Development of an Automated SNP Analyzer
by Ken Tsukii *, Akihiro Murakami *, Toru Takahashi * and Jie Xu *As we enter the “post-genome sequencing” era, great importance attaches totechniques for analyzing single-nucleotide polymorphisms (SNPs), which are
expected to make a crucial contribution to achieving tailor-made medical treatment. This makes it
desirable to realize a system that fully automates the process of SNP analysis.
The Production Engineering Development Center has therefore embarked, together with
FI-Techno and Precision Systems Science, on a joint project for development of a fully automated
DNA analyzer, and has achieved a number of significant technological results relating primarily
to DNA detection. By developing a system for transmitting genetic information by means offluorescence-marked microbeads and a proprietary high-sensitivity detection system that takes
advantage of fiber-optic technology, we have developed a unique DNA information analyzer
capable of high-speed and high-sensitivity analysis of SNPs. We are now manufacturing anapparatus capable of fully automated analysis of SNPs that can be used in clinical diagnosis
applications.

ABSTRACT1.

INTRODUCTION

With smaller size and greater precision, data transmission
equipment using optical fibers and fiber-optic components
are being applied in different fields, and specificallyare widely used in optical instruments and signaltransmission. Biotechnology, meanwhile, is entering
a “post-genome” era in which attention is focused on
analyzing the single-nucleotide polymorphisms (SNPs),
which identify the individual DNA differences that will
be a crucial means of realizing tailor-made medical
treatment. In these SNP analyzers, such as DNA chips
and bead arrays, the importance of high-speed, highsensitivity measurement means that methods that use
fluorescence have become dominant. However DNA
chips, which constitute the most common analyticaltechnique for SNPs, are systems that measure surface
reactions. They therefore have disadvantages in termsof low reactivity and a measurement sensitivity thatis degraded proportionally as densities increase, andof being complicated to operate and requiring longer
measurement time. What is more the DNA samples to bemeasured require pre-processing, presenting problems interms of automation, so that at present, fully automatedsystems are not practical. Fully automated analyzers
are particularly desirable in the clinical context, with the*Production Engineering Development Center, Plant andFacilities Div.

further need for high reliability, greater convenience, andimproved economy.
The Production Engineering Development Centerhas embarked, together with FI-Techno and Precision
Systems Science, on a joint project for development ofa fully automated DNA analyzer. In the present work
we have succeeded in producing an apparatus foranalyzing SNPs and other DNA information, in which the
DNA information is loaded onto microbeads, which areadvantageous in terms of automated processing, andthe DNA information on the beads is then measured in
microchannels as fluorescence at high speed and withhigh sensitivity.

2.

DETECTION SYSTEM

2.1 Principle of SNP Analysis
The microbead array method is used under conditions ofsuspension in an aqueous solution fluid, with the resultthat the degree of freedom of reaction and reactive rate
are high, and the cost of manufacturing the arrays canalso be kept extremely low in comparison with DNA
chips. Furthermore, with microbead arrays, the adoptionof Magtration using magnetic beads, which has beendeveloped by Precision Systems Science, makes it
possible to use the same automated processing systemfor both the pre-processing of the samples and for theirmeasurement, making it the analytical method mostsuitable for clinical diagnosis applications.

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Development of an Automated SNP Analyzer

SNP1 (A/G)
Human DNA sample
Synthetic DNA

PCR amplification, etc.

Green fluorescence

Fluorescence-marked magnetic beads
SNP-specific synthetic DNA
T

Magnetic beads marked
with red and yellow
fluorescence

Combining reaction

C
A
T

C

Figure 1

Green fluorescence

Principle of SNP analysis by measurement of fluorescence-marked beads.

This apparatus analyzes the SNP information on thesamples, marking magnetic beads 2.8 μm in diameter by
combining them with several fluorescent dyes in various
proportions, and applying to them probes corresponding
to each of these markers. The labeled samples are
made to react with fluorescent dyes not used in marking
the beads, determining which of the marked beads the
samples are combined with. Figure 1 shows the principle.
2.2

Principle of Measurement Using FluorescenceMarked Beads
In order to implement the analytical procedure describedabove, it is necessary to develop an apparatus for
detecting the optical information on the beads that issuited to automated operation. And to carry out highspeed analysis for a plurality of SNPs, it is also necessary
to measure the multiple types of fluorescent informationon the microbeads for each bead individually.
One type of equipment for measuring fluorescence is

Bead identificationSmthe spectrophotometer, but it is not capable of measuring
beads individually, and flow site meters, which are used incell research for measuring the fluorescence informationof minute particles, are expensive and very hard to reducein size; they are also unsuited to automated operation.
In the apparatus reported here, a microchannel control
system has been developed which assures that the
microbeads in the fluid flow through the microchannel
in a stable manner one by one, and measurements are
made individually for each of the microbeads. In addition,
as described below, the fluorescence detection headand microchannel are of unitized construction, and atechnique was developed for multi-channel measurement
using a fiber array, making possible high-sensitivity
measurement of fluorescence-marked beads that is suited
to clinical applications. Figure 2 shows the mechanismof measurement and identification using fluorescencemarked beads.

Lg

Med

Size

Fluorescence-marked beads
Time

Fiber 1 (size/red)
Fiber 2 (size/yellow)

Lg/1

Sm/2

Med/1

Lg/3

Sm/2

Med/1

Lg/1

Sm/2

Med/1

Lg/3

Sm/3

Lg/2

Fiber 3 (size/blue)
Fiber 4 (size/green)

Lg 1 1 2 1

Figure 2

Sm/2 Med/1

Lg/1

Lg/1

Med/2

Sm/1

Sm 2 2 2 1...

Med 1 1 1 2

Fiber 1

Lg/2

Sm/2

Med/2

Lg/2

Fiber 2

Sm/0

Med/3

Fiber 3

Med/1

Sm/2

Fiber 4

Lg 3 3 1 3

Mechanism of measurement and identification using fluorescence-marked beads.

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Development of an Automated SNP Analyzera) Before normalizationb) After normalization

0.5

0.5
0.45

Cy5

0.4

0.4

0.35

0.35

0.3

Frequency

Frequency

0.45

0.25
0.2

0.3
0.25
0.2
0.15

0.15
0.1

0.1

0.05

0.05

0

Cy5

1

10

100

1000

10000

Intensity

Figure 3

0

1

10

100

1000

10000

Intensity

Effect of normalization using data on bead tracks in the microchannel.

2.2.1

Unitized Construction of Fluorescence Detection
Head and Microchannel
To detect the low-intensity fluorescence of beads withmulti-color markings using a number of fluorescent dyes,
further distinguished by multiple gradations of light anddark, it is necessary to expose each of the microbeads inthe flow accurately to a specific excitation light beam, andmeasure the optical information from each bead with highsensitivity. Specifically, since the beads being measured
are only 2.8 μm across, the smaller the channel the better.
Furthermore the optics are ordinarily configured on the
peripheral surface of a glass channel, so that problems
like the need to adjust the optical axis to the channel andthe occurrence of optical noise due to surface reflection
also require attention.
We therefore designed a configuration in which aplurality of optical fibers are disposed along the wall of a
precision microchannel measuring some several hundredsof micrometers on a side, with a positional accuracy of
1 μm or better. The channel wall is thus formed by theends of the fibers, enabling the direct exposure by anddetection of the excitation beam on the beads by the
optical fiber. This effects a significant reduction in opticalnoise, limiting the point of measurement to a minutearea, and thereby making possible highly sensitive
measurement. The precise positioning and alignment of
the optical fibers was achieved by applying techniques for
manufacturing optical connectors.
This unitized construction of the channel and the lightpath not only increases sensitivity by more efficienttransmission of light energy but also enables moreaccurate positioning of the optical axes with respect to thebead tracks, thereby achieving improvements in rate ofbead detection and eliminating optical axis adjustment.
2.2.2 Multi-channel Design Using Optical Fiber Arrays
Disposing a plurality of optical fibers along the directionof flow of the fluid makes it possible for each of the fibers
to perform measurements individually for a different
marker. For each fiber, measurements are performed byintroducing the excitation beam into the fiber and exposing
a bead to irradiation from the fiber. The fluorescent light
thus excited is detected by the same fiber, subjected
to wavelength selection by an optical filter, and thefluorescence information carried by the bead is measured.
The measured values are digitized by multi-levelcomparison and by correlating the measured informationfrom each measurement point with information on flowrate and time the multiplicity of optical data carried on thebead is measured. Since each measuring point is in adifferent location, it is possible to measure the spectra ofvarious types of fluorescence with high sensitivity. Since
the transmitted light is measured at each measurement
point simultaneously, the passage of each bead can byunambiguously confirmed, and using the values obtained
for transmitted light, bead diameter can be measured andthe tracks of the beads in the channel can be determined.
Measured values for fluorescence show a large
dispersion without pre-processing, but by normalizing
using measured values for transmitted light obtainedat the same time, it is possible to effect a significant
improvement in the signal-to-noise ratio for fluorescencevalues (see Figure 3).
Figure 4 shows data from measurements offluorescence-marked beads using a typical flow site
meter for purposes of calibration. These results confirm
that beads of five levels of fluorescence intensity can be
identified.
Mounting the optical fibers at high density has madeit possible to miniaturize the detection head, and since
the excitation beam and the fluorescence received fromthe beads are transmitted on fine, flexible fibers, it has
been possible to dispose the lasers, photodetectors
and other optical components in a different location andaccommodate them in the unit compactly. Specificallyin the case of multiple colors a problem arises in thatthe constituent optical systems cannot physically be
accommodated compactly in the detection head, but since
the detection head developed here is a fiber array only,
the number of fluorescent dyes measured can be easily

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Development of an Automated SNP Analyzer

300

250
100%

Frequency

200
28%

150

12%
10%

100

0%

50

0
1

100

10

1000

10000

Intensity (FITC)

Figure 4

Result of measurement of fluorescence-marked
beads by flow site meter for purposes of calibration.

No. of fibers

5

3 = 243 (Types of identifiable beads)
No. of fluorescence intensities recognizable on a single fiber

Figure 5

Bead identifying capability.

increased, and as shown in Figure 5, the bead identifying
capability has been significantly expanded.
2.2.3 Controlling Tracks in the Bead Channel
If the flow channel is too large with respect to the sizeof the beads being measured, significant measurement
dispersion can occur, and if it is too small, the beads cancause blockages that impede stable flow. Accordingly a
fluid control technique using sheath flow was introduced,
thereby controlling bead track dispersion in the
microchannel, improving the bead detection ratio, andsuppressing blockage.
In this sheath flow technique, the sample flow including

the beads and a sheath flow enveloping the sample flow
are introduced, each at a different pressure, into thechannel, so that the flow resistance of the sample flow isdrastically reduced, minimizing the gradient of its rate offlow so that the sample flow is squeezed more narrowly
with respect to the channel diameter. The difference
between the pressures set for the sheath flow and thesample flow regulates the diameter of the sample flow,
concentrating the bead tracks at the desired position andreducing the measurement dispersion to the beads. This
reduction in measurement dispersion is confirmed by theexperimental results shown in Figure 6.
Further, the physical diameter of the channel isincreased, thereby effectively addressing the problems ofbead blockage.
2.2.4 Identification of SNPs Using a Prototype System
Decoding genetic information involves distinguishing
the four types of nucleotides --adenine (A), cytosine(C), guanine (G) and thymine (T), and determining thesequence in which the nucleotides are arranged. By
determining the sequence of these four nucleotides
we can obtain the genetic information that will show
the predisposition of individuals to disease. This is the
purpose of analyzing single-nucleotide polymorphisms(SNPs).
Figure 7 shows the protocol used in an experimental
SNP analysis to evaluate the performance of thisapparatus. In this experiment the analysis was performed
on one SNP having two fluorescent dyes; it differsfrom the principle described in section 2.1 in termsof the marking procedure, but is identical in terms of
the principle for measuring SNPs by fluorescencemarking. Fluorescence measurement was done byspectrophotometry, and the SNP site of the specimen
was identified as A/A when the fluorescent dye was FITC
alone, as A/G when the dyes were FITC and Cy5, and as
G/G when the dye was Cy5 alone.
The result of analysis of one SNP using this prototype
apparatus is shown in Figure 8. Bead identification wasb) With sheath flow
6

5

5

4

4

Intensity

Intensitya) Without sheath flow
6

3

3

2

2

1

1

0

0
0

0.1

0.2

0.3

0.4

0.5

0.6

0

0.1

0.2

0.5

0.6

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Figure 6

0.3

0.4

Pass

Decrease in measurement dispersion due to sheath flow.

91

Development of an Automated SNP Analyzer

SNP site A or G
Biotin-ized primer

PCR

Ag

Primer containing regions of recognition of restricted enzymes

Cutting by restricted enzymes(exposure of SNP site)(When SNP site is A)

8.00

A/A

6.00

Adapter T or C

A/G

Tc

Ag

Tc
Cc
Cc

FITC

FITC

Ag

Cy5

4.00
G/G

Ligation to adapter T or C

2.00

Tc
Ag
Tc
Ag

Fixation to magnetic beads
with avidin attached

0.00
0.00

1.00

Tc
Ag
Tc

A/A
A/G

3.00

4.00

5.00

6.00

Cy5

Emission from FITC alone

Result of measurements by spectrophotometer

Ag
Tc
Ag
Cc

2.00

Emission from both Cy5 and FITC

Gg
Cc
Gg
Cc
Gg

G/G

Figure 7

Emission from Cy5 alone

Protocol for SNP analysis by measurement of fluorescence, and results obtained.

10000
C/C
T/C

Cy3

1000

100

T/T

10

1

1

10

100

1000

10000

Cy5

Figure 8

Result of analysis of one SNP using prototype
system.

performed using Cy3 and Cy5 as the fluorescent dyes.
From these results it was possible to confirm that SNP
identification was possible using various combinations offluorescence intensity.
2.2.5 Multiplexing of Measurement
In section 2.2.4 we looked at analysis of one SNP basedon the presence or absence of two types of fluorescentdye, but high-speed, high-throughput analysis can beachieved by causing multiple SNPs to react in the same
solution and multiplexing the measurement. For thispurpose, according to the principles set forth in section
2.1, it is necessary to perform high-speed identificationof a number of types of fluorescence-marked beads.
And under this principle, since the marked beads can bemeasured individually, multiplexing can be realized bymeasuring a suspension containing a number of types ofmarked beads in a single process step. Since at present it
has been confirmed that with a single type of fluorescentdye five levels of intensity can be distinguished, it ispossible, marking with two types of fluorescent dye andwith samples labeled with a single fluorescent dye, toanalyze twelve SNPs simultaneously. Further, the ability
to analyze multiple SNPs from a minute sample makes it
possible to perform analyses at an unprecedentedly low
cost.
2.2.6 Reducing Measurement Times
In an effort to reduce measurement times and copewith large numbers of samples, it was decided toreduce the length of the flow channel by adopting thecommercially available microplate specification for the
sample fluid and using a unitized construction for theintake nozzle and detection head. This configurationmeans that measurement is completed immediately after
nozzle intake, significantly increasing operating speeds,
approaching to approximately 10 seconds for a sample
fluid of one well. Figure 9 is a simplified schematic ofdetection head and Figure 10 shows a photograph.

3.

CONCLUSION

Applying the precision micro-optics technologies for
optical fibers and optical connectors, we have developed
a detection system that offers higher sensitivity, higher
speed and lower cost, and is optimally suited to a
microbead array analysis technique for clinical diagnostic
applications. We are now manufacturing an SNP analyzer
shown in Figure 11, which incorporates this detection
system and is capable of providing fully automatedFurukawa Review, No. 24

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Development of an Automated SNP Analyzer

Detection head makes measurement
moving three-dimensionally over the samples

Optical fiber
Laser diodeTransmitted light detection
Fluorescence detection

Pressurized air
Sheath fluid

Reacted samples

Figure 9

Simplified schematic of detection head.

Sample microchannel

Optical fiber
15mm

Figure 10 Photo of detection head.

operation from DNA extraction to measurement.
The fully automated analyzer shown in Figure 11 is forresearch applications in SNP analysis, but it is our belief
that by means of this technology we come closer to thegenetic diagnostic capability that will make tailor-mademedical treatment a reality. It is our plan that, based on
this system and in response to market needs, we willproduce analyzers of practical use is clinical diagnostic
applications.

Figure 11 Photo of SNP analyzer.

REFERENCES

1) K. Sawakami et al.; Development of a beads detection systemusing a multi-channel fiber sensor, 25th Conference of The
Molecular Biology Society of Japan (in Japanese)
2) PSS Co., Ltd.; PRODUCTS & APPLICATIONS, Rev. 003

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