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`
`Banner & Witcorf
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`B/B/!2014 10:
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`26:14 AM PAGE
`
`2/008
`
`Fax SErver
`
`Pohl & Shik
`
`f Table 1. Examples using digital PCR for molecular analysis in clinical samples.
`
`
`Application
`Findings
`
`
`
`
`Getection oF AWAS mutational statu
`
`id high-
` de ovarian serous carcinome develop tarougn
`fent pathways
`
`18}
`
`(34
`
`123
`
`17
`
`374
`
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`
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`
`33]
`
`natysis oF ARIAS and Alin APC gangs
`
`
`of Aland KRAS mutations
`
`Detection of LOH in APC locus
`
`
`
`detection oP Al of chromosomes Tp, 8p. T5q and 1d
`
`
`raf Al of chromosome T8q
`
`of chromosornes 8p and 8a
`
`
`
`
`iO
`@ONP markers with high
`fraquency of allelic loss in ovarian caricer
`
`Murations in ARAS and APoF APC occur in aapendiceal adenomas
`
`
`
`a high
`
`
`vascular invasion in colorectal carcinomas
`
`
`in Fistopatholog
`3
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`
`LNA
`
`
` tivity in plasma
`
`Al cart oe detecte
`
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`Fromm patients with ovari
`oro Al car be at
`
`iSeful adjunct far the detection of cancer in
`
`{rs IP Ovarian,
`
`
`
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`
`
`
`
`detection of APC gene expression
`
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`
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`
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`
`
`
`APC: Adenomate
`
`used directly on the PCR products in the same wells, are partiou-
`larly well sulted for this purpose (2). Currently, molecular
`beacons are extensively used to detect the PCR products in
`digital PCR assays [3]. For ovutational analysis, a pair of molecu-
`lar beacoris is designed with one hnybridiving ta the wild type
`sequence that harbors the mutation and the other hybridizing to
`the neighboring sequence tGURE 9, Therefore, the mutational
`status of a specific allele in a well is determined by theratio of
`fluorescence intensity of the two beacons in that particular well,
`Ag multiple wells are courted, digttal POR can be used to detect
`mutations present at relatively low levels in the samples to be
`analysed. The sensttivity of mutation detection depends om the
`mumiber of wells that are inchided for analysis and the intrinsic
`mutation rate of ihe polymerase used for anyplification. For
`assessing allelic imbalance, single nucleotide polymorphisms
`iSMPs} are used to represent the paternal or maternal alleles, A
`pair of PCE primers and a pair of molecular beacons are
`designed for each SNP gicure 2. Digital POR is performed
`using a SNP marker for which the patientie heterozygous. The
`resultant PCR prochicts are then analyzed using molecular
`beacon probes to determine allelic representation. The mecha-
`nism of howmolecular beacons discriminate between maternal
`and paternal alleles is briefly summarized. Molecular beacona are
`single-stranded oligonucleotides which contain 3 fluorescent dve
`and 3 quencher on their 5° and 3° ends, respectively (FIGURE 1).
`
`Both beacons are identical except for the nucleotide corre-
`sponding to the SNP and the fhiorescent label {green or ned),
`Molecular beacons include a hairpin structure, which brings the
`fuorephore closer to the quencher, and do not ernit Tuarescence
`wher not hybridized to a PCR product fa], Upon hybridization
`te their camuplirsentary nucleotide semuences, the quencher is
`distanced from the fhiorophore, resulting in increased flunres-
`cence. Therefore, the ratio of fluorescence intensity of two allele-
`specific beacons with either green or red Tuorescence is calcu-
`lated to determine the allele type in one PCR reaction {well}.
`With hundreds or thousands of wells feactions} counted, the
`percentage of mutant alleles or the ratio of maternal and paternal
`alleles can be determined. For allelic siatus, a rigorous statistical
`method is then used to conclude whether allelic iribalance is
`present inthe background of normat DNA [5,4].
`
`Applications of digital PCR
`Mutational analysis
`For a varlety of basic yesearch anc clinical applications, the identi-
`fication of rare mutations is very important. Analysis of the early
`effects in tumorigenesis often depends on the ability to detect
`small populations of mmitant cells [7.8]. Reliable technology to
`demonstrate the presence of erutations in clinical specimens
`holds great pronise for cancer detection, 33 niutations represent
`arodlecular genetic hallmark of neoplastic diseases,
`
`42
`
`Page 1067 of 1237
`
`BapertRoy. Mol. Dison. (3), (2004)
`
`Page 1067 of 1237
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`
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`Danner & WRElOors
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`S/Of AOL LOT eho he
`
`AM
`
`PAGE
`
`& A OU
`
`raNX oGfryer
`
`To address whether digital PCR is useful for mutation detec-
`tion in canrer, Vogelstein and Kireler have analysed the DNA
`from stool specimens in patients with colorectal cancer 11], Their
`study focused on the VA4Sgene mutation, which ts a frequent
`molecular genetic event in colorectal cancer [510 As the stool
`
`Digital PCR
`
`Tn another study, digital PCR has been usedto identify AAAS
`mutations in paraffin tissues of apperdiiceal mucinous adeno-
`mas in identical twins [12]. One of the twins suffered froma rare
`
`disease called sercomntama peritoneal (PMP), which produces
`
`an overwhelming amount of mucin in the intra-abdominal
`cavity 38 a result of the rapture of the appendiceal mucinous
`tumor, As the nvucinous adenoma is a single layer of neoplastic
`cells embedded in abundardstromal cells and mucin,
`tradi-
`ional methods, such as direct reucleotide sequencing, may not
`be sensitive enough to detect AASrnutations, even when laser
`capture microdissection is employed ta enrich the tumor cell
`population, [n this study, the tumor tissue on paraffin sections
`was dissected under an inverted microsrope and genomic DNA
`was purified and subjected to digital PCR, The study demon-
`strated that identical AAAS mutations were detected in the
`
`appendiceal adenoma and peritoneal tumor fram the twin with
`PMP. whereas the adenoma from the other twin harbored a dif-
`ferent mastation, The XFAS mutational analysis supported the
`viewof the authors that PMP is clonally derived from the asso-
`clated appendiceal mucinous adenoma, The different types of
`mutations in ARASin the turnors from both siblings suggested
`that mutation in AKAS occurs somatically in adenomas and is
`independent of the identical genetic background of the twins.
`
`DNA is pool DNA seleased from a mixed-cell population
`including both normal and tumor cells, approximately 1-109
`of the XMAS genes purified fram stool contained mutant alleles
`q. Therefore, digital PCR appears a well-suited technique to
`assess the presence of mutated AAS gene in stool. A 384-well
`digital PCR experiment was established to include positive con-
`irols (48 of the wells contained 25-gename equivalenis of DNA
`froma normal cells) and negative controls (48 wells without
`DNAtemplate!. The other 288 wells contained an appropriate
`ditution of stool DNA. In this study, molecular beacon red fuo-
`rescence indicated that 102 of these 268 experimental wells com-
`tained PCR products, whereas the other 188 wells did not. The
`red/green ratios of the 102 positive wells suggested that Ave con-
`iained mutant NAASalleles. Th determine the mature of the
`mutam, AWAS genes frora stool in the five positive wells, the
`PCR products were sequenced directly to reveal Gly12Ala muta-
`tions {GGT to GCTat endon 12} in four of them, whereas the
`sequence of the other indicated a silent C>T transition at the
`third position of codon13, This transition presumably resulted
`fromm a PCR error during the first productive cycle of amplifies
`tion wom @ wild type teroplate, Thus, approxirnatery 495
`(4/102) of the ARASalleles present in this stool sarnple con:
`tained a Gly1ZAla muiation. The mutantalleles in the stool pre-
`sumably arose from the colorectal cancer of the patient, as direct
`sequencing of PCR products generated from DNA of the cancer
`identified the identical Glyi2Ala mutation ff,
`
`i fl
`
`2, Quencher:
`
`Assessing allelic imbalancein issue
`Geneuc UIStability B a molecular signahure oF most numan
`cancers [13] and at the ranlemiar fevel
`is characterized by
`allelic imbalance (AN, representing losses or gains of defined
`chromosomal regions. Analysis of Al is important in elucidating
`the molecular basis in the development of cancer. There are,
`lhowever, at least Dbwo major problems associated with the current
`
`
`
`
`Wild tyoe KAAS...
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`
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