`
`‘
`
`IIIIIIIIIIIIIIIIIIIIIIIIIIII
`
`PTO/SB/16 (6-95)
`Approved for use through 04/1 1/98. 0MB 0651-0037
`
`“s“e,0/
`
`ThisIs a request for filing a PROVISIONAL APPLICATION under 37 CFR 1. 53(c)(2).
`
`Docket Number
`
`01 107_81418
`
`Type a plus sign (+)
`inside this box -'
`
`LAST NAME
`VOGELSTEIN
`
`KINZLER
`
`INVENTORISIIAPPLICANTis)
`FIRST NAME “ RESIDENCE (CITY and either STATE or COUNTRY)
`Bert - Baltimore, Maryland
`
`W.
`
`Belair, Maryland
`
`Kenneth
`
`
`
`
`TITLE OF THE INVENTION (280 characters max)
`
`DIGITAL ANIPLIFICATION
`
`CORRESPONDENCE ADDRESS
`
`BANNER & WlTCOFF, LTD.
`Eleventh Floor
`
`1001 G Street, N.W.
`
`0
`
`,m —.
`
`‘4\
`
`ii
`
`STATE Washinoton, D.C.
`
`ZIP 20001-4597
`
`
`
`COUNTRY USA
`
`ENCLOSED APPLICATION PARTS (check all that apply)
`
`-—' Number of Pa-es -- Small Entity Statement
`-— Nemeeeee seeeee
`Other eeeeify)
`CLAIMS
`
`64
`
`METHOD OF PAYMENT (check one)
`- A check or money order is enclosed to cover the Provisional filing fee
`The Commissioner is hereby authorized to
`PROVISIONAL
`charge filing fees and credit Deposit Account
`FILING FEE
`Numb”-
`AMOUNT ($)
`
`19_0733
`
`
`
`The invention was made by an agency of the United States Government or under a contract with an
`agency of the United States Government.
`NO
`
`YES, the name of the U. S. Government agency and the Government contract number are:
`X
`h4
`
`Respectfully submitted, @I'l
`
`TYPED or PRINTED NAMEjamhAfim ._
`
`REG. NO. Iifappropriate}
`
`Additional inventors are being named on separately numbered sheets attached hereto
`
`PROVISIONAL APPLICA T/ON FILING ONL Y
`
`Ambry Exhibit 1026 - Page 1
`
`Ambry Exhibit 1026 - Page 1
`
`
`
`D][GITAL AMPLIFICATION
`
`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 fi‘om the National Institutes of Health.
`
`IEQHNICALEIELILQEIHEJNXENIIQN
`
`This invention is related to diagnostic genetic analyses. In particular
`
`it relates to detection of genetic changes and gene expression.
`
`WWW
`
`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, 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
`
`
`
`15
`
`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
`
`possible at a stage When the primary tumors are still curable and the patients
`
`20
`
`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,
`
`l
`
`Ambry Exhibit 1026 - Page 2
`
`Ambry Exhibit 1026 - Page 2
`
`
`
`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
`
`5
`
`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 ofthe cells analyzed, but the signal to noise ratio
`
`distinguishing mutant and Wild-type (WT) templates is variable. The use of
`
`10
`
`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.
`
`SHMMARYQETHEJNXENIIQN
`
`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
`
`25
`
`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
`
`30
`
`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
`
`Ambry Exhibit 1026 - Page 3
`
`Ambry Exhibit 1026 - Page 3
`
`
`
`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.
`
`5
`
`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
`
`10
`
`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 molecules such 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 fluorescent 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 which has a Tm
`
`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
`
`25
`
`having a fluorescent 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 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.
`
`30
`
`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.
`
`Ambry Exhibit 1026 - Page 4
`
`Ambry Exhibit 1026 - Page 4
`
`
`
`WW5
`
`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
`
`5
`
`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.
`
`10
`
`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,
`
`whether it is WT or mutant at the queried codons. NIB-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
`
`Beacon analysis were purified and directly sequenced.
`
`In the cases with
`
`Glyl2Cys and Gly12Arg mutations, contaminating non-neoplastic cells within
`
`the tumor presumably accounted for the relatively low ratios.
`
`In the cases
`
`with Gly12Ser and GlylZAsp, there were apparently two or more alleles of
`
`25
`
`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 G1y12Asp or Gly13Asp mutations of the c—Ki—Ras gene. Wells with
`
`values >10,000 are shaded yellow. Polyacrylamide gel electrophoretic
`
`3O
`
`analyses of the PCR products from selected wells are shown. Wells with
`
`Ambry Exhibit 1026 - Page 5
`
`Ambry Exhibit 1026 - Page 5
`
`
`
`fluorescence values <35 00 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
`
`5
`
`molecular weight markers used to determine the size of fragments indicated
`
`on the lefi (in base pairs).
`
`FIG. 4. Discriminating W'T from mutant PCR products obtained in Dig-PCR.
`
`RED/GREEN ratios were determined from the fluorescence of MB-RED and
`
`MB-GREEN as describe-d in Materials and Methods. The wells shown are the
`
`10
`
`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 <3500 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.
`
`25
`
`
`DEIAILEDDESQRUPTIQN QF THE INYENTIQN.
`
`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
`
`Ambry Exhibit 1026 - Page 6
`
`Ambry Exhibit 1026 - Page 6
`
`
`
`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
`
`5
`
`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
`
`10
`
`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 ofanalyte. At least l/lO, 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.
`
`25
`
`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
`
`30
`
`allowing sensitivities for mutation detection at the ~O.l% level.
`
`It is also
`
`possible that Digital Amplification can be performed in microarray format,
`
`potentially increasing the sensitivity by another order of magnitude. This
`
`Ambry Exhibit 1026 - Page 7
`
`Ambry Exhibit 1026 - Page 7
`
`
`
`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
`
`5
`
`(such as a G to T transversion at the second position of codon 12 of c—Ki—Ras),
`
`are expected to occur in < 1 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 of
`
`the putative mutants in the positive wells, by direct sequencing as performed
`
`10
`
`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
`
`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
`
`fiaction of alternatively spliced or mutant transcripts from a gene can be easily
`
`determined using fluorescent 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
`
`25
`
`transcript expressed constitutively as well as primers specific for the
`
`experimental transcript. One fluorescent probe would then be used to detect
`
`PCR products from the reference transcript and a second fluorescent 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
`
`30
`
`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.
`
`Ambry Exhibit 1026 - Page 8
`
`Ambry Exhibit 1026 - Page 8
`
`
`
`To distinguish whether one variant is present in each allele (vs. 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
`
`5
`
`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
`
`10
`
`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.)
`
`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
`
`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
`
`25
`
`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
`
`an amplification product. More preferably the dilution will be to between 0.1
`
`30
`
`and 0.6, more preferably to between 0.3 and 0.5 of the assay samples yielding
`
`an amplification product.
`
`Ambry Exhibit 1026 - Page 9
`
`Ambry Exhibit 1026 - Page 9
`
`
`
`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
`
`5
`
`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
`
`10
`
`the method an in situ, or “one-pot” method.
`
`
`
`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
`
`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
`
`25
`
`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.
`
`Biological samples which can be used as the starting material for the
`
`30
`
`analyses may be fi'om 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.
`
`Ambry Exhibit 1026 - Page 10
`
`Ambry Exhibit 1026 - Page 10
`
`
`
`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
`
`electrophoresis, hybridization with other types ofprobes, including TaqManTM
`
`5
`
`(dual—labeled 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
`
`complete understanding can be obtained by reference to the following specific
`
`examples which are provided herein for purposes of illustration only, and are
`
`10
`
`not intended to limit the scope of the invention.
`
`EXAMPLE].
`
`
`32:94:11:i
`
`21:131:211#—
`
`KJ‘I
`
`
`
`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 PCR
`
`plates (RPI). The composition of the reactions was: 67 mM Tris, pH 8.8, 16.6
`
`mM NH4SO4’ 6.7 mM MgC12, 10 mM B-mercaptoethanol, 1 mM dATP, 1 mM
`
`dCTP, 1 mM dGTP, 1 rnM TTP, 6% DMSO, 1 uM primer F1, 1 uM primer
`
`R1, 0.05 units/ul Platinum Taq polymerase (Life Technologies, Inc), and
`
`“one-half genome equivalent" of DNA. To determine the amount 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 pg of total DNA, was defined as "one—half
`
`genome equivalen " and used in each well of subsequent Digital Amplification
`
`experiments. Fifty ul light mineral oil (Sigma M—3516) was added to each
`
`25
`
`well and reactions performed in a HybAid Thermal cycler at the following
`
`temperatures: denaturation at 94° for one min; 60 cycles of 94° for 15 sec, 55 °
`
`for 15 sec., 70° for 15 seconds; 70° for five minutes. Reactions were read
`
`immediately or stored at room temperature for up to 36 hours before
`
`fluorescence analysis.
`
`30
`
`EXAMBLELZ
`
`10
`
`Ambry Exhibit 1026 - Page 11
`
`Ambry Exhibit 1026 - Page 11
`
`
`
`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 NH4SO4,
`
`6.7 mM MgC12, 10 mM lS-mercaptoethanol, 1 mM dATP, 1 mM dCTP, 1 mM
`
`dGTP, 1 mM TTP, 6% DMSO, 5 uM primer INT, 1 uM MB-GREEN, 1 uM
`
`5
`
`MB—RED, 0.1 units/ul Platinum Taq polymerase. The plates were centrifuged
`
`for 20 seconds at 6000 g and fluorescence read at excitation/emission
`
`wavelengths of 485 HID/530 nm for MB—GREEN and 530 uni/590 nm for
`
`MB-RED. This fluorescence in wells without template was typically 10,000
`
`to 20,000 fluorescence "units", with about 75% emanating from the
`
`10
`
`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 94° for 15 sec,
`
`55° for 15 sec., 70° for 15 seconds; 60° for five minutes. The plates were
`
`then incubated at room temperature for at least 20 minutes and fluorescence
`
`measured as described above. The fluorescence readings obtained were stable
`
`for several hours. Specific fluorescence was defined as the difference in
`
`fluorescence before and after the asymmetric amplification. RED/GREEN
`
`ratios were defined as the specific fluorescence of MB-RED divided by that
`
`of MB—GREEN. RED/GREEN ratios were normalized to the ratio exhibited
`
`by the positive controls (25 genome equivalents of DNA from normal cells,
`
`as defined in Materials and Methods). We found that the ability of MB probes
`
`to discriminate between WT and mutant sequences under our conditions could
`
`not be reliably determined from experiments in which they were tested by
`
`hybridization
`
`to
`
`relatively
`
`short
`
`complementary
`
`single
`
`stranded
`
`25
`
`oligonucleotides, and that actual PCR products had to be used for validation.
`
`EXAMELEJ
`
`Oligonucleotides
`
`and
`
`DNA
`
`sequencing.
`
`Primer
`
`F1:
`
`5'—CATGTTCTAATATAGTCACATTTTCA-3';
`
`Primer
`
`R1:
`
`30
`
`5'-TCTGAATTAGCTGTATCGTCAAGG—3';
`
`Primer
`
`INT:
`
`5'—TAGCTGTATCGTCAAGGCAC—3‘;
`
`MB-RED:
`
`5'—Cy3-CACGGGCC‘TGCTGAAAATGACTGCGTG-Dabcyl-3';
`
`11
`
`Ambry Exhibit 1026 - Page 12
`
`Ambry Exhibit 1026 - Page 12
`
`
`
`M
`
`B
`
`—
`
`G
`
`R
`
`E
`
`E
`
`N
`
`5'-F1uorescein-CACGGGAGCTGGTGGCGTAGCGTG—Dabcyl-3'.
`
`Molecular Beacons were synthesized by Midland Scientific and other
`
`5
`
`oligonucleotides were synthesized by Gene Link. All were dissolved at 50 uM
`in TE (10 mM Tris, pH 8.0/ 1 mM EDTA) and kept frozen and in the dark
`
`until use. PCR products were purified using QIAquick PCR purification kits
`
`(Qiagen).
`
`In the relevant experiments described in the text, 20% of the
`
`product from single wells was used for gel electrophoresis and 40% was used
`
`for each sequencing reaction.
`
`The primer used for sequencing was
`
`10
`
`5'—CATTATTTTTATTATAAGGCCTGC-3'. Sequencing was performed
`
`using fluorescently—labeled ABI Big Dye terrninators and an ABI 377
`
`automated sequencer.
`
`
`
`EXAMBLEA
`
`Principles underlying experiment. The experiment is outlined in Fig. 1A.
`
`First, the DNA is diluted into multiwell plates so that there is, on average, one
`
`template molecule per two wells, and PCR is performed.
`
`Second,
`
`the
`
`individual wells are analyzed for the presence of PCR products of mutant and
`
`WT sequence using fluorescent probes.
`
`As the PCR products resulting from the amplification of single
`
`template molecules should be homogeneous in sequence, a variety of standard
`
`techniques could be used to assess their presence. Fluorescent probe-based
`
`25
`
`technologies, which can be performed on the PCR products "in situ" (i.e., in
`
`the same wells) are particularly well-suited for this application. We chose to
`
`explore the utility of one such technology, involving Molecular Beacons
`
`(MB), for this purpose. MB probes are oligonucleotides with stem—loop
`
`structures that contain a fluorescent dye at the 5' end and a quenching agent
`
`30
`
`(Dabcyl) at the 3' end (Fig. 1B). The degree of quenching Via fluorescence
`
`energy resonance transfer is inversely proportional to the 6th power of the
`
`12
`
`Ambry Exhibit 1026 - Page 13
`
`Ambry Exhibit 1026 - Page 13
`
`
`
`distance between the Dabcyl group and the fluorescent dye. After heating and
`
`cooling, MB probes reform a stem-loop structure which quenches the
`
`fluorescent signal from the dye.
`
`If a PCR product whose sequence is
`
`complementary to the loop sequence is present during the heating/cooling
`
`5
`
`cycle, hybridization of the MB to one strand of the PCR product will increase
`
`the distance between the Dabcyl and the dye, resulting in increased
`
`fluorescence.
`
`A schematic of the oligonucleotides used for Digital Amplifications
`
`shown in Fig. 1C. Two] unmodified oligonucleotides are used as primers for
`
`10
`
`the PCR reaction. Two MB probes, each labeled with a different fluorophore,
`
`are used to detect the PCR products. MB-GREEN has a loop region that is
`
`complementary to the portion of the WT PCR product that is queried for
`mutations. Mutations within the corresponding sequence of the PCR product
`
`should significantly impede the hybridization of it to the lVEB probe. MB-RED
`
`has a loop region that is complementary to a different portion of the PCR
`
`product, one not expected to be mutant.
`
`It thus should produce a signal
`
`whenever a well contains a PCR product, whether that product is WT or
`
`mutant in the region queried by MB—GREEN. Both MB probes are used
`
`together to simultaneously detect the presence of a PCR product and its
`
`mutational status.
`
`
`
`
`
`
`
`Practical Considerations. Numerous conditions were optimized to define
`
`conditions that could be reproducibly and generally applied. As outlined in
`
`Fig. 1A, the first step involves amplification from single template molecules.
`
`25
`
`Most protocols for amplification from small numbers of template molecules
`
`use a nesting procedure, wherein a product resulting from one set of primers
`
`is used as template in a second reaction employing internal primers. As many
`
`applications of digital amplification are expected to require hundreds or
`
`thousands of separate amplifications, such nesting would be inconvenient and
`
`30
`
`could lead to contamination problems. Hence, conditions were sought that
`
`would achieve robust amplification without nesting. The most important of
`
`these conditions involved the use of a polymerase that was activated only after
`
`13
`
`Ambry Exhibit 1026 - Page 14
`
`Ambry Exhibit 1026 - Page 14
`
`
`
`heating and optimized concentrations of dNTP’s, primers, buffer components,
`
`and temperature. The conditions specified in Examples 1—3 were defined after
`
`individually optimizing each of these components and proved suitable for
`
`amplification of several different human genomic DNA sequences. Though
`
`5
`
`the time required for PCR was not particularly long (~2.5 hr), the number of
`
`cycles used was high and excessive compared to the number of cycles
`
`required to amplify the “average" single template molecule. The large cycle
`
`number was necessary because the template in some wells might not begin to
`
`be amplified until several PCR cycles had been completed. The large number
`
`10
`
`of cycles ensured that every well (not simply the average well) would generate
`
`a substantial and roughly equal amount of PCR product if a template molecule
`
`
`
`
`
`were present within it.
`
`The second step in Fig 1A involves the detection of these PCR
`
`products.
`
`It was necessary to considerably modify the standard MB probe
`
`approach in order for it to fimction efficiently in Digital Amplification
`
`applications. Theoretically, one separate MB probe could be used to detect
`
`each specific mutation that might occur within the queried sequence. By
`
`inclusion of one ME; corresponding to WT sequence and another
`
`corresponding to mutant sequence, the nature of the PCR product would be
`
`revealed. Though this strategy could obviously be used effectively in some
`
`situations, it becomes complex when several different mutations are expected
`
`to occur Within the same queried sequence. For example, in the c-Ki-Ras gene
`
`example explored here,
`
`twelve different base substitutions resulting in
`
`missense mutations could theoretically occur within codons 12 and 13, and at
`
`25
`
`least seven of these are observed in naturally—occurring human cancers. To
`
`detect all twelve mutations as well as the WT sequence with individual
`
`Molecular Beacons would require 13 different probes.
`
`Inclusion of such a
`
`large number ofMB probes would not only raise the background fluorescence
`
`but would be expensive. We therefore attempted to develop a single probe
`
`30
`
`that would react with WT sequences better than any mutant sequence within
`
`the queried sequence. We found that the length of the loop sequence, its
`
`melting temperature, and the length and sequence of the stern were each
`
`14
`
`Ambry Exhibit 1026 - Page 15
`
`Ambry Exhibit 1026 - Page 15
`
`
`
`important in determining the efficacy of such probes. Loops ranging from 14
`
`to 26 bases and stems ranging from 4 to 6 bases, as well as num