Single-Cell Genetic Analysis
Single-Cell Multiplex Gene Detection and Sequencing
with Microfluidically Generated Agarose Emulsions**
Martyn T. Smith, and Richard A. Mathies*
? 2011 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimAngew. Chem. Int. Ed. 2011, 50, 390–395
Genetic assays, such as polymerase chain reaction (PCR),
typically report on multiple cells or mixtures of genomic
DNA. As a result, they cannot properly characterize the
genetic heterogeneity of a cell population or detect the
cooccurrence of different mutations within a single cell; these
factors are key to understanding the development, progres-
sion, and treatment of cancers.[1,2]In particular, since initial
mutagenesis occurs inherently at the single-cell level, the
detection and characterization of carcinogenesis will be
dramatically facilitated by analytical techniques with single-
Cytometric sorting, limiting dilution, and micromanipu-
lation have been previously used to perform single-cell PCR
assays in 96-well PCR plates, but these approaches are not
ideal for large-scale screening applications.Microfluidic
technology offers fundamentally new capabilities for the
manipulation of fluids, molecules, and cells that are very
pertinent for the development of high-throughput single-cell
analysis methods.[4–7]Microfluidic droplet technology is
particularly advantageous for single-cell/molecule analysis
because it facilitates the rapid statistical compartmentaliza-
tion of targets for massively parallel pico- to nanoliter-scale
assays.[8–10]In particular, microfluidic emulsion PCR (ePCR)
enables high-fidelity digital single-molecule counting owing
to its unique ability to ensure equal population sampling and
To date, most reported single-cell genomic analyses have
been carried out on bacterial samples.[13–15]For mammalian
cells, droplet-based genetic analyses have predominantly
implemented reverse transcriptase PCR for phenotypic
profiling.[11,16]A difficulty in single-cell PCR is the persistent
technical challenge of integrating a robust and scalable DNA-
extraction method.[13,14]The relative lack of suitable technol-
ogies for single-cell genomic analysis combined with the
significant genetic heterogeneity associated with cancer
underscores the importance of developing new microdroplet
methodologies that integrate robust single-cell genome
preparation with multiplex PCR.
To address these challenges, we have developed an
agarose-droplet-based platform that leverages emulsion-gen-
genetic analysis.[4,12]Single cells were microfluidically encap-
sulated together with primer-functionalized beads in agarose-
gel droplets for subsequent SDS lysis and proteinase K
digestion to release genomic DNA. With the coencapsulated
primer beads and purified genomes, we demonstrate multi-
locus single-cell sequencing of the control gene b-actin and
the chromosomal translocation t(14;18), a mutation associ-
ated with 85–90% of cases of follicular lymphoma.[1,17]The
coupling of our robust and high-throughput single-cell DNA-
purification method with the sequencing of multiple gene
targets within single cells will enable detailed studies of
mutation cooccurrence and synergy during carcinogenesis.
The underlying principle of our highly parallel cell-
digestion and DNA-purification method is the microfluidic
encapsulation of cells in agarose droplets (Figure 1) to
maintain single-genome fidelity during cell lysis and DNA
purification as well as efficient multiplex emulsion PCR
target amplification for subsequent analysis. Single lympho-
blast cells were encapsulated along with primer-functional-
ized beads in 1.5% low-melting-point agarose by using a four-
channel microfluidic-emulsion-generator array (MEGA; Fig-
ure 2a).The MEGA platform is very versatile: micropump
Figure 1. Workflow diagram showing the use of agarose-emulsion
droplets for the genetic analysis and multilocus sequencing of single
mammalian cells. a) Single cells are microfluidically encapsulated
together with primer-functionalized beads in agarose-gel droplets.
b) The genomes of single cells are released in the gel droplets upon
SDS lysis and digestion with proteinase K according to a standard
protocol. c) The agarose droplets are equilibrated in PCR buffer
containing fluorescent forward primers, emulsified with oil by mechan-
ical agitation, and thermally cycled. d) Following multiplex PCR amplifi-
cation, primer beads are released by breaking the emulsion and
melting the agarose. The fluorescent beads are then rapidly quantified
by flow cytometry or further subjected to PCR amplification for the
sequencing of target genes. SDS=sodium dodecyl sulfate.
[*] R. Novak,[+]Dr. Y. Zeng,[+]Dr. J. Shuga, Prof. M. T. Smith,
Prof. R. A. Mathies
Center for Exposure Biology, University of California, Berkeley
307 Lewis Hall, Berkeley, CA 94720 (USA)
R. Novak,[+]G. Venugopalan, Prof. D. A. Fletcher, Prof. R. A. Mathies
UC Berkeley/UC San Francisco Joint Graduate Group in
University of California, Berkeley (USA)
Dr. Y. Zeng,[+]Prof. R. A. Mathies
Department of Chemistry, University of California, Berkeley (USA)
Dr. J. Shuga, Prof. M. T. Smith
School of Public Health, University of California, Berkeley (USA)
[+] These authors contributed equally.
[**] This research was supported by the trans-NIH Genes, Environment
and Health Initiative, Biological Response Indicators of Environ-
mental Systems Center Grant U54 ES016115-01 to M.T.S. and
R.A.M., and by Superfund Basic Research Program NIEHS Grant
P42 ES004705 to M.T.S. R.N. is supported by an NSF Graduate
Research Fellowship, and J.S. is supported by the Canary Founda-
tion and ACS Early Detection Postdoctoral Fellowship.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2011, 50, 390–395? 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
actuation was optimized to account for the increased viscosity
of molten agarose without modification of the microfluidic
design. Droplet generation at approximately 408 8C for 30 min
resulted in the encapsulation of approximately 18000 cells at
up to 0.3 cells per droplet (cpd) on average. Figure 2b shows
the generation of uniform 3 nL agarose droplets containing
primer-functionalized beads. The inset highlights an example
of cell and bead coencapsulation in a single droplet.
Encapsulation in agarose droplets enables reproducible
single-cell DNA extraction and isolation through the adapta-
tion of robust DNA-purification protocols. An example of an
agarose droplet in which a bead and a cell were coencapsu-
lated is shown in Figure 2c. Cell lysis and digestion of the
DNA-binding histone proteins upon overnight incubation of
the gel droplets in SDS lysis buffer containing proteinase K
led to DNA release. The void left by the cell in the agarose
was occupied by brightly fluorescent genomic DNA, which
exhibited minimal diffusion into the surrounding gel (Fig-
ure 2d) as a result of the relatively small pore size of 1.5%
agarose (ca. 130 nm).An incubation temperature of 528 8C
facilitated enzymatic protein digestion while preserving the
integrity of the agarose droplets. By staining with propidium
iodide, we were able to visualize single high-molecular-weight
DNA strands protruding from the relatively small agarose
droplets (see Figure S1D in the Supporting Information). This
result indicated that the majority of nuclear proteins were
removed by the combination of proteinase K and SDS. The
agarose droplets were stable for at least one week when
stored in ethanol at 48 8C, as determined by confocal imaging
of DNA diffusion radii.
A key benefit of agarose encapsulation is the ability to
mechanically manipulate the isolated genomic DNA without
mixing the genetic content of different cells. Agarose droplets
equilibrated with PCR mix were reemulsified by mechanical
agitation in dispersing oil to produce uniform nanoliter-
droplet “reactors” for massively parallel single-cell PCR
analysis. Excess PCR mix produces microfines (emulsions less
than 1 mm in diameter), which enhance emulsion stability
during thermal cycling.The agarose droplets melt during
the hot start phase of PCR and remain liquid throughout the
amplification process, maximizing reagent and amplicon
diffusion rates. We varied the initial heating rate and tested
various concentrations of Triton X-100 as well as combina-
tions of Abil em90 and Span 80 detergents in oil. A slow
temperature ramp profile (0.18 8Cs?1) resulted in improved
short-term stability, and the addition of BSA (4 mgmL?1) to
the PCR mix and 0.8% Triton X-100 to the emulsion oil
minimized droplet merger over the course of PCR thermal
cycling (Figure 2e). Fluorescently labeled amplicons bound
to the primer-functionalized beads could be seen inside the
agarose droplets following PCR (Figure 2 f). The 34 mm cross-
linked beads were selected for their ability to amplify targets
exceeding 1 kb in amounts of at least 100 amol per bead.
The absence of fluorescence from beads in droplets without
genomes indicated that genomic targets were not transferred
between agarose droplets.
To demonstrate highly parallel genotyping with single-cell
resolution, we performed a multiplex PCR assay of cancer
cells harboring the t(14;18) translocation at various mutant-
to-wild type (RL/TK6) cell ratios. Labeling of the control
gene product, b-actin, with the cyanine dye Cy5 enabled the
quantification of total cell frequency, whereas the transloca-
tion t(14;18) product labeled with carboxyfluorescein (FAM)
spanned the bcl-2 and IgH genes across their breakpoint
regions and could be used to determine mutation frequency.
A representative flow cytometric profile of beads following
multiplex PCR amplification with 50% RL cells at an average
cell frequency of 0.3 cpd (Figure 3a) demonstrated distinct
populations of negative beads, positive beads with Cy5-
Figure 2. Microfluidic agarose encapsulation and genetic analysis of
single mammalian cells. a) Four-layer glass–polydimethylsiloxane
(PDMS) microfluidic-emulsion-generator array used for agarose-drop-
let formation. A three-valve micropump actuates an agarose–cell–bead
suspension from the sample inlet toward cross-channel nozzles, where
droplet generation occurs. b) Agarose-emulsion generation. Single
cells are shown encapsulated along with primer-labeled beads in
uniform 3 nL droplets. The inset highlights the presence of a cell
(circled) and a bead in one droplet. c) Differential interference contrast
(DIC) image of a cell (arrow) at the edge of an agarose droplet after
protease digestion. d) Projection of the confocal micrograph in (c)
showing DNA stained with acridine orange confined in the agarose
after release from the cell (arrow). e) Following PCR amplification of
the chromosomal translocation t(14;18) and b-actin, agarose droplets
containing beads (arrows) remained intact and did not merge.
f) Epifluorescence micrograph of the sample in (e) showing that
primer-labeled beads (arrows) become fluorescent if a t(14;18)+RL
cell is present in the same droplet, but otherwise remain dark. Scale
bars are 100 mm in (b), (e), and (f) and 10 mm in (c) and (d).
? 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2011, 50, 390–395
labeled b-actin only, and double-positive beads with FAM-
labeled t(14;18) and Cy5-labeled b-actin. Beads containing
t(14;18)only were never observed,which further indicates the
conservation of single-genome integrity during cell lysis and
PCR. By maintaining a constant total cell density in the
single-cell regime (0.1–0.3 cpd on average) and varying the
relative concentrations of mutant RL and wild-type TK6 cells,
we generated a standard curve (Figure 3b) to confirm that
amplification originated from single cells. In this stochastic
regime, the linearity (r=0.993) of the measured concentra-
tion of mutant RL cells with respect to the input in the
0–100% RL-cell-frequency range tested indicated successful
genetic analysis of single cells. Importantly, in the subset of
samples further tested, the ratio of total amplicon-positive
bead frequency determined by flow cytometry to the micro-
scopically observed cell-encapsulation frequency indicated
high PCR efficiency (113?24%).
For the single-cell sequencing of both target gene loci, the
template bound to single beads diluted to the stochastic limit
in a standard 96-well plate was reamplified, and the products
were separated from individual wells by gel electrophoresis.
Gel analysis further confirmed amplification from single
genomes, and frequencies of b-actin single-positive to double-
positive events matched flow cytometry frequency results in
the samples tested. Fluorescence-based sorting can be applied
to amplicon-labeled beads to sequence only populations of
interest and thereby increase efficiency. Size-separated prod-
ucts from single beads were excised from the gel for
sequencing. The expected t(14;18) and b-actin sequences
were recovered (Figure 4; see also Figures S2 and S3 in the
Supporting Information). The random nucleotide insertion
sequence in t(14;18) matched the unique translocation
“fingerprint” determined by sequencing of RL cells in bulk.
The reamplification step enables the integration of single-
cell PCR with standard molecular-biology techniques for the
sequencing of multiple genetic loci in individual cells at a rate
required for meaningful population analysis. Using the single-
bead reamplification and size-separation approach, it is
possible to sequence multiple target genes from hundreds of
single cells in a single experiment and to perform meaningful
statistical analysis of gene-sequence variation in a cell
population. Furthermore, the number of target loci per cell
is limited only by the ability to excise individual bands from a
gel, provided the multiplex PCR has been validated. Adap-
tation of our agarose cell-encapsulation and emulsion PCR
method to next-generation sequencing technologies has the
potential to provide additional gains in throughput for single-
cell sequencing. Although alternative single-cell-resolution
approaches to genetic analysis, such as fluorescence in situ
hybridization[20,21]and in situ PCR,[22,23]are utilized for
offered by these methods is limited, and the lack of amplicon
recovery prevents the detection of unknown mutations. In the
case of follicular lymphoma, identification of the breakpoint
sequence along the IgH and bcl-2 genes can help in the
elucidation of the mechanisms of erroneous genetic recombi-
nation that cause the translocation t(14;18), whereas the
random insertion sequence between the two chromosomes is
a unique identifier of distinct mutation events.
Encapsulation enables robust parallel cell lysis and DNA
purification of cell types that are difficult to screen by using
other single-cell PCR methods. The method can be adapted in
future applications to encapsulate live single cells for cell
culture and subsequent analysis of clonal populations.
Although a similar approach was demonstrated recently for
the screening and amplification of alginate-encapsulated
Escherichia coli colonies containing plasmid libraries,our
approach enables the robust genome purification of mamma-
lian cells, requires 1% of the reagent volume for emulsion
PCR, does not require droplet sorting, and maintains single-
cell segregation throughout all downstream analyses as a
result of the incorporation of primer-functionalized beads as
amplicon substrates. Furthermore, single-cell sequencing
techniques generally involve cell types that are amenable to
lysis during PCR and the sorting of single cells into 96-well
PCR plates.[25,26]However, PCR amplification directly from
single cells has been hindered by the lack of a DNA-
purification step that would remove histones and other
nuclear components that inhibit polymerase activity.[25,27]In
our approach, the incorporation of a highly parallel single-
Figure 3. High-throughput digital genetic analysis of cancer cells.
a) Representative flow cytometry plot and gated populations of beads
from a sample containing 50% RL lymphoblast cells (n=1206) at a
frequency of 0.3 cells per droplet. The observed fraction of total
positive events (26.3%) compares favorably with an expected fre-
quency of 25.9% based on the Poisson distribution. b) Standard curve
for the detection of t(14;18)+RL cells from mixtures of mutant RL and
wild-type TK6 cells. Experimental quantification by multiplex emulsion
PCR and flow cytometry exhibits a linear response between the 10 and
100% mutant-cell frequencies tested.
Angew. Chem. Int. Ed. 2011, 50, 390–395? 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
cell-lysis and DNA-purification step resulted in near 100%
amplification efficiency. Finally, the ability to purify genomic
DNA from single cells in a supporting matrix opens up the
possibility of single-cell epigenetic analysis, such as the
methylation-specific PCR of gene regulatory sequences
following conversion with bisulfite.
Cancer is an evolving disease driven by genetic instability,
which results in a constant clonal divergence of the tumor cell
population. The accumulation of mutations in the genes
coding for cellular pathways plays an important role in
carcinogenesis, metastasis, and therapeutic resistance.[28–30]
Significant cellular heterogeneity may therefore exist in
tumors; this heterogeneity changes dynamically at different
stages of disease progression.We have developed a robust
agarose-microdroplet method for detecting and sequencing
multiple genetic targets from single
cells in a scalable manner. Whereas
oncogene mutations, such as TP53,
CDKN2A, CDKN2B, and blc-6, have
been demonstrated to correlate indi-
vidually with poor prognosis in hem-
atopoetic cancers,[31–35]the ability to
single cells with high throughput will
facilitate investigation of the syner-
gistic effects of mutation cooccur-
rence and their impact on disease
progression and treatment. Further-
more, the screening of large cell
populations will uncover potential
tumor heterogeneity at the single-
nucleotide level that is otherwise
obscured by the ensemble average.
By overcoming the many problems
associated with single-cell genetic
cell lysis, DNA release and amplifi-
technology provides the throughput,
robustness, and resolution required
for probing the stochastic mecha-
nisms of carcinogenesis, progression,
and response to chemotherapy.
Received: September 29, 2010
Published online: December 3, 2010
gene sequencing · microemulsions ·
microfluidics · single-cell analysis
Keywords: cancer mutations ·
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