42
Micro¯uidic devices for DNA sequencing: sample preparation
and electrophoretic analysis
Brian M Paegel, Robert G Blazej and Richard A Mathiesy
Modern DNA sequencing `factories' have revolutionized biology
by completing the human genome sequence, but in the race
to completion we are left with inef®cient, cumbersome, and
costly macroscale processes and supporting facilities. During
the same period, microfabricated DNA sequencing, sample
processing and analysis devices have advanced rapidly toward
the goal of a `sequencing lab-on-a-chip'. Integrated micro¯uidic
processing dramatically reduces analysis time and reagent
consumption, and eliminates costly and unreliable macroscale
robotics and laboratory apparatus. A microfabricated device for
high-throughput DNA sequencing that couples clone isolation,
template ampli®cation, Sanger extension, puri®cation, and
electrophoretic analysis in a single micro¯uidic circuit is now
attainable.
Addresses
Department of Chemistry and UCB/UCSF Joint Bioengineering
Graduate Group, University of California, Berkeley, CA 94720, USA
y
e-mail: [email protected]
Current Opinion in Biotechnology 2003, 14:42±50
This review comes from a themed issue on
Analytical biotechnology
Edited by Norm Dovichi and Dan Pinkel
0958-1669/03/$ ± see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0958-1669(02)00004-6
Abbreviations
BAC
bacterial arti®cial chromosome
CAE
capillary array electrophoresis
lCAE microfabricated capillary array electrophoresis
JGI
Joint Genome Institute
LPA
linear polyacrylamide
PCR
polymerase chain reaction
PRS
polymorphism ratio sequencing
RTD
resistive temperature detector
Introduction
The initial goals of the Human Genome Project are
rapidly approaching completion [1±3]. While this project
is just ®nishing, the completion of a myriad of other
model organisms [4] and the understanding of
sequence-dependent organismal variation [5] will be
more feasible once methods for decreasing cost, analysis
time, and reliance on cumbersome and expensive robotics
have been developed. In addition to increasing sequencing capacity, this new objective will also liberate the
technology to address more diverse comparative sequencing and polymorphism discovery and screening needs.
Current Opinion in Biotechnology 2003, 14:42±50
However, the routine use of electrophoretic sequencing
in these higher-throughput arenas is currently impossible
because of the cost of the macro-process, estimated to be
$0.10±0.50 per reaction depending on scale.
The Joint Genome Institute (JGI), which is representative of streamlined modern production sequencing facilities, has developed an automated process for preparing,
purifying, and analysing DNA sequencing samples.
Figure 1 presents their sequencing chemistry ¯ow chart,
illustrating the volume and time scale involved at each
step. The chart starts with the transformation step; electro-competent cells are transformed with a pUC19
sequencing vector containing a 1 kbp insert from a
large-clone library, such as a bacterial arti®cial chromosome (BAC). The transformed cells are plated and grown
for 18 h before automated colony picking. Inserts are
ampli®ed isothermally in a 10 ml reaction using rolling
circle ampli®cation [6], sequenced in a 10 ml reaction
using Sanger dideoxy dye-terminator chemistry [7], puri®ed from template and free dye-terminators on magnetic
carboxyl beads [8], and analysed on a MegaBACE capillary array electrophoresis (CAE) sequencer.
This robotically driven aÁ la carte sequencing process is
problematic in terms of space and maintenance, and
sample transfer reliability. Furthermore, the enzymemediated reactions, including Sanger sequencing and
template ampli®cation, consume costly reagents at high
volume to meet liquid handling limitations. If these
limitations were to be removed, how far could we scale
down the volumes in the Sanger sequencing process in
principle? Starting with a single transformed Escherichia
coli cell containing ®ve copies of the sequencing vector
(a conservative assumption), 30 cycles of PCR result in
109-fold ampli®cation, generating 10 fmol of template; a
500 nl Sanger sequencing reaction would require only
1 fmol of ampli®ed plasmid DNA. Assuming a minimum yield of 50% for 30 sequencing thermal cycles,
150 fmol of product is generated. When concentrated
to 10 mM (corresponding to 10 nM per peak for a
sequencing ladder of 1000 bands), this amount of
ready-to-inject product occupies 15 nl. The average plug
volume injected on a capillary is only 1 nl. We therefore
conclude that sequencing a vector contained in a single
transformed cell is feasible. Can this ambitious goal be
achieved in practice?
The nanoliter volumes intrinsic to such a micro-sequencing process are inaccessible to current CAE-based
sequencing platforms (Figure 1). Nanoliters are, however,
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Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 43
Figure 1
10 cm
Transformation
25 µL
2h
Plate and grow cells
300 mL
20 h
2m
Pick colonies
10 mL
10 min
30 cm
DNA amplification
10 µL
18 h
Sanger extension
10 µL
120 min
40 cm
Product purification
30 µL
60 min
1m
Electrophoresis
15 µL
150 min
Current Opinion in Biotechnology
A flowchart for conventional macroscale DNA sequencing sample
processing and electrophoretic analysis. Instrumentation, including
scale bars to indicate approximate instrument size, is shown on the left
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the ideal volume for a microchip sequencer that performs
on-chip enzymatic reactions, sample transport, and electrophoretic analysis. Here, we review recent demonstrations of microchip sample analysis and processing in the
context of enabling this micro¯uidic `sequencing lab-ona-chip'.
Microfabricated electrophoretic DNA
sequencing separation devices
In 1995, Woolley and Mathies [9] demonstrated that
narrow sample zone injection on a microdevice greatly
reduces the column length required to separate DNA
sequencing fragments; 200 bases were called from a fourcolour sequencing sample using a 3 cm long microchannel
in only 9 min. Presently, work on single-channel microchip electrophoresis applied to DNA sequencing has
focused on improving data quality and read length by
incorporating well-characterized, high-molecular weight
linear polyacrylamide (LPA) sieving matrices, ef®cient
sequencing chemistries utilizing energy-transfer (ET)
¯uorescent reagents, and microchannels with increased
separation length. By extending the channel separation
length from conventional microchip values of 3 to
7 cm and synthesizing sequencing fragments with
improved ET reagents, 500 bases (99% accuracy) were
acquired in only 20 min [10]. Similar experimental conditions applied to an 11.5 cm long channel produced an
average of 505 bases in 27 min from human chromosome
17 samples [11]. Further improvement of single-channel
separation devices was accomplished by implementing a
blended, emulsion-polymerized LPA sieving matrix [12]
on long, straight channels. The basic channel schematic
and results for these devices are summarized in Figure 2.
A standard cross-injector with an offset of 150 mm is used
to increase the amount of sample injected (Figure 2a). An
11.5 cm long version of this device produced 580 bases
(98.5% accuracy) in 18 min [13]. For longer read
lengths, the channel length can be increased to 40 cm
to acquire 800 bases, although the run time is substantially increased to 80 min [14]. Plots of resolution versus
base position for both devices are presented in Figures 2b
and 2c. Bases with a resolution >0.5 were considered to be
of high data quality, although phred (base-calling and data
accuracy evaluation software) analysis is now used exclusively to describe sequencing data quality [15,16].
An even more attractive advantage of the microfabricated
platform is the ease with which intricate, ultra-dense
channel arrays can be constructed. Although conventional CAE-based sequencers are substantially faster
than slab-gel-based instrumentation, their predominant
contributions to genomic sequencing lie in their capacity
to provide a higher degree of automation and analysis
of each process step. Space measured in many square metres is
required to accommodate the supporting instrumentation. (Protocols
taken from http://www.jgi.doe.gov/Internal/prots_index.html.)
Current Opinion in Biotechnology 2003, 14:42±50
44 Analytical biotechnology
Figure 2
Sample
(a)
Cathode
In 2000, a prototype microfabricated capillary array electrophoresis (mCAE) DNA sequencing device consisting of
an array of sixteen 7 cm long channels was presented [19].
The 16 cross-channels were fanned out on a 100 mm
diameter wafer, each channel having its own cathode,
sample, and waste wells. All channels converge on a
common high-voltage anode. An average of 457 bases
per lane (99% accuracy) was acquired in 15 min. Other
array structures for DNA sequencing were also presented
around this time, but lacked the cross-injector, a requirement for high-performance, high-speed microchip DNA
sequencing [20,21].
Waste
(b)
1.2
Leff = 11.5 cm
18 min
Resolution
1.0
0.8
0.6
0.4
0.2
100
300
500
700
Base number
(c)
1.2
Leff = 40 cm
78 min
Resolution
1.0
0.8
0.6
0.4
0.2
0
200
400
600
Base number
700
1000
Straight-channel DNA sequencing chip design and resolution results.
(a) The standard cross-injector sample and waste channel arms are
offset to increase the injected plug length. (b,c) Plots of sequencing
fragment resolution versus base position show device performance; a
resolution of >0.5 is considered high quality. Increasing the device
length from (b) 11.5 cm to (c) 40 cm increases DNA sequencing read
length at the expense of increased analysis time. Reproduced with
permission from [13] and [14].
Current Opinion in Biotechnology 2003, 14:42±50
multiplexing. Microfabrication removes limitations associated with assembling multi-capillary arrays, allowing
micron-precision, scalable control of channel layout and
arrangements [17,18] in addition to the integrated reactor
structures to be discussed later.
A 96-lane mCAE DNA sequencing bioprocessor was
introduced in 2002 [22], the ®rst massively parallel
micro¯uidic circuit design (Figure 3) exploiting microchip injectors for high-performance separation. The chip
is radially symmetric, composed of 48 doublets containing
unique sample wells and common cathode and waste
wells [23]. The serpentine channel leading toward the
common, high-voltage anode at the centre of the device
contains four hyperturns and provides the necessary
separation length for performing DNA sequencing in
LPA. The hyperturns minimize geometric band dispersion introduced by turns in the separation path [24±26].
The separation length is extended from 5.5 cm for a
straight channel on a 150 mm diameter wafer to 15.9 cm.
Filling long channels with gel-sieving matrix required the
use of a high-pressure loading and cleaning device [27],
although novel copolymer matrices exhibiting thermoresponsive viscosity may alleviate this requirement [28].
Detection is accomplished using a rotary confocal ¯uorescence scanner that interrogates all lanes sequentially
[23]. The 96-lane mCAE device acquired 41 000 bases
(99% accuracy) in only 24 min [22], a sequencing
throughput ®vefold higher than currently deployed systems. The same bioprocessor was subsequently used in
the development and implementation of a novel, sequencing-based polymorphism identi®cation and screening
assay called polymorphism ratio sequencing (PRS)
[29]. In this study, the entire human mitochondrial
genome was screened by PRS in a 30 min run of the
sequencing bioprocessor that identi®ed 30 known and six
previously unknown polymorphisms. This work demonstrated the versatility of the mCAE processor as well as the
value of a comprehensive sequencing-based assay.
Micro¯uidic DNA puri®cation
Electrophoretic analysis of the DNA sequencing products, although challenging from the standpoint of separation science, is only the ®nal step in an involved series
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Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 45
of chemical processing steps, as presented in Figure 1.
Puri®cation of DNA sequencing products, the step just
before electrophoretic analysis, is critical. Residual background ions (buffer, chloride, unincorporated dideoxynucleotides) present in the sequencing extension reaction
cocktail after thermal cycling reduce injection ef®ciency,
signi®cantly degrading sequence signal-to-noise [30].
Frequently, either ethanol precipitation or more rigorous
spin-column methods are used to purify the extension
products [31,32], although for high-throughput operations, both pose serious automation problems because
centrifugation is required. The ideal microchip puri®cation circuit should be fully integrated, low volume, and,
most importantly, eliminate manual sample transfer and
centrifugation steps.
Figure 3
(a)
(b)
Sample
Cathode
Waste
(c)
Anode
The 96-lane DNA sequencing bioprocessor. (a) Forty-eight doublet
structures (96 samples) are radially arrayed on a 150 mm glass wafer
around a common, central anode. (b) The injector region of a doublet
contains two unique sample wells, and common cathode and waste
wells. Cross-injection is achieved by applying a positive potential to the
waste well while grounding the sample well, which fills the 1 nl
intersection with DNA. Switching to grounded cathode, small positive
potentials on sample and waste wells, and high voltage at the
anode draws a small plug of DNA down the serpentine channel for
separation. (c) A tapered turn, or hyperturn, allows the incorporation of
serpentine channel geometries without introducing geometric
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Capillary-based systems containing serial separation
steps provided initial indications of how puri®cation
might be coupled to electrophoretic analysis of DNA
sequencing products. In a study from Yeung's group
[33], capillary zone electrophoresis was used to isolate
extension fragments in free solution. The isolated band
was then directly injected onto a manually coupled
DNA sequencing capillary ®lled with gel sieving matrix.
However, much like bundling capillaries to form arrays,
joining capillaries is problematic because it requires
individual intricate glass joints, and also this method
requires complex timing for band extraction. Microfabricated systems offer more precise electrophoretic control of analyte, and band extraction channels can easily
be fabricated to accomplish micro-preparative fraction
collection, although band extraction timing is still challenging [34].
In other work toward the goal of chemistry integration,
Tian et al. [35] developed a low-volume ¯ow cell containing a variety of silica resins for solid-phase extraction of
DNA from cell lysate followed by off-chip PCR veri®cation. Their silica-resin-containing 500 nl bed had a recovery ef®ciency of 70% for DNA collected from white
blood cells, removing >80% of contaminating proteins in
10 min. This work was later expanded to a microfabricated ¯ow cell containing sol-gel/bead composites with
similar DNA extraction ef®ciencies and analysis times
[36]. Another intriguing puri®cation method relying on
analyte focusing was demonstrated in which the analyte
electrophoretic migration is modulated spatially by establishing a temperature gradient along the path of travel.
Since electrophoretic mobility is temperature dependent
and bulk electroosmotic ¯ow is constant, a point in the
microchannel exists such that the analyte electrophoretic
velocity is exactly countered by the bulk ¯ow, resulting in
analyte concentration. A variety of ¯uorescently tagged
dispersion from the turn. The separation channel shown here contains
four hyperturns with a total separation length of 16 cm. Reproduced
with permission from [22].
Current Opinion in Biotechnology 2003, 14:42±50
46 Analytical biotechnology
molecules, including DNA, were isolated and concentrated as high as 10 000-fold in this manner [37].
An alternative approach for on-chip puri®cation is hybridization-mediated capture, which is perhaps the most
effective and stringent isolation method for nucleic acids.
Capture probes immobilized in a gel-phase support can
be used for size- and sequence-selective isolation of
nucleic acids. Olsen and coworkers at NIST [38] have
demonstrated the use of DNA hydrogel plugs polymerized inside a microchannel for selective capture and 100fold concentration of a 150 nM solution of TAMRAlabeled oligonucleotide complementary to the hydrogel
in 25 min. A similar concept was recently utilized in our
group [39] to develop the integrated microdevice for
DNA sequencing shown in Figure 4. A 60 nl chamber is
coupled to a microchip injector and serpentine sequencing channel. The chamber is ®lled with a replaceable
acrylamide-methacryl-DNA capture copolymer. The
gel-immobilized capture probe sequence is chosen such
that it is complementary only to sequencing extension
fragments, thus providing selective capture. Sample is
electrophoretically driven through the chamber, preconcentrating and desalting only the extension products
while passing excess non-complementary primer and
ionic contaminants. The product is electrophoretically
washed with fresh buffer, and then released by raising
the temperature of the chip to the sequencing process
temperature. Standard cross injection is followed by
separation. The total chamber volume is more than
100-fold lower than macroscale puri®cation reactions
and results in a 200-fold volumetric concentration of
sample. Puri®cation of the sequencing products is accomplished in 120 s, a tenfold decrease in puri®cation time
over conventional methods. Furthermore, this process
requires no centrifugation, integrates sample transfer
steps, and generates sequencing peaks that are 85% of
the intensity of ethanol-precipitated sequencing product
[39].
Submicroliter PCR and Sanger sequencing
Several strategies for performing low-volume Sanger
cycle sequencing in capillaries have been presented that
build on the previously established feasibility of thermally cycling premixed Sanger sequencing reagents in
sealed capillaries using fast, convection-based cyclers
[40,41]. Hadd and coworkers [42] demonstrated that
500 nl dye-terminator reactions prepared in a highthroughput 96 capillary reactor array could be rapidly
cycled, blown out of the capillary reactor, precipitated,
and analysed on a commercial sequencer to yield results
comparable to the macroscale sequencing process. Xue
and coworkers [43] demonstrated even lower volume
(100 nl) reactions inside a capillary. These studies establish the feasibility of nanoliter-scale sample preparation,
but because of manual coupling, their integration is
problematic.
Current Opinion in Biotechnology 2003, 14:42±50
Figure 4
(a)
Inject
Bind
Separate
+
+
S
+
+
(b)
(c)
(d)
5
10
15
20
Time (min)
25
30
Integrated nanoliter affinity extraction sample cleanup and
preconcentration followed by electrophoretic analysis. (a) The sample
purification circuit contains a 60 nl extraction chamber coupled to
standard cross injection fluidics. Crude product (well `S') is
electrophoretically driven through the chamber, which contains an
affinity extraction acrylamide matrix. Extension products are selectively
bound and washed with electrophoresis buffer. For cross injection, the
sample is brought to the sequencing process temperature
(T 678C > TM ), melting the matrix-product duplex, and injection
proceeds as described in Figure 3. (b±d) The C terminations from
four-colour sequencing runs illustrate the difference between sample
purified by (b) on-chip affinity extraction, (c) off-chip
ethanol-precipitated sample, and (d) directly injected crude product.
The excess primer and first four terminations (highlighted in black)
have no specificity for the capture matrix and are therefore
absent from the on-chip-purified sample. Reproduced with permission
from [39].
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Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 47
On-chip template ampli®cation has been presented in a
variety of formats based primarily on PCR. Recent work
has explored alternative methods for thermally cycling
ultra-low volumes with expanded temperature control
and increased thermal ramping rates. Liu and coworkers
[44] showed that as little as 12 nl of PCR mix could be
peristaltically pumped through three thermal zones on a
polydimethylsiloxane (PDMS) device by activating onchip valves in a rotary fashion. PCR ampli®cation was
successful, although the starting template concentration
of 2 ng/ml was relatively high for a micro¯uidic reactor
(approximately 3 million template copies in the reactor).
Similarly, contactless heating using an infrared diode laser
provided a means for cycling only 5 nl with heating times
of >508C/s [45].
The ®rst monolithic, nanoliter-scale DNA ampli®cation
system integrated with electrophoretic analysis was presented by Lagally et al. [46]. The system consists of a
300 nl reactor ®tted with a polyimide foil heater and
thermocouple for reactor temperature control, and
coupled directly to a high-speed microchip electrophoresis circuit to provide integrated analysis of the PCRampli®ed product. Twenty thermal cycles were performed in 10 min, and the product was injected, separated, and detected in 90 s. The reactor ampli®ed as few
as 20 starting copies of template with an extrapolated limit
of detection of two copies [46]. Subsequent elaboration of
the device design included the incorporation of microfabricated thin-®lm heaters and platinum resistive temperature detector (RTD) temperature control elements. A
schematic of this system is presented in Figure 5, where
the chamber with microfabricated heating elements and
RTD elements is shown in the magni®ed inset. The heater
is fabricated on the outer surface of the device whereas the
RTD is fabricated so that it is in direct contact with the
¯uid inside the nanoliter reactor, thus providing exquisite
control over process temperature [47]. The device is
capable of stochastic single-template ampli®cation [48],
greatly exceeding the reaction sensitivity requirements for
on-chip template preparation from a single transformed
cell.
Figure 5
(a)
Heater leads (backside)
RTD leads
4-Lead
RTD
S
C
W
V
Reactor
Heating
coils
A
(b)
RTD leads
Reactor
Heater
A thermal cycling reactor integrated with microchip electrophoresis
circuitry. (a) The reactor (green) addressed on the right by
microfabricated heating elements (red) and 4-lead RTD (blue) for
sensing and controlling process temperature. The reactor volume is
sealed by a valve at the top (S) and a hydrophobic vent at the bottom
(V). The left side of the reactor is coupled to an injection cross with
cathode (C), anode (A), and waste (W) wells. (b) The side view shows
the layer structure of the device with the RTD (blue) inside the glass
(hatched) reactor. The heater (red) is fabricated on the back of the
chip. Adapted from [47].
Integrated cell sorting
The front end of DNA sequencing, requiring lengthy
culture growth steps and colony picking with expensive
and macroscale robotics, represents the most time-inef®cient and complicated portion of the process. Sorting
individual transformants directly from the electroporated
culture to sequence ampli®cation reactors obviates these
constraints, but will require precisely driven ¯uidics
capable of sensing single transformed cells and must
be compatible with culture media.
Microfabricated cell sorters generally consist of a ¯uidic
driving mechanism (electrokinetic or hydrodynamic)
leading to a T- or Y-shaped intersection, which connects
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waste and collection reservoirs. Electrokinetically driven
¯uidics sort by switching the driving voltage source
between the target and waste arms [49]. Hydrodynamically driven ¯uidics (Figure 6) sort using valves [50] on
each arm to determine the destination well. Pressuredriven systems are preferable for cell sorting because
buffers used in electrokinetically driven systems may
be incompatible with the culture, and the high ionic
strength of culture media does not provide a favourable
double-layer for establishing effective electroosmotic
¯ow. In addition to eliminating buffer compatibility
issues, bi-directional peristaltic pumping can be used
Current Opinion in Biotechnology 2003, 14:42±50
48 Analytical biotechnology
Figure 6
Figure 7
1 cm
Collection
Waste
V2
Fluorescence
V1
Transformation
25 µL
2h
Sort cells
5 µL
20 min
V2 open
V1 open
Time
Peristaltic
pump
1 cm
DNA amplification
300 nL
10 min
PDMS
valve
Sanger extension
300 nL
20 min
Input
culture
A PDMS cell sorting device with integrated valves. The culture to be
sorted is introduced at the bottom of the structure. A channel leads to a
Y intersection with output arms to collection and waste reservoirs.
Pneumatically actuated PDMS valves V1 and V2 control flow through
the arms. Cells are driven through the structure by peristaltic pumping
accomplished by serially actuating a series of valves before the sorting
intersection. The inset shows fluorescence signal and corresponding
valve states for a fluorescence-activated cell sorting strategy. Adapted
from [51].
to trap a cell in the detection volume for signal con®rmation, demonstrating the precision single-cell handling
capabilities necessary for sorting individual sequencing
transformants [51]. Integration of these all-PDMS
devices with electrophoretic analysis may be problematic
as PDMS is not well characterized for electrophoresis,
DNA sample loss through absorption could be considerable, and polymer auto¯uorescence will signi®cantly
increase detection limits. Recently, Grover and coworkers
[52] presented new fabrication methods for reliably producing valves to control ¯uid ¯ow in all-glass CE channels
using PDMS membrane multi-layer structures. The biggest challenges to the microsequencing paradigm proposed here lie in these initial cell-handling steps. The
success of its evolution will hinge on the development of
integrated and scalable sorting strategies.
Conclusions
We have dissected the modern DNA sequencing production line into its most basic and necessary elements. Each
Current Opinion in Biotechnology 2003, 14:42±50
1 cm
Product purification
60 nL
2 min
15 cm
µCAE
10 nL
24 min
Current Opinion in Biotechnology
A microfabricated `sequencing factory-on-a-chip'. An individual
subclone is sorted from a transformed culture and directed to a PCR
amplification chamber. Single-cell amplification is followed by template
purification. Purified template is moved to a thermal cycling reactor for
generation of sequencing extension fragments. The extension
fragments are isolated, preconcentrated, and injected into an
electrophoretic analysis microchannel. The total estimated process
volume from cell to called bases is 1 ml.
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Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 49
part can be executed on much lower volume and time
scales when integrated into a micro¯uidic device. We
anticipate the development of an integrated processor
that can take a single transformed cell directly to called
bases using less than 1 ml of reagent in total. This microsequencing process, shown schematically in Figure 7, will
exhibit entirely on-chip sample transport between steps.
Nanoliter-scale enzymatic reactions that are completed in
minutes and linked directly to product puri®cation, followed by high-speed microchip electrophoretic analysis
will be all employed. Analysis times can be expected to
drop an order of magnitude and reaction volumes can be
expected to drop two or more orders of magnitude. The
development of such a microfabricated `sequencing factory-on-a-chip' will bring high-throughput DNA sequencing to the everyday biology laboratory of tomorrow.
Acknowledgements
This work was supported by grants from the National Institutes of Health
(HG01399) and from the Director, Of®ce of Science, Of®ce of Biological and
Environmental Research of the US Department of Energy under contract
DEFG91ER61125. BMP was supported by a NIH trainee fellowship from
the Berkeley Programme in Genomics (T32 HG00047).
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Current Opinion in Biotechnology 2003, 14:42±50
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