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New publications:
The Significance of Digital Gene Expression Profiles Stephane Audic &Jean-Michel Claverie Laboratory of Structural and Genetic Information, Centre National de la Recherche Scientifique – E.P.91, Marseille 13402, France GENOMER ESEARCH (1997) 7: 986–995 Genes differentially expressed in different tissues, during development, or during specific pathologies are of foremost interest to both basic and pharmaceutical research. ‘‘Transcript profiles’’ or ‘‘digital Northerns’’ are generated routinely by partially sequencing thousands of randomly selected clones from relevant cDNA libraries. Differentially expressed genes can then be detected from variations in the counts oft heir cognate sequence tags. Here we present the first systematic study on the influence of random fluctuations and sampling size on the reliability of this kind of data. We establish a rigorous significance test and demonstrate its use on publicly available transcript profiles. The theory links the threshold of selection of putatively regulated genes (e.g., the number of pharmaceutical leads) to the fraction of false positive clones one is willing to risk. Our results delineate more precisely and extend the limits within which digital Northern data can be used. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation Alexandra S. Whale1, Jim F. Huggett1,*, Simon Cowen1, Valerie Speirs2, Jacqui Shaw3, Stephen Ellison1, Carole A. Foy1 and Daniel J. Scott1 1LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, 2Leeds Institute of Molecular Medicine, University of Leeds, St. James’s University Hospital, Leeds LS9 7TF and 3Cancer Studies & Molecular Medicine, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK Nucl. Acids Res. (2012) First published online: February 28, 2012 One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR. National Measurement Institute , Lindfield, New South Wales, Australia. Anal Chem. 2012 84(2): 1003-1011 Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies. Further Improvement in Quantifying Male Fetal DNA in Maternal Plasma. Jin S, Michelle Lin X, Law H, Kwek KY, Yeo GS, Ding C. Growth, Development and Metabolism Program, Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore; Clin Chem. 2012 BACKGROUND: Cell-free fetal DNA (cffDNA) in maternal plasma can be clinically useful for detecting prenatal disorders and pregnancy monitoring. More sensitive, specific, and quantitative detection of cffDNA in maternal plasma may expand the clinical utility of such measurements. METHODS: We developed a quantitative real-time PCR (qPCR) assay [Y chromosome repetitive sequence (YRS) assay] based on a highly repetitive short sequence specific for the Y chromosome. Both standard qPCR and digital qPCR were performed to compare the sensitivity and specificity of this new assay against already established male DNA-specific assays. RESULTS: The YRS assay was at least 10-fold more sensitive than the currently most sensitive DYS14 assay. The YRS assay was able to detect 0.5 genome equivalents (GE) per PCR reaction when fetal DNA was present at 0.2% of the total DNA. The background noise for the YRS assay was much lower than for the DYS14 assay in analyses of plasma samples from pregnancies with female fetuses. CONCLUSIONS: The YRS assay is a substantial improvement for quantifying rare male fetal DNA in maternal plasma. The higher sensitivity and specificity may expand the clinical and research utility of cffDNA. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW. Bio-Rad Laboratories, Inc., Pleasanton, California 94566, United States. Anal Chem. 2011 Nov 15;83(22): 8604-8610 Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.  Multiplexed
quantification of nucleic acids with large dynamic range using
multivolume digital RT-PCR on a rotational SlipChip tested with HIV and
hepatitis C viral load.
Shen F, Sun B, Kreutz JE, Davydova EK, Du W, Reddy PL, Joseph LJ, Ismagilov RF. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, United States. J Am Chem Soc. 2011 Nov 9;133(44): 17705-17712 In this paper, we are
working toward a problem of great importance to global health:
determination of viral HIV and hepatitis C (HCV) loads under
point-of-care and resource limited settings. While antiretroviral
treatments are becoming widely available, viral load must be evaluated
at regular intervals to prevent the spread of drug resistance and
requires a quantitative measurement of RNA concentration over a wide
dynamic range (from 50 up to 10(6) molecules/mL for HIV and up to 10(8)
molecules/mL for HCV). "Digital" single molecule measurements are
attractive for quantification, but the dynamic range of such systems is
typically limited or requires excessive numbers of compartments. Here
we designed and tested two microfluidic rotational SlipChips to perform
multivolume digital RT-PCR (MV digital RT-PCR) experiments with large
and tunable dynamic range. These designs were characterized using
synthetic control RNA and validated with HIV viral RNA and HCV control
viral RNA. The first design contained 160 wells of each of four volumes
(125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range of 5.2
× 10(2) to 4.0 × 10(6) molecules/mL at 3-fold resolution.
The second design tested the flexibility of this approach, and further
expanded it to allow for multiplexing while maintaining a large dynamic
range by adding additional wells with volumes of 0.2 nL and 625 nL and
dividing the SlipChip into five regions to analyze five samples each at
a dynamic range of 1.8 × 10(3) to 1.2 × 10(7) molecules/mL
at 3-fold resolution. No evidence of cross-contamination was observed.
The multiplexed SlipChip can be used to analyze a single sample at a
dynamic range of 1.7 × 10(2) to 2.0 × 10(7) molecules/mL at
3-fold resolution with limit of detection of 40 molecules/mL. HIV viral
RNA purified from clinical samples were tested on the SlipChip, and
viral load results were self-consistent and in good agreement with
results determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1
Test. With further validation, this SlipChip should become useful to
precisely quantify viral HIV and HCV RNA for high-performance
diagnostics in resource-limited settings. These microfluidic designs
should also be valuable for other diagnostic and research applications,
including detecting rare cells and rare mutations, prenatal
diagnostics, monitoring residual disease, and quantifying copy number
variation and gene expression patterns. The theory for the design and
analysis of multivolume digital PCR experiments is presented in other
work by Kreutz et al. 2011
Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR. Kreutz JE, Munson T, Huynh T, Shen F, Du W, Ismagilov RF. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United States. Anal Chem. 2011 Nov 1;83(21): 8158-8168 This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for "digital" (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12,000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, <500 molecules/mL to >1,000,000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011), this approach is used to design and test digital RT-PCR devices for quantifying RNA.  Digital PCR provides absolute quantitation of viral load for an occult RNA virus. White RA 3rd, Quake SR, Curr K. Department of Bioengineering at Stanford University and Howard Hughes Medical Institute, Stanford, CA 94305, USA; Department of Biology at California State University-East Bay, Hayward, CA 94544, USA. J Virol Methods. 2012 179(1): 45-50 Using a multiplexed
LNA-based Taqman assay, RT-digital PCR (RT-dPCR) was performed in a
prefabricated microfluidic device that monitored absolute viral load in
native and immortalized cell lines, overall precision of detection, and
the absolute detection limit of an occult RNA virus GB Virus Type C
(GBV-C). RT-dPCR had on average a 10% lower overall coefficient of
variation (CV, a measurement of precision) for viral load testing than
RT-qPCR and had a higher overall detection limit, able to quantify as
low as three 5'-UTR molecules of GBV-C genome. Two commercial
high-yield in vitro transcription kits (T7 Ribomax Express by Promega
and Ampliscribe T7 Flash by Epicentre) were compared to amplify GBV-C
RNA genome with T7-mediated amplification. The Ampliscribe T7 Flash
outperformed the T7 Ribomax Express in yield of full-length GBV-C RNA
genome. THP-1 cells (a model of monocytic derived cells) were
transfected with GBV-C, yielding infectious virions that replicated
over a 120h time course and could be infected directly. This study
provides the first evidence of GBV-C replication in monocytic derived
clonal cells. Thus far, it is the only study using a microfluidic
device that measures directly viral load of mammalian RNA virus in a
digital format without need for a standard curve.
1-Million droplet array with wide-field fluorescence imaging for digital PCR. Hatch AC, Fisher JS, Tovar AR, Hsieh AT, Lin R, Pentoney SL, Yang DL, Lee AP. Department of Biomedical Engineering, University of California-Irvine, Irvine, CA 92697, USA. Lab Chip. 2011 11(22): 3838-3845 Digital droplet reactors
are useful as chemical and biological containers to discretize reagents
into picolitre or nanolitre volumes for analysis of single cells,
organisms, or molecules. However, most DNA based assays require
processing of samples on the order of tens of microlitres and contain
as few as one to as many as millions of fragments to be detected.
Presented in this work is a droplet microfluidic platform and
fluorescence imaging setup designed to better meet the needs of the
high-throughput and high-dynamic-range by integrating multiple
high-throughput droplet processing schemes on the chip. The design is
capable of generating over 1-million, monodisperse, 50 picolitre
droplets in 2-7 minutes that then self-assemble into high density
3-dimensional sphere packing configurations in a large viewing chamber
for visualization and analysis. This device then undergoes on-chip
polymerase chain reaction (PCR) amplification and fluorescence
detection to digitally quantify the sample's nucleic acid content.
Wide-field fluorescence images are captured using a low cost
21-megapixel digital camera and macro-lens with an 8-12 cm(2)
field-of-view at 1× to 0.85× magnification, respectively.
We demonstrate both end-point and real-time imaging ability to perform
on-chip quantitative digital PCR analysis of the entire droplet array.
Compared to previous work, this highly integrated design yields a
100-fold increase in the number of on-chip digitized reactors with
simultaneous fluorescence imaging for digital PCR based assays.
Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield. Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS. NE Thames Regional Molecular Genetics Laboratories, Great Ormond Street Hospital for Children, London, United Kingdom. PLoS One. 2011;6(10): e25202 OBJECTIVE: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. METHODS: Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples. RESULTS: Total cell-free DNA quantity increased significantly with time in samples stored in K(3)EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4 °C did not prevent these changes. CONCLUSION: When samples can be processed within eight hours of blood draw, K(3)EDTA tubes can be used. Prolonged transfer times in K(3)EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests. Megapixel digital PCR. Heyries KA, Tropini C, Vaninsberghe M, Doolin C, Petriv OI, Singhal A, Leung K, Hughesman CB, Hansen CL. Nat Methods. 2011 8(8): 649-651 We present a microfluidic 'megapixel' digital PCR device that uses surface tension-based sample partitioning and dehydration control to enable high-fidelity single DNA molecule amplification in 1,000,000 reactors of picoliter volume with densities up to 440,000 reactors cm(-2). This device achieves a dynamic range of 10(7), single-nucleotide-variant detection below one copy per 100,000 wild-type sequences and the discrimination of a 1% difference in chromosome copy number. Evaluation of Digital PCR for Absolute DNA Quantification. Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA. Anal Chem. 2011 Aug 1. [Epub ahead of print] LGC, Queens Road, Teddington, Middlesex, UK The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design. Probing individual environmental bacteria for viruses by using microfluidic digital PCR. Tadmor AD, Ottesen EA, Leadbetter JR, Phillips R. Science. 2011 333(6038): 58-62. Department of Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, CA 91125, USA Viruses may very well be the most abundant biological entities on the planet. Yet neither metagenomic studies nor classical phage isolation techniques have shed much light on the identity of the hosts of most viruses. We used a microfluidic digital polymerase chain reaction (PCR) approach to physically link single bacterial cells harvested from a natural environment with a viral marker gene. When we implemented this technique on the microbial community residing in the termite hindgut, we found genus-wide infection patterns displaying remarkable intragenus selectivity. Viral marker allelic diversity revealed restricted mixing of alleles between hosts, indicating limited lateral gene transfer of these alleles despite host proximity. Our approach does not require culturing hosts or viruses and provides a method for examining virus-bacterium interactions in many environments. Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. Zhong Q, Bhattacharya S, Kotsopoulos S, Olson J, Taly V, Griffiths AD, Link DR, Larson JW. Lab Chip. 2011 11(13): 2167-2174 RainDance Technologies, Inc., 44 Hartwell Ave., Lexington, MA 02150, USA Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented. Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array. Qi Z, Ma Y, Deng L, Wu H, Zhou G, Kajiyama T, Kambara H. Analyst. 2011 136(11): 2252-2259 Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China. To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the β-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC. Genomic evidence of pre-invasive clonal expansion, dispersal and progression in bronchial dysplasia. McCaughan F, Pipinikas CP, Janes SM, George PJ, Rabbitts PH, Dear PH. J Pathol. 2011 224(2): 153-159 MRC Laboratory of Molecular Biology, Cambridge, UK The term 'field cancerization' is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression. Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Pekin D, Skhiri Y, Baret JC, Le Corre D, Mazutis L, Salem CB, Millot F, El Harrak A, Hutchison JB, Larson JW, Link DR, Laurent-Puig P, Griffiths AD, Taly V. Lab Chip. 2011 11(13): 2156-2166 Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 allée Gaspard Monge, BP 70028, F-67083 Strasbourg Cedex, France. Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment. Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA. Tsui NB, Kadir RA, Chan KC, Chi C, Mellars G, Tuddenham EG, Leung TY, Lau TK, Chiu RW, Lo YM. Blood. 2011 117(13): 3684-3691 Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, Hong Kong, China. Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families. Statistical considerations for digital approaches to non-invasive fetal genotyping. Chu T, Bunce K, Hogge WA, Peters DG. Bioinformatics. 2010 26(22): 2863-2866 Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA. MOTIVATION: A growing body of literature has demonstrated the potential for non-invasive diagnosis of a variety of human genetic diseases using cell-free DNA extracted from maternal plasma samples in early gestation. Such methods are of great significance to the obstetrics community because of their potential use as clinical standard of care. Proof of concept for such approaches has been established for aneuploidy and paternally inherited dominant traits. Although significant progress has recently been made, the non-invasive diagnosis of monogenic diseases that segregate in a recessive mendelian fashion is more problematic. Recent developments in microfluidic digital PCR and DNA sequencing have resulted in a number of recent advances in this field. These have largely, although not exclusively, been used for the development of diagnostic methods for aneuploidy. However, given their prevalence, it is likely that such methods will be utilized towards the development of non-invasive methods for diagnosing monogenetic disorders. RESULTS: With this in mind, we have undertaken a statistical modeling of three contemporary (digital) analytical methods in the context of prenatal diagnosis using cell free DNA for monogenic diseases that segregate in a recessive mendelian fashion. We provide an experimental framework for the future development of diagnostic methods in this context that should be considered when designing molecular assays that seek to establish proof of concept in this field. Prenatal diagnosis of fetal aneuploidies: post-genomic developments. Hahn S, Jackson LG, Zimmermann BG. Genome Med. 2010 2(8):50 Department of Biomedicine, University Women's Hospital, University Clinics Basel, Hebelstrasse 20, CH-4031, Switzerland. ABSTRACT : Prenatal diagnosis of fetal aneuploidies and chromosomal anomalies is likely to undergo a profound change in the near future. On the one hand this is mediated by new technical developments, such as chromosomal microarrays, which allow a much more precise delineation of minute sub-microscopic chromosomal aberrancies than the classical G-band karyotype. This will be of particular interest when investigating pregnancies at risk of unexplained development delay, intellectual disability or certain forms of autism. On the other hand, great strides have been made in the non-invasive determination of fetal genetic traits, largely through the analysis of cell-free fetal nucleic acids. It is hoped that, with the assistance of cutting-edge tools such as digital PCR or next generation sequencing, the long elusive goal of non-invasive prenatal diagnosis for fetal aneuploidies can finally be attained. Methylation analysis DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight Weisenberger DJ, Trinh BN, Campan M, Sharma S, Long TI, Ananthnarayan S, Liang G, Esteva FJ, Hortobagyi GN, McCormick F, Jones PA, Laird PW. Nucleic Acids Res. 2008 Aug;36(14): 4689-98 Department of Surgery, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA 90033, USA. Alterations in
cytosine-5 DNA methylation are frequently observed in most types of
human cancer. Although assays utilizing PCR amplification of
bisulfite-converted DNA are widely employed to analyze these DNA
methylation alterations, they are generally limited in throughput
capacity, detection sensitivity, and or resolution. Digital PCR, in
which a DNA sample is analyzed in distributive fashion over multiple
reaction chambers, allows for enumeration of discrete template DNA
molecules, as well as sequestration of non-specific primer annealing
templates into negative chambers, thereby increasing the
signal-to-noise ratio in positive chambers. Here, we have applied
digital PCR technology to bisulfite-converted DNA for single-molecule
high-resolution DNA methylation analysis and for increased sensitivity
DNA methylation detection. We developed digital bisulfite genomic DNA
sequencing to efficiently determine single-basepair DNA methylation
patterns on single-molecule DNA templates without an interim cloning
step. We also developed digital MethyLight, which surpasses traditional
MethyLight in detection sensitivity and quantitative accuracy for low
quantities of DNA. Using digital MethyLight, we identified
single-molecule, cancer-specific DNA hypermethylation events in the CpG
islands of RUNX3, CLDN5 and FOXE1 present in plasma samples from breast
cancer patients.
Thomas Mikeska, Ida LM Candiloro, & Alexander DobrovicThe implications of heterogeneous DNA methylation for the accurate quantification of methylation Epigenomics 2010, Vol. 2: 561-573 DNA methylation based
biomarkers have considerable potential for molecular diagnostics, both
as tumor specific biomarkers for the early detection or
post-therapeutic monitoring of cancer as well as prognostic and
predictive biomarkers for therapeutic stratification. Particularly in
the former, the accurate estimation of DNA methylation is of compelling
importance. However, quantification of DNA methylation has many traps
for the unwary, especially when heterogeneous methylation comprising
multiple alleles with varied DNA methylation patterns (epialleles) is
present. The frequent occurrence of heterogeneous methylation as
distinct from a simple mixture of fully methylated and unmethylated
alleles is generally not taken into account when DNA methylation is
considered as a cancer biomarker. When heterogeneous DNA methylation is
present, the proportion of methylated molecules is difficult to
quantify without a method that allows the measurement of individual
epialleles. In this article, we critically assess the methodologies
frequently used to investigate DNA methylation, with an emphasis on the
detection and measurement of heterogeneous DNA methylation. The
adoption of digital approaches will enable the effective use of
heterogeneous DNA methylation as a cancer biomarker.
Candiloro IL, Mikeska T, Hokland P, Dobrovic A.Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene Epigenetics Chromatin. 2008 Nov 3;1(1): 7 Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria 8006, Australia. BACKGROUND: Methylation-sensitive
high resolution melting (MS-HRM) methodology is able to recognise
heterogeneously methylated sequences by their characteristic melting
profiles. To further analyse heterogeneously methylated sequences, we
adopted a digital approach to MS-HRM (dMS-HRM) that involves the
amplification of single templates after limiting dilution to quantify
and to determine the degree of methylation. We used this approach to
study methylation of the CDKN2B (p15) cell cycle progression inhibitor
gene which is inactivated by DNA methylation in haematological
malignancies of the myeloid lineage. Its promoter region usually shows
heterogeneous methylation and is only rarely fully methylated. The
methylation status of CDKN2B can be used as a biomarker of response to
treatment. Therefore the accurate characterisation of its methylation
is desirable.
RESULTS: MS-HRM was used to assess
CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the
AML samples that were methylated at the CDKN2B promoter (40/93) showed
varying degrees of heterogeneous methylation. Six representative
samples were selected for further study. dMS-HRM was used to
simultaneously count the methylated alleles and assess the degree of
methylation. Direct sequencing of selected dMS-HRM products was used to
determine the exact DNA methylation pattern and confirmed the degree of
methylation estimated by dMS-HRM.
CONCLUSION: dMS-HRM is a powerful
technique for the analysis of methylation in CDKN2B and other
heterogeneously methylated genes. It eliminates both PCR and cloning
bias towards either methylated or unmethylated DNA. Potentially complex
information is simplified into a digital output, allowing counting of
methylated and unmethylated alleles and providing an overall picture of
methylation at the given locus. Downstream sequencing is minimised as
dMS-HRM acts as a screen to select only methylated clones for further
analysis.
GMO analysis Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction Corbisier P, Bhat S, Partis L, Xie VR, Emslie KR. Anal Bioanal Chem. 2010 396(6): 2143-2150 European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium Quantitative analysis of
genetically modified (GM) foods requires estimation of the amount of
the transgenic event relative to an endogenous gene. Regulatory
authorities in the European Union (EU) have defined the labelling
threshold for GM food on the copy number ratio between the transgenic
event and an endogenous gene. Real-time polymerase chain reaction (PCR)
is currently being used for quantification of GM organisms (GMOs).
Limitations in real-time PCR applications to detect very low number of
DNA targets has led to new developments such as the digital PCR (dPCR)
which allows accurate measurement of DNA copies without the need for a
reference calibrator. In this paper, the amount of maize MON810 and hmg
copies present in a DNA extract from seed powders certified for their
mass content and for their copy number ratio was measured by dPCR. The
ratio of these absolute copy numbers determined by dPCR was found to be
identical to the ratios measured by real-time quantitative PCR (qPCR)
using a plasmid DNA calibrator. These results indicate that both
methods could be applied to determine the copy number ratio in MON810.
The reported values were in agreement with estimations from a model
elaborated to convert mass fractions into copy number fractions in
MON810 varieties. This model was challenged on two MON810 varieties
used for the production of MON810 certified reference materials (CRMs)
which differ in the parental origin of the introduced GM trait. We
conclude that dPCR has a high metrological quality and can be used for
certifying GM CRMs in terms of DNA copy number ratio.
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