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`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Patent No.: 6,440,706
`
`Inventor: Bert Vogelsteinetal.
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`Assignee: Johns Hopkins University
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`Issued: August 27, 2002
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`Application No.: 09/613,826
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`EXHIBIT 1
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`Page 11 of 1224
`
`

`

`a2) United States Patent
`US 6,440,706 B1
`(0) Patent No.:
`Vogelstein et al.
`Aug. 27, 2002
`(45) Date of Patent:
`
`US006440706B1
`
`(54) DIGITAL AMPLIFICATION
`
`(75)
`
`Inventors: Bert Vogelstein, Baltimore; Kenneth
`W. Kinzler, BelAir, both of MD (US)
`
`(73) Assignee: Johns Hopkins University, Baltimore,
`MD (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/613,826
`
`(22)
`
`Filed:
`
`Jul. 11, 2000
`
`(60)
`
`Related U.S. Application Data
`Provisional application No. 60/146,792, filed on Aug. 2,
`1999.
`
`(51)
`
`Int. CR vec cecseeecceeesseeeeee C12P 19/34; C120 1/68;
`CO0O7H 21/02; CO7H 21/04; CO7H 19/00
`(52) U.S. Ch wee 435/91.2; 435/60; 435/7.1;
`435/91 .1; 536/22.1; 536/23.1; 536/24.3;
`536/24.31; 536/24.32; 536/24.33
`(58) Field of Search 2.0.0.0... 435/6, 7.1, 91.1,
`435/91.2; 536/22.1, 23.1, 24.3, 24.31, 24.32,
`24.33
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`9/1997 Lapidusetal.
`5,670,325 A
`9/1998 Grucnertctal.
`5,804,383 A
`1/1999 Nisson et al.
`5,858,603 A
`7/1999 ‘lyagi et al. oo. 435/6
`5,925,517 A *
`7/1999 Lapidusetal.
`5,928,870 A
`2/2000 T.apidusetal.
`6,020,137 A
`3/2000 Tyagi et al. oo... 435/6
`6,037,130 A *
`11/2000 Brownetal.
`6,143,496 A
`FOREIGN PATENT DOCUMENTS
`
`EP
`WwO
`WO
`
`0643140 A
`WO 95/13399
`WO 99/13113
`
`3/1995
`5/1995
`3/1999
`
`OTHER PUBLICATIONS
`
`Newton Essential PCR pp. 51-52 1995.*
`Darren G. Monckton, et al., “Minisatellite “Isoallele” Dis-
`crimination in Pseudohomozygotes by Single Molecule
`PCR and Variant Repeat Mapping”, Genomics 11, pp.
`465-467, 1991.
`Gualberto Ruano, et al., “Haplotype of Multiple Polymor-
`phisms Resolved by Enzymatic Amplification of Single
`DNA Molecules’, Proc. National Science USA, 1990 vol. 87
`pp 6296-6300.
`W. Navidi, et al., “Using PCR in Preimplantation Genetic
`Disease Diagnosis”, Human Reproduction, vol. 6, No. 6, pp.
`836-849, 1991.
`Hongua Li, et al., “Amplification and Analysis of DNA
`Sequences in Single Human Sperm and Diploid Cells”,
`Nature, vol. 335, Sep. 29, 1988 pp. 414-417.
`Ramon Parsons, et al., “Mismatch Repair Deficiency in
`Phenotypically Normal Human Cells”, Science, vol. 268,
`May 5, 1995 pp. 738-740.
`
`Page 12 of 1224
`
`Lin Zhang, et al., “Whole Genome Amplification from a
`Single Cell:
`Implications for Genetic Analysis”, Proc.
`National Science USA, vol. 89, pp. 5847-5851, Jul. 1992.
`David Sidransky, et al., “Clonal Expansion of p53 Mutant
`Cells in Associated with Brain Tumor Progression”, Nature,
`Feb. 27, 1992 vol. 355, pp. 846-847.
`Alec J. Jeffreys, et al., “Mutation Processes at Human
`Minisatellites”, Electophoresis, pp. 1577-1585, 1995.
`C. Schmitt, et al., “High Sensitive DNA Typing Approaches
`for
`the Analysis of Forensic Evidence: Comparison of
`Nested Variable Number of Tandem Repeats (VNTR)
`Amplification and a Short Tandem Repeats (STR) Polymor-
`phism”, Forensic Science
`International, vol.
`66, pp.
`129-141, 1994.
`Paul M. Lizardi, et al., “Mutation Detection and Single—M-
`olecule Counting Using Isothermal Rolling—Circle Ampli-
`fication”, Nature Genetics, vol. 19, Jul. 1988 pp. 225-232.
`A. Piatek et al., “Molecular Beacon Sequence Analysis for
`Detecting Drug Resistance in Mycobacterium Tuberculo-
`sis”, Nature Biotechnology, Apr. 1998, pp. 359-363, vol. 16,
`No. 4.
`
`S. Tyagi et al., “Multicolor Molecular Beacons for allele
`discrimination”, Nature Biotechnology, pp. 303-308, Jan.
`1998, vol. 16, No. 1.
`
`J. A.M. Vet et al., “Multilex Detection of Four Pathogenic
`Retroviruses Using Molecular Beacons”, Proceedings of the
`National Academyof Sciences of the United States, May 25,
`1999, pp. 6394-6399, vol. 96, No. 11.
`S. Tyagiet al., “Molecular Beacons: probes that Fluoresce
`Upon Hybridization”, Nature Biotechnology, 1996, pp.
`303-308, vol. 14, No. 3.
`
`W.P. Halford et al., “The Inherent Quantitative Capacity of
`the Reverse Transcription—Polymerase Chain Reaction”,
`Analytical Biochemistry, Jan. 15, 1999, pp. 181-191, vol.
`266, No. 2.
`
`B. Vogelstein et al., “Digital PCR”, Proceedings of the
`National Academy of Sciences of the United States, Aug. 3,
`1999, pp. 9236-9241, vol. 96, No. 16.
`K. D.E. Everett et al, “Identification of nine species of the
`Chlamydiaceae Uisng PCR-RFLP”’, Apr.
`1999,
`pp.
`803-813, vol. 49, No. 2.
`
`* cited by examiner
`
`Primary Examiner—Jcfirey Sicw
`(74) Attorney, Agent, or Firm—Banner & Witcoff, Ltd.
`
`(57)
`
`ABSTRACT
`
`The identification of pre-defined mutations expected to be
`present in a minorfraction of a cell population is important
`for a variety of basic 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 ana-
`lyzed for the presence of mutations. The process provides a
`reliable and quantitative measure of the proportion of variant
`sequences within a DNA sample.
`
`64 Claims, 7 Drawing Sheets
`
`Page 12 of 1224
`
`

`

`U.S. Patent
`
`Aug. 27, 2002
`
`Sheet 1 of 7
`
`US 6,440,706 B1
`
`FIG. 1A
`DNA
`
`eee
`eee
`eoece
`
`ee
`>.
`ee
`
`°
`
`oo
`
`STEP 1 | DILUTETO~ 1/2COPY/
`
`
`WELL PCR
`
`@
`e
`e@
`
`@
`e@
`@
`
`ece
`eee
`eee
`
`e©
`©
`©
`
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`«©
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`Ons
`CS
`ES
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`co ee
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`«©
`
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`e
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`
`ADD FLUORESCENT PROBES
`
`FLUOQROMETRY oe
`
`ee
`
`OQ) QDeoee
`S Ores
`
`= NO PCR PRODUCT
`S = WILD TYPE PCR PRODUCT
`Q — MUTANT PCR PRODUCT
`
`Page 13 of 1224
`
`Page 13 of 1224
`
`

`

`Aug. 27, 2002
`
`Sheet 2 of 7
`
`US 6,440,706 B1
`
`U.S. Patent
`
`FLUORESCENT
`
`$7NL=oo”
`
`FIG.1B
`
`NON-FLUORESCENT
`
`Page 14 of 1224
`
`
`
` FLUORESCENTQUENCHER\
`
`/ DYE
`
`Page 14 of 1224
`
`

`

`Sheet 3 of 7
`
`US 6,440,706 B1
`
`INT
`
`
`
`QUERIEDCODONS
`
`MB-GREEN
`
`MB-RED
`
`U.S. Patent
`
`Aug. 27, 2002
`
`FIG.1€
`
`Page 15 of 1224
`
`Page 15 of 1224
`
`

`

`
`
`GCTGGTOGGCGTAINT
`
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`PLL
`<7SN 2
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`SSSSS]_ =UT
`
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`i—
`PI
`UZZ/ZESSS
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`poeYY =
`SmEE
`Ss
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`RWNTMTa
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`PyZLEliE
`RSS[S{SSsKKK
`
`
`WILDTYPE
`
`
`
`>
`Ss
`
`U.S. Patent
`
`Aug. 27, 2002
`
`Sheet 4 of 7
`
`US 6,440,706 B1
`
`SSS
`UT
`LLLLALLA
`BSSay
`Eph
`
`KXKKK
`
`|
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`Mut
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`
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`
`Page 16 of 1224
`
`Page 16 of 1224
`
`

`

`U.S. Patent
`
`Aug.27, 2002
`
`Sheet 5 of 7
`
`US 6,440,706 B1
`
`SPU
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`U.S. Patent
`
`Aug.27, 2002
`
`Sheet 6 of 7
`
`US 6,440,706 B1
`
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`Page 18 of 1224
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`

`

`U.S. Patent
`
`Aug.27, 2002
`
`Sheet 7 of 7
`
`US 6,440,706 B1
`
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`Page 19 of 1224
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`

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`US 6,440,706 B1
`
`1
`DIGITAL AMPLIFICATION
`
`This application claims the benefit of U.S. Ser. No.
`60/146,792, filed Aug. 2, 1999.
`The U.S. governmentretains certain rights in this inven-
`tion 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 inventionis 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 important for understanding disease. With the
`realization that somatic mutations are the primary cause of
`cancer (1), and may also play a role in aging (2,3), new
`genetic principles have arisen. These discoveries have pro-
`vided 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 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 tumorsare still curable and the patients asymptom-
`atic. 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 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 carcino-
`gens is often dependent on the ability to detect small
`populations of mutant cells (15-17).
`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 mutations present in a minor
`proportionof the cells analyzed, but the signal to noise ratio
`distinguishing mutant and wild-type (WI) 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
`quantitate the fraction of mutant molcculcs in the starting
`population with these techniques (23-28). Other innovative
`approachesfor the detection of somatic mutations have been
`reviewed (29-32). A general problem with these methodsis
`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.
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`It is another object of the present invention to provide
`molecular beacon probes useful in the method of the inven-
`tion.
`
`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 ampli-
`fied to form a population of amplified moleculesin the assay
`samples of the set. The amplified molecules in the assay
`samples of the set are then analyzed to determine a first
`oumber 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
`oumber 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 second numberto 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 oligo-
`nucleotide with a stem-loop structure having a photolumi-
`nescent dye at one of the 5’ or 3' ends and a quenching agent
`al the opposite 5' or 3' end. The loop consists of 16 base pairs
`and has a T,,, of 50-51° C. 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 hasa T,,,
`of 54—-56° C. The stem consists of 4 base pairs having a
`sequence 5'-CACG-3'.
`Another embodimentprovides 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
`
`FIGS. 1A, 1B, 1C. Schematic of experimental design. (A)
`The basic two steps involved: PCR on diluted DNA samples
`is followed by addition of fluorescent probes which dis-
`criminate between WT and mutant alleles and subsequent
`fluorometry. (B) Principle of molecular beacon analysis. In
`the stem-loop configuration, fluorescence from a dyeat 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 fluo-
`rescence. Modified from Marraset al. (C) Oligonucleotide
`desigo. 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
`
`Page 20 of 1224
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`

`US 6,440,706 B1
`
`3
`Methods). MB-RED is a Molecular Beacon which detects
`any appropriate PCR product, 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 ofcells 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 Beacon analysis were purified
`and directly sequenced. In the cases with Gly12Cys (SEQ ID
`NO: 11) and Gly12Arg (SEQ ID NO: 10) mutations, con-
`laminating non-neoplastic cells within the tumor presum-
`ably accounted for the relatively low ratios. In the cases with
`Gly12Ser (SEQ ID NO: 8) and Gly12Asp (SEQ ID NO: 12),
`there were apparently two or more alleles of mutant c-Ki-
`Ras for every WT allele (SEQ ID NO: 7); both these tumors
`were aneuploid. Analysis of the Gly13Asp mutation is also
`shown (SEQ ID NO: 9).
`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. Polyacryla-
`mide gel electrophoretic analyses of the PCR products from
`selected wells are shown. Wells with fluorescence values
`<3500 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 numberof cycles required for Dig-PCR did
`not affect the fluorescence analysis. M1 and M2 are molecu-
`lar 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 same as those illustrated in FIG. 3. The sequences of
`PCR products from the indicated wells were determined as
`described in Materials and Mcthods. The wells with RED/
`GREENratios >3.0 each contained mutant sequences while
`those with RED/GREEN ratios of ~1.0 contained WT
`sequences. WT c-Ki-Ras (SEQ ID NO: 7), Gly12Asp (SEQ
`ID NO: 13), and Gly13Asp (SEQ ID NO: 9) were analyzed.
`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 stan-
`dard deviation). The wells colored yellow contained no
`template DNA and each was negative with MB-RED (i.e.,
`fluorescence <3500 fluorescence units.). The other wells
`contained diluted DNA from the stool sample. Those reg-
`istering as positive with MB-RED were colored either red or
`green, depending on their RED/GREENratios. Those reg-
`istering negative with MB-RED were colored white. PCR
`products from the indicated wells were used for automated
`sequence analysis. The sequence of WT c-Ki-Ras in well K1
`(SEQ ID NO: 7), and mutant c-Ki-Ras in wells C10, E11,
`M10, and L12 (SEQ ID NO: 14), and well F21 (SEQ ID NO:
`15) were analyzed.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The method devised by the present inventors involves
`separately amplifying small numbers of template molecules
`
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`4
`so that the resultant products have a proportionof the analyte
`sequence whichis 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 ampli-
`fied 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.
`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 amplifiable template molecules. Desirably each
`assay sample prior to amplification will contain less than a
`hundred or Iess than ten template molecules.
`Digital amplification can be used to detect mutations
`presentat 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 per-
`formed under our conditions was <0.3%,
`i.e., in control
`experiments with DNA from normalcells, 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 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/
`GREENratios). 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 prospective mutation: a signifi-
`cant fraction of the mutations found in individual wells
`should be identical if the mutation occurred in vivo. Sig-
`nificance can be established through rigorous statistical
`analysis, as positive signals should be distributed according
`to Poisson probabilities. Moreover, the error rate in particu-
`lar Digital Amplification experiments can be precisely deter-
`mined through performance of Digital Amplification on
`DNAtemplates from normal cells.
`Digital Amplification is as easily applied to RT-PCR
`products generated from RNA templates as it is to genomic
`
`Page 21 of 1224
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`Page 21 of 1224
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`

`

`US 6,440,706 B1
`
`5
`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 spe-
`cific 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 numberof wells in which the
`test transcript is amplified divided by the numberof wells in
`which the reference transcript is amplified provides a quan-
`titative 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. both occurring in one allele),
`cloning of PCR products is generally performed. The
`approach described here would simplify the analysis by
`eliminating the need for cloning. Other potential applica-
`tions of D