U.S. UTILITY Patent Application
`o. 1.V
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`PATENT DATE
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`PATENT NUM
`
`84407
`
`PTO-2040
`12/99
`
`ORIGINAL
`
`iSSUING CLASSIFICATION
`CROSS REFERENCE(S)
`
`CLASS
`
`SUBCLASS
`
`CLASS
`
`SUBCLASS (ONE SUBCLASS PER BLOCK)
`
`___
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`INTERNATIONAL CLASSIFICATION
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`___________
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`L1 Continued on issue Slip Inside File Jacket
`
`oTERMINAL
`DISCLAIMER
`
`Shes Drwg.
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`DRAWINGS
`Figs. Drwg.
`
`Print Fig.
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`Total Claims
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`_Rb
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`E: The term of this patent
`subsequent to _______
`has been disclaimed.
`EU The term of this patent shall
`not extend beyond the expiration date
`of U.S Patent. No. _______
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`-months of
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`Possession outside the U.S. Patent & Trademark Office is restricted to authorized employees and contractors only.
`FILED WITh ThS-CiiS(CRF)
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`E]FICHE El CD-ROM
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`(Attached In po"*a on right inside flhp)
`
`Form PTO-438A
`(Rev. 6119)
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`Ambry Exhibit 1002 - Page 1
`
`The Johns Hopkins University Exhibit JHU2015 - Page 1 of 206
`
`

`

`ENT APPLICATION
`09613826Q9636
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`
`P,
`
`\CONTENTS
`Date Received
`(Incl. C. lot M.)
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`INITIALS,4..
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`
`Ambry Exhibit 1002 - Page 2
`
`The Johns Hopkins University Exhibit JHU2015 - Page 2 of 206
`
`

`

`-n
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`I
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`(RIGHT OUTSIDE)
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`Ambry Exhibit 1002 - Page 3
`
`The Johns Hopkins University Exhibit JHU2015 - Page 3 of 206
`
`

`

`Ft
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`ISSUE SLIP STAPLE AREA (for additional cross references)
`
`INDEX OF CLAIMS
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`If more than 150 claims or 10 actions
`staple additional sheet here
`
`(LEFT INSIDE)
`
`Ambry Exhibit 1002 - Page 4
`
`The Johns Hopkins University Exhibit JHU2015 - Page 4 of 206
`
`

`

`UNITED STATES PATENT AND 71;VDEMARK OMGE
`
`IInIIIIIIIEurnuI0IulII11
`
`Bib Data Sheet
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`Page 1 of I
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`COMMISSIONER FOR RWETtS
`UNITED STATES PATENT AND TRADEMARK OFFICE
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`CONFIRMATION NO. 9893
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`PILING DATE
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`RULE
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`435
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`GROUP ART UNIT
`1632
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`ATTORNEY
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`
`APPLICANTS
`Bert Vogelstein, Baltimore, MD;
`Kenneth W. Kinzler, BelAir, MD;
`
`*CONTINUING
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`DATA*********************
`THIS APPLN CLAIMS BENEFIT OF 60/146,792 08/02/1 999
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`STATE OR SHES
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`CLAIMS
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`VifEE
`Acko ed
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`FEES Auhoityha bee gieMnDae
`t hrecedtDPSTACON
`N.__
`
`1
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`oe AllFee
`17 Fee (Pocssn Ext of
`
`ie
`
`896 o. _______forfollwin:
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`01.18 Fees ('isue)
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`Ambry Exhibit 1002 - Page 5
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`http://nw:8000/preexm/JavaProxY/sp/bibdata/transforr
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`COMMISSIONER FOR PATIENTS
`UNITED STATES PATENT AND TRADEMARK OFICE
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`DTOKEYO
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`UNiTED STATES PATENT AND 7k nADMM OFncGE
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`Sib D;-
`eat
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`SERIAL NUMBER
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`FILING DATE
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`RULE
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`APPLICANTS
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`DRAWING
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`64
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`CLAIMS
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`ADDRESS
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`TITLE
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`Ambry Exhibit 1002 - Page 6
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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
`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
`measure of the proportion of variant sequences within a DNA sample.
`
`5
`
`10
`
`0J
`
`L
`
`Ambry Exhibit 1002 - Page 7
`
`The Johns Hopkins University Exhibit JHU2015 - Page 7 of 206
`
`

`

`I
`
`I
`
`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
`of its support of the underlying research, supported by grants CA 43460, CA
`57345, and CA 62924 from the National Institutes of Health.
`TEIVETO
`TEINIA.IEDO
`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
`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
`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
`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
`
`43 5
`
`10
`
`15
`
`20
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`Ambry Exhibit 1002 - Page 8
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`The Johns Hopkins University Exhibit JHU2015 - Page 8 of 206
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`

`

`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).
`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 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
`extrmelysensitive mtosfrdetecting such mutations, but it is difficult t
`quantitate the fraction of mutant molecules in the starting population with
`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.
`
`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
`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
`
`5
`
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`LU
`
`15
`
`0
`
`20
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`25
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`30
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`
`Ambry Exhibit 1002 - Page 9
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`The Johns Hopkins University Exhibit JHU2015 - Page 9 of 206
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`

`

`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. 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 agent at the opposite 5' or 3' end. The loop consists of 16 base
`pairs and has a Tm of 50-5S1i'.
`The stem consists of 4 base pairs having a
`sequence 5'-CACG-3'.
`A second type of molecular beacon probe is provided in another
`It comprises an oligonucleotide with a stem-loop structure
`embodiment.
`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 TIM of 54-56 0C. The stem consists of 4 base pairs having a sequence 5'-
`CACG-T'.
`Another embodiment provides the two types of molecular beacon
`probes, either mixed together or provided in a divided container as a kit.
`
`5
`
`10
`
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`11!
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`
`Ambry Exhibit 1002 - Page 10
`
`The Johns Hopkins University Exhibit JHU2015 - Page 10 of 206
`
`

`

`5
`
`10
`
`the art with the means to obtain
`thus provides
`The invention
`quantitative assessments of particular DNA or RNA sequences in mixed
`populations of sequences using digital (binary) signals.
`BRIEF DESCRIPTION OF THE DRAWINGS
`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
`which discrimina
`ciple of molecular beacon analysis. In the stem-loop
`(B)
`it'fluorometry.
`configuration, fluores ence from a dye at the 5' end of the oligonucleotide
`probe is quenched by a abcyl group at the 3Yend. 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.
`plify the genomic region of interest. Primer
`Primers FI and RI are used to
`ded DNA from the original PCR products
`is used to produce single s
`during a subsequent asymmetric CR step (see Materials and Methods).
`detects any appropriate PCR product,
`MB-RED is a Molecular Beacon whi
`whether it is WT or mutant at the ueried codons. MB-GREEN is a
`Molecular Beacon which preferentially d tects the WT PCR product.
`
`11,INT
`
`15
`
`!j
`
`:3
`
`20
`
`25
`
`2. Discrimination between WT and mutant PCR products by Molecular
`Ten separate PCR products, each generated from -50 genome
`Beacon
`A of cells containing the indicated mutations of c-Ki-Ras,
`equivalents o
`Molecular Beacon probes described in the text.
`were analyzed with
`e PCR products used for Molecular Beacon
`Representative examples o
`In the cases with Glyl2Cys
`quenced.
`analysis were purified and directly
`-neoplastic cells within the tumor
`and Gly12Arg mutations, contaminating n
`In the cases with
`atios.
`presumably accounted for the relatively lo
`ore alleles of mutant
`Glyl2Ser and Glyl2Asp, there were apparently two o
`c-Ki-Ras for every WIT allele; both these tumors were aneu oid.
`
`Fib. 3. Detecting Dig-PCR products with MB-RED. Specific Fluorescence
`units of representative wells from an experiment employing colorectal cancer
`
`30
`
`4
`
`Ambry Exhibit 1002 - Page 11
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`The Johns Hopkins University Exhibit JHU2015 - Page 11 of 206
`
`

`

`5
`
`cells with Glyl2Asp or Glyl3Asp mutations of the c-KM-Ras gene. Wells with
`values >10,000 are shaded yellow. Polyacrylamide 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 number of cycles
`required for Dig-PCR 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).
`
`1criminating WIT from mutant PCR products obtained in Dig-PCR.
`GG.4.
`RED/GREEN r I s were determined from the fluorescence of MB-RED and
`e llh w r h
`Te
`Mth o s
`e a s d
`M B- R E E N as d s r ba
`indicaEN welsw derined asdItdi Materials and Methods. Thewelshnarte
`
`Jj ~
`
`~
`
`samthoe withos RED/RENratio oFi 1.0 cotan sequences. rdcsfo h
`rEE ratos >3. 0 ech co
`e mutanseucswhl
`1 wel
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`G. Dig-PCR of DNA from a stool sample. The 384 wells used in the
`are displayed. Those colored blue contained 25 genome
`A from normal cells. Each of these registered positive with
`equivalents of
`/GREEN ratios were 1.0 +/- 0.1 (mean +/- 1 standard
`MB-RED and the
`deviation). The wells c ored yellow contained no template DNA and each
`i.e., fluorescence <3500 fluorescence units.).
`was negative with MB-RE
`ted DNA from the stool sample prepared
`The other 288 wells contained
`RioTechniques 25:588-592.)
`(Rubeck et
`by alkaline extraction.
`were colored either red or green,
`Those registering as positive with MB-
`se registering negative with
`depending on their RED/GREEN ratios.
`the indicated wells were
`MB-RED were colored white. PCR products fro
`used for automated sequence analysis.
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`,1998,
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`The method devised by the present inventors involves separately
`amnplifying 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.
`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 1110, 1/5, 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
`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
`4"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
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`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
`i.e., in
`error rate in PCR as performed under our conditions was <0.3%,
`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
`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
`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
`be precisely determined through performance of Digital Amplification on
`DAtemplates from normal cells.
`Digital Amplification is as easily applied to RT-PCR products
`generated from RNA templates as it is to gen6mic 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
`transcript and a second
`the reference
`from
`detect PCR products
`photoluminescent probe used for the test transcript. The number of wells in
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`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.
`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 applications of Digital Amplification are
`listed in Table 1. When the goal is the quantitation of the proportion of two
`relatively common alleles or transcripts rather than the detection of rare
`alleles, techniques such as those employing TaqMan and real time PCR
`provide an excellent alternative to use of molecular beacons. Advantages of
`real time PCR methods include their simplicity and the ability to analyze
`multiple samples simultaneously. However, Digital Amplification may prove
`useful for these applications when the expected differences are small, (e.g.,
`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 PCR to a linear one. It should
`thereby prove useful for experiments requiring the investigation of individual
`alleles, rare variants/mutations, or quantitative analysis of PCR 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
`If the analysis method
`genetic sequence can be used for this determination.
`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
`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
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`an amplification product. More preferably 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
`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
`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.
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`The number of different situations in which the digital amplification
`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
`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
`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
`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
`often result from a disease state. These can be detected using digital
`amplification.
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`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.
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`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
`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
`'allowed" transition. Photoluminescence also includes phosphorescence
`which denotes emission accompanying descent from an excited triplet or
`singlet state to a lower state of different multiplicity, i.e., a quantum
`mechanically "forbidden" transition. Compared to "allowed" transitions,
`"forbidden" transitions are associated with relatively longer excited state
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`lifetimes.
`The quenching of photoluminescence may be analyzed by a variety of
`methods which vary primarily in terms of signal transduction. Quenching
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`may be transduced as changes in the intensity of photoluminescence or as
`changes in the ratio of photoluminescence intensities at two different
`wavelengths, or as changes in photoluminescence lifetimes, or even as
`changes in the polarization (anisotropy) of photoluminescence. Skilled
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`practitioners will recognize that instrumentation for the measurement of
`these varied photoluminescent responses are known. The particular
`ratiometric methods for the analysis of quenching in the instant examples
`should not be construed as limiting the invention to any particular form of
`signal transduction. Ratiometric measurements of photoluminescence
`intensity can
`include
`the measurement of changes
`in
`intensity,
`photoluminescence lifetimes, or even polarization (anisotropy).
`Although the working examples demonstrate the use of molecular
`beacon probes as the means of analysis of the amplified dilution samples,
`other techniques can be used as well. These include sequencing, gel
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`TaqManTm (dual-labeled fluorogenic) probes (Perkin Elmer Corp./Applied
`Biosystems, Foster City, Calif), pyrene-labeled probes, and other
`biochemical assays.
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`The above disclosure generally describes the present invention. A
`more complete understanding can be obtained by reference to the following
`specific examples which are provided herein for purposes of illustration
`only, and are not intended to limit the scope of the invention.
`
`EXAMPEBI
`
`Step 1: PCR amplifications. The optimal conditions for PCR described
`in this section were determined by varying the parameters described in the
`Results. PCR was performed in 7 ul volumes in 96 well polypropylene
`Thle
`(Marsh Biomedical Products, Rochester, NY).
`PCR plates
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`composition of the reactions was: 67mrM Tris, pH 8.8, 16.6 MM NH4S0 4,
`6.7 MMv MgCl2, 10 mM P-mercaptoethanol, 1 mM dATP, 1 mM dCTP, 1
`mM dGTP, 1 mM T-'P, 6% DMSO, 1 uM primer Fl, 1 uM primer Ri, 0.05
`units/ul Platinum Taq polymerase (Life Technologies, Inc.), and "one-half
`To determine the amount of DNA
`genome equivalent" of DNA.
`corresponding to one-half genome equivalent, DNA samples were serially
`diluted and tested via PCR. The amount that yielded amplification
`products in half the wells, usually -1.5 pg of total DNA, was defined as
`"one-half genome equivalent' and used in each well of subsequent Digital
`Amplification experiments. Fifty ul light mineral oil (Sigma M-35 16) was
`added to each well and reactions performed in a HybAid Thermal cycler
`at the following temperatures: denaturation at 940 for one min; 60 cycles
`of 940* for 15 sec, 550* for 15 sec., 700* for 15 seconds; 70' for five minutes.
`Reactions were read immediately or stored at room temperature for up to
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`36 hours before fluorescence analysis.
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`Step 2: Fluorescence analysis. 3.5 ul of a solution with the following
`composition was added to each well: 67 mM Tris, pH 8.8, 16.6 mM
`NH14S04.6.7 mM MgCl2, 10 mM P-mercaptoethanol, 1 mM dATP, 1 mM
`dCTP, 1mM dGTP,1nmM TrP, 6%DMSO, 5uM primer INT,l1uM
`MB-GREEN, 1 uM MB-RED, 0. 1 units/ul Platinum Taq polymerase. The
`plates were centrifuged for 20 seconds at 6000 g and fluorescence read at
`excitation/emnission wavelengths of 485 nm/530 nm for MB-GREEN and
`530 nm/590 im for MB-RED. The fluorescence in wells without template
`was typically 10,000 to 20,000 fluorescence "units", with about 75%
`emanating from the fluorometer background and the remainder from the
`MB probes. The plates were then placed in a thermal cycler for asymmetric
`amplification at the following temperatures: 94' for one minute; 10 - 15
`cycles of 940 for 15 sec, 55' for 15 sec., 700 for 15 seconds; 940 for one
`minute; and 600 for five minutes. The plates were then i