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`
`|
`
`for following:
`
`Te —
`Beri Vogelstein, Baltimore, MD;
`Kenneth W.Kinzler, BelAlr, MD;
`
`‘
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`
`APPLICANTS
`
`Bert Vogelstein, Baltimore, MD;
`
`Kenneth W.Kinzler, BetAlr, MD;
`
`CONTINUING DATA St*tet#tettansseeseraavas
`THIS APPLN CLAIMS BENEFIT OF 60/146,782 08/02/1999
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`

`DIGITAL AMPLIFICATION
`
`ABSTRACT
`
`The identification of pre-defined mutations expected to be presentin
`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; cach product is then separately analyzed for the
`presence of mutations: The process providesa reliable and quantitative
`measure of the proportion of variant sequences within a DNA sample.
`
`10
`
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`
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`
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`32
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`Page 358 of 1365
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`
`
`DIGITAL AMPLIFICATION
`
`This application claims the benefit ofU.S. Serial No. 60/146,792,filed
`
`August 2, 1999.
`
`The U.S. governmentretains certain rights in this invention by virtue
`ofits support of the underlying research, supported by grants CA 43460, CA
`
`
`
`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 managementas wellas for
`basic research into the pathogenesis of neoplasia. However, many of these
`opportunities hinge upon detection of a sniall 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
`
`DNAofneoplastic cells have also been found in the blood of cancer patients
`(9-11). The detectionofresidual disease in lymph nodes or surgical margins
`
`10
`
`15
`
`1 D
`
`ind
`
`mn
`fod
`LJ
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`n
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`al
`
`ed
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`Page 359 of 1365
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`||
`\||''
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`||'i
`
`a m
`
`ay 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 ofthe 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,butit is difficult to
`
`quantitate the fraction of mutant molecules 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
`
`methods is that it is difficult or impossible to independently confirm the
`
`existence of any mutationsthat are identified.
`Thus there is a need in the art for methods for accurately and
`
`quantitatively detecting genetic sequences in mixed populations ofsequences.
`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
`
`10
`
`15
`
`[as
`
`25
`
`genetic sequences.
`
`It is another object of the present invention to provide molecular
`beacon probes useful in the methodofthe invention.
`These and other objects of the invention are achieved by providing a
`
`30
`
`method for determiningthe presence of a selected genetic sequence in a
`population ofgenetic 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
`
`1)
`
`Page360of1365
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`Page 360 of 1365
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`

`

`5
`
`10
`
`‘amplifiedmolecules in the assay samples of the set. The amplified molecules
`in the assay samples of the set are then enalyzed to determinea first number
`
`of assay samples which contain the selected genetic sequence and a second
`
`numberof assay samples which contain a reference genetic sequence. The
`
`first number is then compared to the second numberto ascertain a ratio which
`
`reflects the composition of the biological sample.
`
`Another embodimentofthe inventionis 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
`
`15
`
`second number to ascertain a ratio which reflects the composition of the
`
`20
`
`25
`
`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 agentat 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
`
`It comprises an oligonucleotide with a stem-loop structure
`embodiment.
`having @ photoluminescent dye at one of the 5' or 3' ends and a quenching
`
`agentat the opposite 5' or 3' end. The loop consists of 19-20 basepairs and
`has a T,, 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
`
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`probes, either mixed together or provided inadivided container as a kit.
`
` Page 361 of 1365
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`Page 361 of 1365
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`

`

`
`
`
`
` t| | ||
`
`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.
`
`BRIGE DESCRIPTION OF
`THE DRAWINGS
`Fic. 1. Schemdtic of experimental design. (A) The basic twosteps involved:
`PCR on diluted NA samples is followed by addition of fluorescent probes
`which discriminate
`between WT and mutant alleles and subsequent
`
`In the stem-loop
`ciple of molecular beacon analysis.
`fluorometry. (B)
`configuration, fluoreskence from a dye at the 5' end of the oligonucleotide
`probe is quenched by a Qabcyl group at the 3‘ end. Upon hybridization to a
`template, the dye is sepayated from the quencher, resulting in increased
`fluorescence. Modified
`Marras ef al. . (C) Oligonucleotide design.
`Primers FI and R1 are used to
`4mplify the genomic region of interest. Primer
`INT is used to produce single stranded DNA from the original PCR products
`during a subsequent asymmetric
`step (see Materials and Methods).
`
`whether it is WI or mutant at the
`
`queried codons. MB-GREENis a
`
`be
`i
`
`15
`
`datects the WT PCR product.
`
`Molecular Beacon which preferentially
`
`
`
`Gly12Ser and Gly12Asp,there were apparently two ofgore alleles ofmutant
`c-Ki-Ras for every WT allele; both these tumors were aneuploid.
`
`Fic. 3. Detecting Dig-PCR products with MB-RED.Specific Fluorescence
`Avaits ofrepresentative wells from an experiment employing colorectal cancer
`
`30
`
`Page 362 0f 1365.
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`Page 362 of 1365
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`

`

`
`
`
`
`
`
`
`
`cells with Gly12Asp or Gly13Asp mutations ofthe c-Ki-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 productof the correct size while wells
`with fluorescence values >10,000 SFU always contained PCR products of 129
`
`bp. Non-specific products generated during the large number of cycles
`required for Dig-PCR did not affect the fluorescence analysis. M1 and M2 are
`molecular weight markers used to determine the size of fragments indicated
`
`on the left (in base pairs).
`
`20
`
`25
`
`equivalents of BNA from normal cells. Each of these registered positive with
`MB-REDand the RED/GREENratios were 1.0 +/- 0,1 (mean +/- 1 standard
`
`deviation). The wells cajored yellow contained no template DNA and each
`was negative with MB-RED\(i.c., fluorescence <3500 fluorescence units.),
`The other 288 wells contained diiyted DNA from the steol sample prepared
`
`(Rubeck et al, 1998, BioTechniques 25:588-592.)
`by alkaline extraction.
`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 frota the indicated wells were
`
`used for automated sequence analysis.
`
`
`
`Page 363 of 1365
`
`Page 363 of 1365
`
`

`

`The method devised by the present inventors involves separately
`
`amplifying small numbers oftemplate molecules so that the resultant products
`have a proportion of the analyte sequence which is detectable by the detection
`
`means chosen. Atits 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 mutantalleles in the
`analyzed population.
`The biological sampleis diluted to a point at which a practicafly usable
`
`number of the diluted samples contain a proportion of the selected genetic
`
`sequence (analyte) relative to total template molecules such that the analyzing
`
`10
`
`ad
`
`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 1/50 of the diluted samples have a detectable
`
`proportion of analyte, Atleast 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
`aloss of economy, as many samples will be analyzed and provide no signal,
`
`A particularly prefered degree ofdilution is to a point where each of the assay
`
`samples has on average one-half of a template. The dilution can be performed
`
`from more concentrated samples. Alternatively, dilute sources of template
`nucleic acids can be used. All of the samples may contain amplifable
`template molecules, Desirably each assay sample prior to amplification will
`
`contain less than a hundred or less than ten template molecules.
`
`Digital amplification can be used to detect mutations present at
`
`25
`
`30
`
`relatively low levels in the samples to be analyzed. Thelimit 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
`
`eeeteee,
`
`i
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`Page 364 of 1365
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`

`

`§
`
`10
`
`15
`
`20
`
`25
`
`30
`
`commercially available and 1536 well plates are on the horizon,theoretically
`allowing sensitivities for mutation detection at the ~0.1% level. It is also
`possible that Digital Amplification can be performed in microarray format,
`potentially increasing the sensitivity by another order of magnitude. This
`sensitivity may ultimately be limited by polymerase errors. The effective
`error rate in PCR as performed under our conditions was <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
`of c-Ki-ras) is expected to occur in <I 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 mutarits 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
`DNAtemplates 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 mutanttranscripts 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 photoluminescentprobe 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
`
`ad
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`Page365°6f T3657 *
`
`
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`Page 365 of 1365
`
`

`

`1
`
`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 examplesinvolves the investigations of allelic
`status when two mutations are observed upon sequence analysis of a standard
`DNA sample. To distinguish whether one variantis present in each allele (vs.
`both occurringin oneallele), cloning ofPCR products is generally performed.
`The approach described here would simplify the analysis by eliminating the
`need for cloning. Other potential applications of Digital Amplification are
`listed in Table 1, When the goal is the quantitation of the proportion of two
`relatively commonalleles or transcripts rather than the detection of rare
`alleles, techniques such as those émploying 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,(¢.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 exponentialnatureofPCR. to a linear one. It should
`therebyproveuseful forexperiments requiringtheinvestigation ofindividual
`alleles, rare variants/mutations, orquantitativeanalysisofPCRproducts,
`In one preferred embodiment each diluted sample has on average one
`halfa template molecule. This is the same as dne halfofthediluted samples
`having one template molecule. This can be empirically determined by
`amplification. Hither the analyte (selected genetic sequence) or the reference
`genetic sequence can be used for this determination. Ifthe analysis method
`being used can detect analyte when presentat a level of 20%, then one must
`dilute such that a significant numberof diluted essay 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 ofthe assay samplesyield
`
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`Page 366 of 1365
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`Page 366 of 1365
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` I|| ||: || ||i:
`
`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 atoplification 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 numberof 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 ace performed in the same receptacle. This makes
`
`the method an in situ, or “one-pot” method,
`
`The numberof 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. Chromosomaltranslocations 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.
`
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`
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`
`20
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`
`Similarly, using cDNA made from mRNA one can determinerelative levels
`of transcription of two different genes. One can usedigital amplification to
`
`distinguish betweena situation where oneallele 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.
`
`Thése can be detected using digital
`
`preOTEECTEFETEre
`
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`amplification.
`
`Page 367 of 1365
`
`Page 367 of 1365
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`

`

`Biological samples which can be used as the starting material for the
`analyses may be from any fissue or body sample fram 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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`Page 368 of 1365
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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 photonoflight, Such processes include fluorescence,
`
`which denotes emission accompanying descent from an excitedstate with
`
`paired electrons (a "singlet" state) or unpaired electrons {a "triplet" state)
`
`to a lower state with the same multiplicity, i.¢., a quantum-mechanically
`“allowed” transition. Photoluminescence also includes phosphorescence
`
`which denotes emission eccompanying descent from an excited triplet or
`
`singlet state to a lower state of different multiplicity, 1.2, a quantum
`mechanically "forbidden" transition. Compared to “allowed"transitions,
`“forbidden” transitions are associated with relatively longer excited state
`lifetimes.
`Thequenching ofphotoluminescencemay be analyzedbya variety of
`methods which vary primarily in terms of signal transduction. Quenching
`may be transduced as changes in the intensity ofphotoluminescence or as
`changes in the ratio of photoluminescence intensities at two different
`wavelengths, or as chatiges in photoluminescence lifetimes, or even as
`changes in the polarization (anisotropy) of photoluminescence. Skilled
`practitioners will recognize that instrumentation for the measurement of
`these varied photoluminescent responses are known. The particular
`
`tatiometric methods for the analysis of quenching in the instant examples
`
`25
`
`should not be construed as limiting the invention to any particular form of
`
`signal transduction. Ratiometric measurements of photoluminescence
`
`in intensity,
`intensity can include the measurement of changes
`photoluminescence lifetimes, or even polarization (anisotropy).
`
`Although the working examples demonstrate the use of molecular
`
`30
`
`beacon probes as the meansof analysis of the amplified dilution samples,
`other techniques can be used as well. These include sequencing, gel
`
`12
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`Page 370 of 1365
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`

`

`TagMan™ (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 ere provided herein for purposes of illustration
`only, and are not intended to limit the scope of the invention.
`
`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 (Marsh Biomedical Products, Rochester, NY).
`The
`composition of the reactions was: 67 mM Tris, pH 8.8, 16.6 mM NH,SO,
`6.7 mM MgCl,, 10 mM B-mercaptoethanol, 1 mM dATP, 1 mM dCTP,1
`mM dGTP, 1 mM TTP, 6% DMSO,1 uM primer F1, 1 uM primer RI, 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 genomeequivalent, 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-3516) was
`added to each 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.
`
`10
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`led
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`Page 371 of 1365
`
`13
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`I
`
`EXAMPLE1
`
`
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`Page 371 of 1365
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`

`

`EXAMPLE2
`
`
`
`
`
`EXAMPLE3
`
`
`
`creatntae
`
`||
`
`|
`
`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
`NH,SO,6.7 mM MgCl, 10 mM §-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 MB-RED,0,1 units/u] Platinum Taq polymerase. The
`
`plates were centrifuged for 20 seconds at 6000 g and fluorescence read at
`excitation/emission wavelengths of 485 nm/530 nm for MB-GREEN and
`
`530 nm/590 nm 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 94° for 15 sec, 55° for 15 sec., 70° for 15 seconds; 94° for one
`minute; and 60° for five minutes. The plates were then incubated atroom
`femperaturé for ten to sixty minutes and fluorescence measured as
`described above. Specific fluorescence was defined as the difference in
`fluorescence before and after the asymmetric amplification. RED/GREEN
`ratios were defined as the specific fluoreacence 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 above in Example 1). We found that the ability of
`
`MB probesto 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 oligonucleotides, and that actual PCR products had to be used for
`validation.
`
`5
`
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`Oligonucleotid
`
`and
`
`DNA
`
`sequencing.
`
`Primer
`
`§'-CATGTTCTAATATAGTCACATTTICA-3;
`
`Primer
`
`FI:
`
`RI:
`
`5'-TCTGAATTA
`
`GTATCGTCAAGG-3';
`
`Primer
`
`INT:
`
`Page 372 of 1365
`
`14
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`a
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`Page 372 of 1365
`
`

`

`5'-TAGCTGTATCGTCAAGGCAC-3';
`
`MB-RED:
`
`§'.Cy3-CACGGGCCTGCTGAAAATGACTGCGTG-Dabcyl-3';
`M
`B
`-
`G
`R
`EB
`EB
`N
`
`5'-Fluorescein-CACGGGAGCTGGTGGCGTAGCGTG-Dabcyl-3'.
`
`Molecular Beacons (33,34) were synthesized by Midland Scientific and
`
`other oligonucleotides were synthesized by Gene Link (Thornwood, NY).
`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
`QlAquick PCR purification kits (Qiagen).
`In the relevant experiments
`describedin the text, 20% ofthe product from single wells was used for gel
`
`electrophoresis and 40% was used for each sequencing reaction. The
`primer
`used
`for
`sequencing
`was
`5-CATTATTITTATTATAAGGCCTGC-3'. Sequencing was performed
`
`using fluorescently-labeled ABI Big Dye terminators and an ABI 377
`
`automated sequencer.
`
`10
`
`15
`
`wt
`
`EXAMPLE4
`
`Principles underlying experiment. The experimentis 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 ofPCR 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 technologies, which can be performed on the PCR products
`“in situ" (i.e. in the same wells) are particularly well-suited for this
`application (31, 33-40). We chose to explore the utility of one such
`technology, involving Molecular Beacons (MB),for this purpose (33,34).
`MB probes are oligonucleotides with