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abbreviations: digital PCR - DigitalPCR - dPCR - dePCR
Introduction
Definition: Digital PCR (dPCR) is a
refinement of conventional PCR methods that can be used to directly
quantify and clonally amplify nucleic acids (including DNA, cDNA,
methylated DNA, or RNA). The key difference between dPCR and
traditional PCR lies in the method of measuring nucleic acids amounts,
with the former being a more precise method than PCR. PCR carries out
one reaction per single sample. dPCR also carries out a single reaction
within a sample, however the sample is separated into a large number of
partitions and the reaction is carried out in each partition
individually. This separation allows a more reliable collection and
sensitive measurement of nucleic acid amounts. The method has been
demonstrated as useful for studying variations in gene sequences - such
as copy number variants, point mutations, and
it is routinely used for clonal amplification of samples for
"next-generation sequencing."
PCR Basics: The PCR method is used
to quantify nucleic acids by amplifying a nucleic acid molecule with
the enzyme DNA polymerase. Conventional PCR is based on the theory that
amplification is exponential. Therefore, nucleic acids may be
quantified by comparing the number of amplification cycles and amount
of PCR end-product to those of a reference sample. However, many
factors complicate this calculation, creating uncertainties and
inaccuracies.
These factors include the following:
Digital PCR overcomes
the difficulties of conventional PCR. With
dPCR, a sample is partitionedso that individual nucleic acid molecules
within the sample are localized and concentrated within many separate
regions. The partitioning of the sample allows one to count the
molecules by estimating according to Poisson. As a result, each part
will contain "0" or "1" molecules, or a negative or positive reaction,
respectively. After PCR amplification, nucleic acids may be quantified
by counting the regions that contain PCR end-product, positive
reactions.
In conventional PCR,
starting copy number is proportional to the number of PCR amplification
cycles. dPCR, however, is not dependent on the number of amplification
cycles to determine the initial sample amount, eliminating the reliance
on uncertain exponential data to quantify target nucleic acids and
providing absolute quantification.
Development: The dPCR concept was
conceived in 1992 by Sykes et al. using nested PCR. An important
development occurred in 1995 with co-inventions by Brown at Cytonix and
Silver at the National Institutes of Health of single-step
quantitization and sequencing methods employing nano-scale arrays and
localized clonal colonies using capillaries, gels, affinity
surfaces/particles and immiscible fluid containments, resulting in a
1997 U. S. Patent (U. S. Patent 6,143,496) and subsequent
divisional and continuation patents.
Vogelstein and Kinzler further developed the concept by quantifying
KRAS mutations in stool DNA from colorectal cancer patients. Digital
PCR has been shown to be a promising surveillance tool for illnesses
such as cancer. Significant additional developments have included using
emulsion beads for digital PCR by Dressman and colleagues. Digital PCR
has many other applications, including detection and quantitization of
low-level pathogens, rare genetic sequences, gene expression in single
cells, and the clonal amplification of nucleic acids (cPCR or clonal
PCR) for the identification and sequencing of mixed nucleic acids
samples or fragments. It has also proved useful for the analysis of
heterogeneous methylation.
In 2006
Fluidigm
introduced the first commercial system for digital PCR based on
integrated fluidic circuits (chips) having integrated chambers and
valves for partitioning samples. In March 2010, a patent was published
for digital PCR based on emulsions.
Digital PCR has many
potential applications, including the detection and quantification of
low-level pathogens, rare genetic sequences, copy number variations,
and relative gene expression in single cells. Clonal amplification
enabled by single-step digital PCR is a key factor in reducing the time
and cost of many of the "next-generation sequencing" methods and hence
enabling personal genomics.
Application of digital PCR for Absolute Quantitation Digital PCR is quantitative PCR method that can be used to measure absolute quantitation. In this technique, the number of positive and negative amplification reactions is used to the determine precise measurement of target concentration. dPCR Applications:
Reference: http://en.wikipedia.org
Absolute Quantification: digtal-PCR vs. classical standard curve based 'absolute' Quantification by Life Technologies When calculating the "absolute" results of your real-time PCR (qPCR) experiment, you can use either digital PCR method or classical standard curve based "absolute quantification". More info at Life Technologies
Absolute Quantification Using the Digital PCR MethodDigital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.
The
standard curve method for absolute quantification is similar to the
standard curve method for relative quantification, except the absolute
quantities of the standards must first be known by some independent
means.
The guidelines below are critical for proper use of the standard curve method for absolute quantification:
It is generally not possible to use DNA as a standard for absolute quantification of RNA because there is no control for the efficiency of the reverse transcription step. StandardsThe absolute quantities of the standards must first be known by some independent means. Plasmid DNA and in vitro transcribed RNA are commonly used to prepare absolute standards. Concentration is measured by A260 and converted to the number of copies using the molecular weight of the DNA or RNA. Digital PCR Using the OpenArray® Real-Time PCR System More info at Life Technologies Digital PCR is a new approach to nucleic acid detection and quantification, which is a different method of absolute quantification and rare allele detection relative to conventional qPCR. Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls. The OpenArray® Real-Time PCR System enables digital PCR experiments at a scale previously unattainable—in a single day, one user can generate >36,000 digital PCR data points on the OpenArray® Real-Time PCR System, without the use of robotics. Other features of the system include:
Digital PCR - A breakthrough in quantitative PCR by Bio-Rad The QX100 Droplet Digital PCR system is the third generation of PCR technology. Droplet Digital™ PCR (ddPCR™) provides an absolute measure of target DNA molecules with unrivaled accuracy, precision, and sensitivity. Applications include copy number variation, rare sequence detection, mutation detection, and gene expression analysis. The QX100 ddPCR system lets you:
Digital PCR: a powerful new tool for noninvasive prenatal diagnosis? Zimmermann BG, Grill S, Holzgreve W, Zhong XY, Jackson LG, Hahn S. Prenat Diagn. 2008 28(12): 1087-1093. Fluidigm Corporation, South San Francisco, USA. Recent reports have indicated that digital PCR may be useful for the noninvasive detection of fetal aneuploidies by the analysis of cell-free DNA and RNA in maternal plasma or serum. In this review we provide an insight into the underlying technology and its previous application in the determination of the allelic frequencies of oncogenic alterations in cancer specimens. We also provide an indication of how this new technology may prove useful for the detection of fetal aneuploidies and single gene Mendelian disorders. Non-invasive prenatal diagnosis by single molecule counting technologies Chiu RW, Cantor CR, Lo YM. Trends Genet. 2009 Jul;25(7): 324-331 Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong SAR, China. Non-invasive prenatal
diagnosis of fetal chromosomal aneuploidies and monogenic diseases by
analysing fetal DNA present in maternal plasma poses a challenging
goal. In particular, the presence of background maternal DNA interferes
with the analysis of fetal DNA. Using single molecule counting methods,
including digital PCR and massively parallel sequencing, many of the
former problems have been solved. Digital mutation dosage assessment
can detect the number of mutant alleles a fetus has inherited from its
parents for fetal monogenic disease diagnosis, and massively parallel
plasma DNA sequencing enables the direct detection of fetal chromosomal
aneuploidies from maternal plasma. The analytical power of these
methods, namely sensitivity, specificity, accuracy and precision,
should catalyse the eventual clinical use of non-invasive prenatal
diagnosis.
Digital polymerase chain reaction; new diagnostic opportunities European Pharmaceutical Review - Genomics page 7-9; published 22 February 2010 Jim Huggett 1 & Daniel J Scott 2 1 Molecular and Cell Biology, LGC; 2 Project Manager, Research and Technology Division, LGC LGC is an international science-based company located in South West London. A progressive and innovative enterprise, LGC operatesin socially responsible fields underpinning the health, safety and security of the public, and regulation and enforcement for UKgovernment departments and blue chip clients. Our products and services enable our customers to have a sound basis on whichto base their scientific and commercial decisions or conformity to international statutory and regulatory standards. DNA diagnostics gets digitized by Mikael Kubista and Anders Stahlberg Drug Discovery Wold - Fall 2011 Quantitative real-time PCR (qPCR) has during the last two decades emerged as the preferred technology for nucleic acid analysis in routine as well as in research. qPCR has the sensitivity to detect a single molecule, the specificity to differentiate targets by a single nucleotide, and, because of its exponential nature, virtually unlimited dynamic range. PCR’s next frontier Nathan Blow reports. PCR - the workhorse of modern molecular biology - is charging forward using both conventional and digital methods to explore single cells and even single molecules. Follow this report! Emerging real-timePCR applications Mikael Kubista Drug Discovery World Summer 2008 Since its introduction on the commercial market little more than 10 years ago,real-time PCR has become the main technical platform for nucleic aciddetection in research and development, as well as in routine diagnostics. In2007 the real-time PCR market revenue in the US was estimated at $740million with annual growth of more than 10%. In this article latestdevelopments and future expectations are presented. Video on Digital PCR by TATAA Biocenter The video on Digital PCR describing latest platform at TATAA Biocenter. http://www.youtube.com/watch?v=qdFOpRbrYE0&noredirect=1 For more information how to do dPCR at TATAA Biocenter see http://www.tataa.com/Services/Projects.html#Digital Concept of the limiting dilution assay Limiting dilution analysis: from frequencies to cellular interactions Dozmorov I, Eisenbraun MD, Lefkovits I. Immunol Today. 2000 Jan;21(1):15-8. Dept of Pathology, University of Michigan, Ann Arbor, MI 48109, USA. Limiting dilution
analysis (LDA)1has gained widespread accept-ance as a tool for
quantifyingcells that possess observablefunctional activities.
Thoroughly plannedtitration experiments can produce straight-forward
and interpretable single-hit kinetics,whereas analyses of
unfractionated cellpopulations over a broader dilution rangeresult in
data that deviate from linearity anddo not adhere to all-or-none
functionality(e.g. virgin and memory CD4T cells2andothers3–10).
However, by studying the factorsthat cause the deviation from
linearity, theinteractions between different cell types inthe
population can be identified and charac-terized. As a corollary, it
follows that, alongwith quantification of desired cells, LDA al-lows an
analysis of the regulatory processesthat underlie an observed activity.
End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: performance under optimal and suboptimal conditions Ramachandran S, Xia GL, Ganova-Raeva LM, Nainan OV, Khudyakov Y. J Virol Methods. 2008 Aug;151(2): 217-224 Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. An approach for
determination of hepatitis C virus (HCV) quasispecies by end-point
limiting-dilution real-time PCR (EPLD-PCR) is described. It involves
isolation of individual coexisting sequence variants of the
hypervariable region 1 (HVR1) of the HCV genome from serum specimens
using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak
study provided insights into the epidemiological relationships between
incident and chronic cases. When applied to samples from a longitudinal
study of infected patients, HVR1 sequences from each sampling
time-point were observed to group as distinct phylogenetic clusters.
Melting peak analysis conducted on EPLD-PCR products generated from
these patients could be used for evaluation of HVR1 sequence
heterogeneity without recourse to clonal sequencing. Further, to better
understand the mechanism of single-molecule PCR, experiments were
conducted under optimal and suboptimal annealing temperatures. Under
all temperature conditions tested, HVR1 variants from the major
phylogenetic clusters of quasispecies could be amplified, revealing
that successful HVR1 quasispecies analysis is not contingent to
dilution of starting cDNA preparations to a single-molecule state. It
was found that EPLD-PCR conducted at suboptimal annealing temperatures
generated distributions of unique-sequence variants slightly different
from the distribution obtained by PCR conducted at the optimal
temperature. Hence, EPLD-PCR conditions can be manipulated to access
different subpopulations of HCV HVR1 quasispecies, thus, improving the
range of the quasispecies detection. Although EPLD-PCR conducted at
different conditions detect slightly different quasispecies
populations, as was shown in this study, the resulted samples of
quasispecies are completely suitable for molecular epidemiological
investigation in different clinical and epidemiological settings.
Digital PCR Digital PCR Vogelstein B, Kinzler KW. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41. The Howard Hughes Medical Institute and the Johns Hopkins Oncology Center, Baltimore, MD 21231, USA The identification of
predefined mutations expected to be present in a minor fraction of a
cell population is important for a variety of basic research and
clinical applications. Here, we describe an approach for transforming
the exponential, analog nature of the PCR into a linear, digital signal
suitable for this purpose. Single molecules are isolated by dilution
and individually amplified by PCR; each product is then analyzed
separately for the presence of mutations by using fluorescent probes.
The feasibility of the approach is demonstrated through the detection
of a mutant ras oncogene in the stool of patients with colorectal
cancer. The process provides a reliable and quantitative measure of the
proportion of variant sequences within a DNA sample.
Nanoliter scale PCR with TaqMan detection Kalinina O, Lebedeva I, Brown J, Silver J. Nucleic Acids Res. 1997 May 15;25(10):1999-2004. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA. We monitored PCR in
volumes of the order of 10 nl in glass microcapillaries using a
fluorescence energy transfer assay in which fluorescence increases if
product is made due to template-dependent nucleolytic degradation of an
internally quenched probe (TaqMan assay). This assay detected single
starting template molecules in dilutions of genomic DNA. The results
suggest that it may be feasible to determine the number of template
molecules in a sample by counting the number of positive PCRs in a set
of replicate reactions using terminally diluted sample. Since the assay
system is closed and potentially automatable, it has promise for
clinical applications.
Pohl G, Shih IeM.Principle and applications of digital PCR Expert Rev Mol Diagn. 2004 Jan;4(1): 41-47 Department of Pathology, 418 North Bond Street, B-315, Baltimore, MD 21231, USA. Digital PCR represents an example of the power of PCR and provides unprecedented opportunities for molecular genetic analysis in cancer. The technique is to amplify a single DNA template from minimally diluted samples, therefore generating amplicons that are exclusively derived from one template and can be detected with different fluorophores or sequencing to discriminate different alleles (e.g., wild type vs. mutant or paternal vs. maternal alleles). Thus, digital PCR transforms the exponential, analog signals obtained from conventional PCR to linear, digital signals, allowing statistical analysis of the PCR product. Digital PCR has been applied in quantification of mutant alleles and detection of allelic imbalance in clinical specimens, providing a promising molecular diagnostic tool for cancer detection. The scope of this article is to review the principles of digital PCR and its practical applications in cancer research and in the molecular diagnosis of cancer. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Dennis Lo YM. Clin Chem. 2008 Oct;54(10): 1664-1672. Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. BACKGROUND: The precise measurement
of cell-free fetal DNA in maternal
plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal
aneuploidies and other applications. We tested the hypothesis that
microfluidics digital PCR, in which individual fetal-DNA molecules are
counted, could enhance the precision of measuring circulating fetal DNA.
METHODS: We first determined whether
microfluidics digital PCR,
real-time PCR, and mass spectrometry produced different estimates of
male-DNA concentrations in artificial mixtures of male and female DNA.
We then focused on comparing the imprecision of microfluidics digital
PCR with that of a well-established nondigital PCR assay for measuring
male fetal DNA in maternal plasma.
RESULTS: Of the tested platforms,
microfluidics digital PCR
demonstrated the least quantitative bias for measuring the fractional
concentration of male DNA. This assay had a lower imprecision and
higher clinical sensitivity compared with nondigital real-time PCR.
With the ZFY/ZFX assay on the microfluidics digital PCR platform, the
median fractional concentration of fetal DNA in maternal plasma was
> or =2 times higher for all 3 trimesters of pregnancy than
previously reported.
CONCLUSIONS: Microfluidics digital
PCR represents an improvement over
previous methods for quantifying fetal DNA in maternal plasma, enabling
diagnostic and research applications requiring precise quantification.
This approach may also impact other diagnostic applications of plasma
nucleic acids, e.g., in oncology and transplantation.
Lo YM, Lun FM, Chan KC, Tsui NB, Chong KC, Lau TK, Leung
TY, Zee BC,
Cantor CR, Chiu RW.Digital PCR for the molecular detection of fetal chromosomal aneuploidy Proc Natl Acad Sci U S A. 2007 7;104(32): 13116-13121 Li Ka Shing Institute of Health Sciences, Department of Chemical Pathology, School of Public Health, The Chinese University of Hong Kong, Sha Tin, New Territories, Hong Kong Special Administrative Region, People's Republic of China. Trisomy 21 is the most
common reason that women opt for prenatal
diagnosis. Conventional prenatal diagnostic methods involve the
sampling of fetal materials by invasive procedures such as
amniocentesis. Screening by ultrasonography and biochemical markers
have been used to risk-stratify pregnant women before definitive
invasive diagnostic procedures. However, these screening methods
generally target epiphenomena, such as nuchal translucency, associated
with trisomy 21. It would be ideal if noninvasive genetic methods were
available for the direct detection of the core pathology of trisomy 21.
Here we outline an approach using digital PCR for the noninvasive
detection of fetal trisomy 21 by analysis of fetal nucleic acids in
maternal plasma. First, we demonstrate the use of digital PCR to
determine the allelic imbalance of a SNP on PLAC4 mRNA, a
placenta-expressed transcript on chromosome 21, in the maternal plasma
of women bearing trisomy 21 fetuses. We named this the digital RNA SNP
strategy. Second, we developed a nonpolymorphism-based method for the
noninvasive prenatal detection of trisomy 21. We named this the digital
relative chromosome dosage (RCD) method. Digital RCD involves the
direct assessment of whether the total copy number of chromosome 21 in
a sample containing fetal DNA is overrepresented with respect to a
reference chromosome. Even without elaborate instrumentation, digital
RCD allows the detection of trisomy 21 in samples containing 25% fetal
DNA. We applied the sequential probability ratio test to interpret the
digital PCR data. Computer simulation and empirical validation
confirmed the high accuracy of the disease classification algorithm.
Lo YM, Chiu RW.Noninvasive prenatal diagnosis of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis Clin Chem. 2008 54(3): 461-466 Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China. BACKGROUND: The discovery of
circulating cell-free fetal nucleic acids
in maternal plasma has opened up new possibilities for noninvasive
prenatal diagnosis. The potential application of this technology for
the noninvasive prenatal detection of fetal chromosomal aneuploidies is
an aspect of this field that is being actively investigated. The main
challenge of work in this area is the fact that cell-free fetal nucleic
acids represent only a minor fraction of the total nucleic acids in
maternal plasma. Methods and
RESULTS: We performed a review of
the literature, which revealed that
investigators have applied methods based on the physical and molecular
enrichment of fetal nucleic acid targets from maternal plasma. The
former includes the use of size fractionation of plasma DNA and the use
of the controversial formaldehyde treatment method. The latter has been
achieved through the development of fetal epigenetic and fetal RNA
markers. The aneuploidy status of the fetus has been explored through
the use of allelic ratio analysis of plasma fetal epigenetic and RNA
markers. Digital PCR has been shown to offer high precision for allelic
ratio and relative chromosome dosage analyses.
CONCLUSIONS: After a decade of work,
the theoretical and practical
feasibility of prenatal fetal chromosomal aneuploidy detection by
plasma nucleic acid analysis has been demonstrated in studies using
small sample sets. Larger scale independent studies will be needed to
validate these initial observations. If these larger scale studies
prove successful, it is expected that with further development of new
fetal DNA/RNA markers and new analytical methods, molecular noninvasive
prenatal diagnosis of the major chromosomal aneuploidies could become a
routine practice in the near future.
Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy Fan HC, Blumenfeld YJ, El-Sayed YY, Chueh J, Quake SR. Am J Obstet Gynecol. 2009 200(5): 543.e1-7 Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, CA, USA. OBJECTIVE: The purpose of this
study was to demonstrate that digital polymerase chain reaction (PCR)
enables rapid, allele independent molecular detection of fetal
aneuploidy.
STUDY DESIGN: Twenty-four
amniocentesis and 16 chorionic villus samples were used for
microfluidic digital PCR analysis. Three thousand and sixty PCR
reactions were performed for each of the target chromosomes (X, Y, 13,
18, and 21), and the number of single molecule amplifications was
compared to a reference. The difference between target and reference
chromosome counts was used to determine the ploidy of each of the
target chromosomes.
RESULTS: Digital PCR accurately
identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases
of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed.
The remaining specimens were determined to have normal ploidy for the
chromosomes tested.
CONCLUSION:
Microfluidic digital PCR allows detection of fetal chromosomal
aneuploidy utilizing uncultured amniocytes and chorionic villus tissue
in less than 6 hours.Digital PCR provides sensitive and absolute calibration for high throughput sequencing White RA 3rd, Blainey PC, Fan HC, Quake SR. BMC Genomics. 2009 Mar 19;10:116. Department of Bioengineering at Stanford University and Howard Hughes Medical Institute, Stanford, CA 94305, USA. BACKGROUND: Next-generation DNA
sequencing on the 454, Solexa, and SOLiD platforms requires absolute
calibration of the number of molecules to be sequenced. This
requirement has two unfavorable consequences. First, large amounts of
sample-typically micrograms-are needed for library preparation, thereby
limiting the scope of samples which can be sequenced. For many
applications, including metagenomics and the sequencing of ancient,
forensic, and clinical samples, the quantity of input DNA can be
critically limiting. Second, each library requires a titration
sequencing run, thereby increasing the cost and lowering the throughput
of sequencing.
RESULTS: We demonstrate the use of
digital PCR to accurately quantify 454 and Solexa sequencing libraries,
enabling the preparation of sequencing libraries from nanogram
quantities of input material while eliminating costly and
time-consuming titration runs of the sequencer. We successfully
sequenced low-nanogram scale bacterial and mammalian DNA samples on the
454 FLX and Solexa DNA sequencing platforms. This study is the first to
definitively demonstrate the successful sequencing of picogram
quantities of input DNA on the 454 platform, reducing the sample
requirement more than 1000-fold without pre-amplification and the
associated bias and reduction in library depth.
CONCLUSION: The digital PCR assay
allows absolute quantification of sequencing libraries, eliminates
uncertainties associated with the construction and application of
standard curves to PCR-based quantification, and with a coefficient of
variation close to 10%, is sufficiently precise to enable direct
sequencing without titration runs.
Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays Wang J, Ramakrishnan R, Tang Z, Fan W, Kluge A, Dowlati A, Jones RC, Ma PC. Clin Chem. 2010
Apr;56(4):623-32. Epub 2010 Mar 5.
Fluidigm Corporation, South San Francisco, CA, USA.BACKGROUND: The EGFR [epidermal
growth factor receptor (erythroblastic leukemia viral (v-erb-b)
oncogene homolog, avian)] gene is known to harbor genomic alterations
in advanced lung cancer involving gene amplification and kinase
mutations that predict the clinical response to EGFR-targeted
inhibitors. Methods for detecting such molecular changes in lung cancer
tumors are desirable.
METHODS: We used a nanofluidic
digital PCR array platform and 16 cell lines and 20 samples of genomic
DNA from resected tumors (stages I-III) to quantify the relative
numbers of copies of the EGFR gene and to detect mutated EGFR alleles
in lung cancer. We assessed the relative number of EGFR gene copies by
calculating the ratio of the number of EGFR molecules (measured with a
6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules
of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit)
(measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each
panel. To assay for the EGFR L858R (exon 21) mutation and exon 19
in-frame deletions, we used the ARMS and Scorpion technologies in a
DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array.
RESULTS: The digital array detected
and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations
(0.02%-9.26% abundance) that were present in formalin-fixed,
paraffin-embedded samples of early-stage resectable lung tumors without
an associated increase in gene copy number. Our results also
demonstrated the presence of intratumor molecular heterogeneity for the
clinically relevant EGFR mutated alleles in these early-stage lung
tumors.
CONCLUSIONS: The digital PCR array
platform allows characterization and quantification of oncogenes, such
as EGFR, at the single-molecule level. Use of this nanofluidics
platform may provide deeper insight into the specific roles of
clinically relevant kinase mutations during different stages of lung
tumor progression and may be useful in predicting the clinical response
to EGFR-targeted inhibitors.
Microdissection molecular copy-number counting (microMCC)--unlocking cancer archives with digital PCR McCaughan F, Darai-Ramqvist E, Bankier AT, Konfortov BA, Foster N, George PJ, Rabbitts TH, Kost-Alimova M, Rabbitts PH, Dear PH. J Pathol. 2008 216(3): 307-316 Centre for Respiratory Research, Department of Medicine, Royal Free and University College Medical School, The Rayne Institute, London WC1E 6JJ, UK. Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics. Single-molecule genomics McCaughan F, Dear PH. J Pathol. 2010 Jan;220(2):297-306. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. The term 'single-molecule genomics' (SMG) describes a group of molecular methods in which single molecules are detected or sequenced. The focus on the analysis of individual molecules distinguishes these techniques from more traditional methods, in which template DNA is cloned or PCR-amplified prior to analysis. Although technically challenging, the analysis of single molecules has the potential to play a major role in the delivery of truly personalized medicine. The two main subgroups of SMG methods are single-molecule digital PCR and single-molecule sequencing. Single-molecule PCR has a number of advantages over competing technologies, including improved detection of rare genetic variants and more precise analysis of copy-number variation, and is more easily adapted to the often small amount of material that is available in clinical samples. Single-molecule sequencing refers to a number of different methods that are mainly still in development but have the potential to make a huge impact on personalized medicine in the future. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria Ottesen EA, Hong JW, Quake SR, Leadbetter JR. Science. 2006 314(5804): 1464-1467 Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment. Concordance among digital gene expression, microarrays, and qPCR when measuring differential expression of microRNAs Pradervand S, Weber J, Lemoine F, Consales F, Paillusson A, Dupasquier M, Thomas J, Richter H, Kaessmann H, Beaudoing E, Hagenbüchle O, Harshman K. Biotechniques. 2010 48(3): 219-222 Genomic Technologies Facility, Center for Integrative Genomics, University of Lausanne, Genopode Building, Lausanne, Switzerland Profiling microRNA (miRNA) expression is of widespread interest given the critical role of miRNAs in many cellular functions. Profiling can be achieved via hybridization-based (microarrays), sequencing-based, or amplification-based (quantitative reverse transcription-PCR, qPCR) technologies. Among these, microarrays face the significant challenge of accurately distinguishing between mature and immature miRNA forms, and different vendors have developed different methods to meet this challenge. Here we measure differential miRNA expression using the Affymetrix, Agilent, and Illumina microarray platforms, as well as qPCR (Applied Biosystems) and ultra high-throughput sequencing (Illumina). We show that the differential expression measurements are more divergent when the three types of microarrays are compared than when the Agilent microarray, qPCR, and sequencing technology measurements are compared, which exhibit a good overall concordance. Amplification-free digital gene expression profiling from minute cell quantities Ozsolak F, Ting DT, Wittner BS, Brannigan BW, Paul S, Bardeesy N, Ramaswamy S, Milos PM, Haber DA. Nat Methods. 2010 7(8):619-21. Epub 2010 Jul 18. Helicos BioSciences Corporation, Cambridge, Massachusetts, USA Generating reliable expression profiles from minute cell quantities is critical for scientific discovery and potential clinical applications. Here we present low-quantity digital gene expression (LQ-DGE), an amplification-free approach involving capture of poly(A)(+) RNAs from cellular lysates onto poly(dT)-coated sequencing surfaces, followed by on-surface reverse transcription and sequencing. We applied LQ-DGE to profile malignant and nonmalignant mouse and human cells, demonstrating its quantitative power and potential applicability to archival specimens. Amplification-free digital gene expression profiling from minute cell quantities. Digital transcriptome profiling from attomole-level RNA samples Ozsolak F, Goren A, Gymrek M, Guttman M, Regev A, Bernstein BE, Milos PM. Genome Res. 2010 Apr;20(4): 519-525 Helicos BioSciences Corporation, Cambridge, MA 02139, USA Accurate profiling of minute quantities of RNA in a global manner can enable key advances in many scientific and clinical disciplines. Here, we present low-quantity RNA sequencing (LQ-RNAseq), a high-throughput sequencing-based technique allowing whole transcriptome surveys from subnanogram RNA quantities in an amplification/ligation-free manner. LQ-RNAseq involves first-strand cDNA synthesis from RNA templates, followed by 3' polyA tailing of the single-stranded cDNA products and direct single molecule sequencing. We applied LQ-RNAseq to profile S. cerevisiae polyA+ transcripts, demonstrate the reproducibility of the approach across different sample preparations and independent instrument runs, and establish the absolute quantitative power of this method through comparisons with other reported transcript profiling techniques and through utilization of RNA spike-in experiments. We demonstrate the practical application of this approach to define the transcriptional landscape of mouse embryonic and induced pluripotent stem cells, observing transcriptional differences, including over 100 genes exhibiting differential expression between these otherwise very similar stem cell populations. This amplification-independent technology, which utilizes small quantities of nucleic acid and provides quantitative measurements of cellular transcripts, enables global gene expression measurements from minute amounts of materials and offers broad utility in both basic research and translational biology for characterization of rare cells. Single-molecule sequencing of an individual human genome Pushkarev D, Neff NF, Quake SR. Nat Biotechnol. 2009 27(9): 847-852 Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, California, USA. Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to approximately 90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28x average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined approximately 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics. Digital PCR on a SlipChip Shen F, Du W, Kreutz JE, Fok A, Ismagilov RF. Lab Chip. 2010 10(20):2666-72. Epub 2010 Jul 1. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 E 57th St, Chicago, Illinois 60637, USA This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout. Somatic deletion of the NF1 gene in a neurofibromatosis type 1-associated malignant melanoma demonstrated by digital PCR Rübben A, Bausch B, Nikkels A. Mol Cancer. 2006 Sep 10;5:36. Department of Dermatology, University Hospital RWTH Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany BACKGROUND: Neurofibromatosis type 1 (NF1) is the most common hereditary neurocutaneous disorder and it is associated with an elevated risk for malignant tumors of tissues derived from neural crest cells. The NF1 gene is considered a tumor suppressor gene and inactivation of both copies can be found in NF1-associated benign and malignant tumors. Melanocytes also derive from neural crest cells but melanoma incidence is not markedly elevated in NF1. In this study we could analyze a typical superficial spreading melanoma of a 15-year-old boy with NF1 for loss of heterozygosity (LOH) within the NF1 gene. Neurofibromatosis in this patient was transmitted by the boy's farther who carried the mutation NF1 c. 5546 G/A. RESULTS: Melanoma cells were isolated from formalin-fixed tissue by liquid coverslip laser microdissection. In order to obtain statistically significant LOH data, digital PCR was performed at the intragenic microsatellite IVS27AC28 with DNA of approx. 3500 melanoma cells. Digital PCR detected 23 paternal alleles and one maternal allele. Statistical analysis by SPRT confirmed significance of the maternal allele loss. CONCLUSION: To our knowledge, this is the first molecular evidence of inactivation of both copies of the NF1 gene in a typical superficial spreading melanoma of a patient with NF1. The classical double-hit inactivation of the NF1 gene suggests that the NF1 genetic background promoted melanoma genesis in this patient. Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution Suzanne Weaver, Simant Dube, Alain Mir, Jian Qin, Gang Sun, Ramesh Ramakrishnan, Robert C. Jones, Kenneth J. Livak Methods. 2010 Apr;50(4):271-6. Epub 2010 Jan 15. Fluidigm Corporation, 7000 Shoreline Court, Suite 100, South San Francisco, CA 94080, USA. This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis. One bacterial cell, one complete genome Woyke T, Tighe D, Mavromatis K, Clum A, Copeland A, Schackwitz W, Lapidus A, Wu D, McCutcheon JP, McDonald BR, Moran NA, Bristow J, Cheng JF. PLoS One. 2010 5(4): e10314 Department of Energy Joint Genome Institute, Walnut Creek, California, USA While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes. Spinning disk platform for microfluidic digital polymerase chain reaction. Sundberg SO, Wittwer CT, Gao C, Gale BK. Anal Chem. 2010 82(4): 1546-1550. University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City, Utah 84112, USA An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.
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