystems, Foster City,
`
`Calif), pyrene-labeled probes, and other biochemical assays.
`
`The above disclosure generally describes the present invention. A more
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`complete understanding can be obtained by reference to the following specific
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`examples which are provided herein for purposes of illustration only, and are
`
`not intended to limit the scope of the invention.
`
`EXAMPLE 1
`
`Step 1: PCR amplifications. The optimal conditions for PCR described in this
`
`section were determined by varying the parameters described in the Results.
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`PCR was performed in 7 ul volumes in 96 well polypropylene PCR plates
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`(RPI). The composition of the reactions was: 67 mM Tris, pH 8.8, 16.6 mM
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`NH4SO4, 6.7 mM MgC12, 10 mM B-mercaptoethanol, 1 mM dATP, 1 mM
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`dCTP, 1 mM dGTP, 1 mM TTP, 6% DMSO, 1 uM primer F1, 1 uM primer
`R1, 0.05 units/u] Platinum Taq polymerase (Life Technologies, Inc.), and
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`“one-half genome equivalent” of DNA. To determine the amount of DNA
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`corresponding to one-half genome equivalent, DNA samples were serially
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`diluted and tested via PCR. The amount that yielded amplification products in
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`half the wells, usually ~1 pg of total DNA, was defined as “one-half genome
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`equivalent” and used in each well of subsequent Digital Amplification
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`experiments. Fifty ul light mineral oil (Sigma M-3516) was added to each well
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`O)
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`and reactions performed in a HybAid Thermal cycler at the following
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`temperatures: denaturation at 94° for one min; 60 cycles of94° for 15 sec, 55°
`
`for 15 sec., 70° for 15 seconds; 70° for