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`PATENT DATE
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`APPUCATION NO.
`09/613826
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`'CONTIPRIOR CLASS
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`435
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`~.Bert 'Vogelstei~
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`Digital amplification
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`~U G ~. 't 2002
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`Page 4 of 206
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`II
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`Page 1 of 1
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`COMMISSIONER f'"OR PATI!NTS
`UNIT~O STAtts PATENT AND TRADEMARK Ol"flCE
`Wt..SHINOlOM, D.C. 20231
`'f/WW,uspto.gOY
`
`CONFIRMATION NO. 9893
`
`CLASS
`435
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`GROUP ART UNIT
`1632
`
`ATTORNEY
`DOCKET NO.
`01107.00031
`
`IIIIIIIIIIIIIIIIIIIIIIIIIUIII
`
`Bib Data Shllt
`
`SERIAL NUMBER
`09/613,826
`
`FILING DATE
`07/1112000
`RULE
`
`APPLICANTS
`Bert Vogelstein, Baltimore, MD;
`Kenneth W. Kinzler, BelAir, MD;
`
`,. CONTINUING DATA ***********111*.111111*#1*******
`THIS APPLN CLAIMS BENEFIT OF 60/146,792 08/02/1999
`
`• FOREIGN APPLICATIONS ••••••••••••••••••••
`
`IF REQUIRED, FOREIGN FILING LICENSE
`~RANTED .. 08/29/2000
`CI yes"
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`TOTAL
`CLAIMS
`64
`
`INDEPENDENl
`CLAIMS
`5
`
`ADDRESS
`~907
`
`TITLE
`
`Digital amplification
`
`'IG FEE FEES: Authority has been given in Paper
`to charge/credit DEPOSIT ACCOUNT
`for following:
`
`No.
`
`I
`
`896
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`I EIVED No.
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`I [::J All Fees
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`Page 5 of 206
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`
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`http://neo:8000/preexamiJavaProxy/jsp/bibdataitransfomi
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`COMMISSIONER FOR PA.TENTS '
`UNITED STATES PATENT AND TRADE:MARK OFFICE
`w..SHINGTON, D.C. 20231
`www.uspto.gov ,
`
`CONFIRMATION NO. 9893
`
`CLASS
`435
`
`GROUP ART UNIT
`1637
`
`ATTORNEY
`DO~KETNO.
`01107.00031
`
`l~~ UNITED STATES PATENT AND ThAoEMARK OFFICE
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`~~-, ... ~~
`
`*BIBnlATASHEET*
`Bib Dc~~
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`
`SERIAL NUMBER
`09/613,826
`
`FILING DATE
`07/11/2000
`
`RULE
`
`APPL,ICANTS
`
`Bert Vogelstein, Baltimore, MD;
`
`Kenneth W. Kinzler, BelAir, MD;
`
`.. CONTINUING DATA *_ •• **._** ••• * •••••••••••
`THIS APPLN CLAIMS BENEFIT OF 60/146,792 08/02/1999
`
`** FOREIGN APPLICATIONS ********************
`
`IF REQUIRED, FOREIGN FILING LICENSE GRANTED •• SMALL ENTITY"
`~. 08/29/2000
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`FOreign Prtortty claimed
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`~DDRESS
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`BANNER & WITCOFF
`1001 G STREET N W
`SUITE 1100
`WASHINGTON, DC
`20001
`.
`
`;
`
`TITLE
`Digital a, nplification
`
`STATE OR
`
`SHEETS
`
`TOTAL
`
`INDEPENDENT
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`COUNTRY DRAWING
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`7
`
`CLAIMS
`64
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`CLAIMS
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`
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`Page 6 of 206
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`
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`DIGITAL AMPLIFICATION
`
`ABSTRACT
`
`The identification of pre-defined mutations expected to be present in
`
`a minor fraction of a cell population is important for a variety of basic
`
`5
`
`research and clinical applications. The exponential, analog nature of the
`
`polymerase chain reaction is transformed into a linear, digital signal
`
`suitable for this purpose. Single molecules can be isolated by dilution and
`
`individually amplified; each product is then separately analyzed for the
`
`presence of mutations. The process provides a reliable and quantitative
`
`10
`
`measure of the proportion of variant sequences within a DNA sample.
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`DIGITAL AMPLIFICATION
`
`This application claims the benefit of U.S. Serial No. 60/146,792, filed
`
`August 2, 1999.
`
`The U.S. government retains certain rights in this invention by virtue
`
`5
`
`of its support of the underlying research, supported by grants CA 43460, CA
`
`57345, and CA 62924 from the National Institutes of Health.
`TECHNICAL FlEW OF THE INVENTION
`This invention is related to diagnostic genetic analyses. In particular
`
`it relates to detection of genetic changes and gene expression.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`In classical genetics, only mutations of the germ-line were considered
`
`important for understanding disease. With the realization that somatic
`
`mutations are the primary cause of cancer (l), and may also playa role in
`
`aging (2,3), new genetic principles have arisen. These discoveries have
`
`15
`
`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 (4), stool (5,6), and sputum (7,8) of patients with
`
`20
`
`cancers of the bladder, colorectum, and lung, respectively. Such detection has
`
`been shown in some cases to be 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
`
`(9-11). The detection of residual disease in lymph nodes or surgical margins
`
`1
`
`Page 8 of 206
`
`
`
`•
`may be useful in predicting which patients might benefit most from further
`
`therapy (12-14). 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 (15-17).
`
`5
`
`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%
`
`(18,19). Mutant-specific oligonucleotides can sometimes be used to detect
`
`10
`
`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
`
`(20-22), 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
`
`15
`
`quantitate the fraction of mutant molecules in the starting population with
`
`20
`
`25
`
`these techniques (23-28). Other innovative approaches for the detection of
`
`somatic mutations have been reviewed (29-32). 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.
`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
`
`30
`
`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
`
`2
`
`Page 9 of 206
`
`
`
`amplified molecules in the assay samples of the set. The amplified molecules
`
`in the assay samples of the set are then analyzed to detennine 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
`
`5
`
`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 detennining 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
`
`10
`
`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. The first number is compared to the
`
`15
`
`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 stern-loop
`
`structure having a photoluminescent dye at one of the 5' or 3' ends and a
`
`20
`
`quenching agent at the opposite 5' or 3' end. The loop consists of 16 base
`
`pairs and has a Tm of 50-51°C. The stern 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 stern-loop structure
`
`25
`
`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 54-56°C. The stern consists of 4 base pairs having a sequence 5'(cid:173)
`
`CACG-3'.
`
`Another embodiment provides the two types of molecular beacon
`
`30
`
`probes, either mixed together or provided in a divided container as a kit.
`
`3
`
`Page 10 of 206
`
`
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`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
`
`5
`
`FIG. 1. Schem tic of experimental design. (A) The basic two steps involved:
`
`PCR on diluted NA samples is followed by addition of fluorescent probes
`
`between WT and mutant alleles and subsequent
`
`fluorometry. (B)
`
`ciple of molecular beacon analysis. In the stem-loop
`
`configuration, fluores ence from a dye at the 5' end of the oligonucleotide
`
`10
`
`probe is quenched by a abcyl group at the 3' end. Upon hybridization to a
`
`template, the dye is sep ated from the quencher, resulting in increased
`
`fluorescence. Modified fr Marras et al . . (C) Oligonucleotide design.
`Primers PI and Rl are used to
`!NT is used to produce single s
`
`pJify the genomic region of interest. Primer
`
`ded DNA from the original PCR products
`
`15
`
`during a subsequent asymmetric
`
`R step (see Materials and Methods).
`
`MB-RED is a Molecular Beacon whi detects any appropriate PCR product,
`
`whether it is WT or mutant at the ueried codons. MB-GREEN is a
`
`Molecular Beacon which preferentially d tects the WT PCR product.
`
`2. Discrimination between WT and mutant PCR products by Molecular
`
`Ten separate PCR products, each generated from -50 genome
`
`A of cells containing the indicated mutations of c-Ki-Ras,
`
`Molecular Beacon probes described in the text.
`
`Representative examples 0
`
`e PCR products used for Molecular Beacon
`
`analysis were purified and directly quenced. In the cases with Gly12Cys
`
`25
`
`and Gly1 2Arg mutations, contaminating n
`-neoplastic cells within the tumor
`presumably accounted for the relatively 10
`
`Glyl2Ser and Glyl2Asp, there were apparently two 0
`
`c-Ki-Ras for every WT allele; both these tumors were aneu oid.
`
`FIG. 3. Detecting Dig-PCR products with MB-RED. Specific Auorescence
`
`30
`
`/Units of representative wells from an experiment employing colorectal cancer
`
`4
`
`Page 11 of 206
`
`
`
`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 PeR products from selected wells are shown. Wells with
`
`fluorescence values <3500 had no peR product of the correct size while wells
`
`5
`
`with fluorescence values> 10,000 SFU always contained PeR products of 129
`
`bp. Non-specific products generated during the large number of cycles
`
`required for Dig-PeR did not affect the fluorescence analysis. Ml and M2 are
`
`molecular weight markers used to determine the size of fragments indicated
`
`on the left (in base pairs).
`
`1~ G.4.
`
`. criminating WT from mutant peR products obtained in Dig-peR.
`
`RED/GREEN r
`
`. s were determined from the fluorescence of MB-RED and
`
`MB-GREEN as describe
`. Materials and Methods. The wens shown are the
`same as those illustrated in Fig.
`
`The sequences of peR products from the
`
`indicated wens were determined as de
`
`'bed in Materials and Methods. The
`
`15
`
`wells with RED/GREEN ratios >3.0 each co
`
`those with RED/GREEN ratios of -1.0 contained
`
`~G' . Dig-peR of DNA from a stool sample. The 384 wens used in the
`C? /e~penme
`
`displayed. Those colored blue contained 25 genome
`
`A from normal cells. Each of these registered positive with
`
`20
`
`. IGREEN ratios were 1.0 +/- 0.1 (mean +/- 1 standard
`
`deviation). The wells cored yellow contained no template DNA and each
`
`i.e., fluorescence <3500 fluorescence units.).
`
`The other 288 wells contained
`
`ted DNA from the stool sample prepared
`
`by alkaline extraction. (Rubeck et
`
`,1998, BioTechniques 25:588-592.)
`
`25
`
`Those registering as positive with MB-
`
`were colored either red or green,
`
`depending on their RED/GREEN ratios.
`
`se registering negative with
`
`MB-RED were colored white. peR products fro
`
`the indicated wells were
`
`used for automated sequence analysis.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`5
`
`" ,'" ""
`
`Page 12 of 206
`
`
`
`The method devised by the present inventors involves separately
`
`amplifying smaIl 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
`
`5 '
`
`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
`
`10
`
`the output providing a digital readout of the fraction of mutant alleles in the
`
`analyzed population.
`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
`
`15
`
`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 1150 of the diluted samples have a detectable
`
`proportion of analyte. At least 1/10, 115,3/10,2/5, 112, 3/5, 7/10,4/5, or 9110
`
`of the diluted samples may have a detectable proportion of analyte. The
`
`20
`
`higher the fraction of samples which will provide useful information. the
`
`25
`
`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 temJ;>late
`I
`nucleic acids can be used. All of the samples may contain amplifable
`(I.
`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
`
`30
`
`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 peR plates are
`
`6
`
`/)
`
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`Page 13 of 206
`
`
`
`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
`
`5
`
`sensitivity may ultimately be limited by polymerase errors. The effective
`
`error rate in PCR as performed under our conditions was <0.3%,
`
`i.e., in
`
`control experiments with DNA from normal cells, none of 340 wells
`
`containing PCR products exhibited RED/GREEN ratios >3.0. Any individual
`
`mutation (such as a G- to C- transversion at the second position of codon 12
`
`10
`
`of c-Ki-ras) is expected to occur in <1 in 50 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 of
`
`the putative mutants in the positive wells, by direct sequencing as performed
`
`here or by any of the other techniques, provides unequivocal validation of a
`
`15
`
`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 can
`
`20
`
`be 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
`
`25
`
`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
`
`30
`
`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
`
`7
`
`Page 14 of 206
`
`
`
`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
`
`5
`
`DNA sample. To distinguish whether one variant is present in each allele (vs.
`
`both occurring in one allele), cloning of peR products is generally perfonned.
`
`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
`
`10
`
`relatively common alleles or transcripts rather than the detection of rare
`
`alleles, techniques such as those employing TaqMan and real time peR
`
`provide an excellent alternative to use of molecular beacons. Advantages of
`
`real time peR methods include their simplicity and the ability to analyze
`
`multiple samples simultaneously. However, Digital Amplification may prove
`
`15
`
`useful for these applications when the expected differences are small, (e.g.,
`
`20
`
`25
`
`only -2-fold, such as occurs with allelic imbalances (55)),
`
`The ultimate utility of Digital Amplification lies in its ability to
`
`convert the intrinsically exponential nature of peR to a linear one. It should
`
`thereby prove useful for experiments requiring the investigation of individual
`alleles, rare variants/mutations, or quantitative analysis of peR products.
`
`In one preferred embodiment each diluted sample has on average one
`
`half a template molecule. This is the same as one half of the diluted samples
`
`having one template molecule. This can be empirically determined by
`
`amplification. Either the analyte (selected genetic sequence) or the reference
`genetic sequence can be used for this determination. If the analysis method
`
`being used can detect analyte when present at a level of 20%, then one must
`
`dilute such that a significant number of diluted assay samples contain more
`
`than 20% of analyte. If the analysis method being used requires 100% analyte
`
`to detect, then dilution down to the single template molecule level will be
`
`30
`
`required.
`
`To achieve a dilution to approximately a single template molecule
`
`level, one can dilute such that between 0.1 and 0.9 of the assay samples yield
`
`8
`
`Page 15 of 206
`
`
`
`an amplification product. More pteferably the dilution will be to between 0.1
`
`and 0.6, more preferably to between 0.3 and 0.5 of the assay samples yielding
`
`an amplification product.
`
`The digital amplification method requires analysis of a large number
`
`5
`
`of samples to get meaningful results. Preferably at least ten diluted assay
`
`samples are amplified and analyzed. More preferably at least 15, 20, 25, 30,
`
`40, 50, 75, 100, 500, or 1000 diluted assay samples are amplified and
`
`analyzed. As in any method, the accuracy of the determination will improve
`
`as the number of samples increases, up to a point. Because a large number of
`
`10
`
`samples must be analyzed, it is desirable to reduce the manipulative steps, .
`
`especially sample transfer steps. Thus it is preferred that the steps of
`
`amplifying and analyzing are performed in the same receptacle. This makes
`
`the method an in situ, or "one-pot" method.
`
`The number of different situations in which the digital amplification
`
`15
`
`method will find application is large. Some of these are listed in Table 1. As
`
`shown in the examples, the method can be used to find a tumor mutation in a
`
`population of cells which is not purely tumor cells. As described in the
`
`examples, a probe for a particular mutation need not be used, but diminution
`
`in binding to a wild-type probe can be used as an indicator of the presence of
`
`20
`
`one or more mutations. Chromosomal translocations which are characteristic
`
`of leukemias or lymphomas can be detected as a measure of the efficacy of
`
`therapy. Gene amplifications are characteristic of certain disease states.
`
`These can be measured using digital amplification. Alternatively spliced
`
`forms of a transcript can be detected and quantitated relative to other forms of
`
`25
`
`the transcript using digital amplification on cDNA made from mRNA.
`
`Similarly, using cDNA made from mRNA one can determine relative levels
`
`of transcription of two different genes. One can use digital amplification to
`
`distinguish between a situation where one allele carries two mutations and one
`
`mutation is carried on each of two alleles in an individual. Allelic imbalances
`
`30
`
`often result from a disease state. These can be detected using digital
`
`amplification.
`
`Iii
`
`9
`
`[ c )
`
`Page 16 of 206
`
`
`
`Biological samples which can be used as the starting material for the
`
`analyses may be from any tissue or body sample from which DNA or mRNA
`
`can be isolated. Preferred sources include stool, blood, and lymph nodes.
`
`Preferably the biological sample is a cell-free lysate.
`
`10
`
`If
`
`Page 17 of 206
`
`
`
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`
`Table 1. Potential Applications ofDig-PCR
`
`Application
`
`Example
`
`Probe 1 Detects:
`
`Probe 2 Detects:
`
`Base substitution
`
`Cancer gene mutations in stool, blood, lymph
`
`mutant or WT alleles
`
`WT PCR products
`
`mutations
`
`Chromosomal
`
`translocations
`
`nodes
`
`Residual leukemia cells after therapy (DNA or
`
`normal or translocated
`
`translocated allele
`
`RNA)
`
`alleles
`
`Gene amplifications
`
`Determine presence or extent of amplification
`
`sequence within amplicon
`
`sequence from another part of
`
`I , ___
`I ,- ,
`Y-..;
`
`Alternatively spliced
`
`Determine fraction of altematively spliced
`
`minor exons
`
`ccomon exons
`
`products
`
`transcripts from same gene (RNA)
`
`Changes in gene
`
`Determine relative levels of expression of two
`
`first transcript
`
`reference transcript
`
`same chromosome arm
`
`expression
`
`genes (RNA)
`
`Allelic discrimination
`
`Two different alleles mutated vs. one mutation
`
`first mutation
`
`second mutation
`
`in each of two alleles
`
`Allelic imbalance
`
`Quantitative analysis with non-polymorphic
`
`marker from test
`
`marker from reference
`
`markers
`
`chromosome
`
`chromosome
`
`-
`
`.
`
`11
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`Page 18 of 206
`
`
`
`Molecular beacon probes according to the present invention can utilize
`
`any photoluminescent moiety as a detectable moiety. Typically these are
`
`dyes. Often these are fluorescent dyes. Photoluminescence is any process
`
`in which a material is excited by radiation such as light, is raised to an
`
`5
`
`excited electronic or vibronic state, and subsequently re-emits that
`
`excitation energy as a photon of light. Such processes include fluorescence,
`
`which denotes emission accompanying descent from an excited state with
`
`paired electrons (a "singlet" state) or unpaired electrons (a "triplet" state)
`
`to a lower state with the same multiplicity, i.e., a quantum-mechanically
`
`10
`
`"allowed" transition. Photoluminescence