Development of a Microfluidic Platform for Integrating
Nanoliter DNA Sequencing Protocols
By
Mayank Kumar
B.Tech. Department of Mechanical Engineering (2004)
Indian Institute of Technology Kanpur, INDIA
Submitted to the Department of Mechanical Engineering
in Partial Fulfillment of the Requirement for the Degree of
Master of Science in Mechanical Engineering
at the
Massachusetts Institute of Technology
February 2007
©2007 Massachusetts Institute of Technology
Signature of Author
Department -Meeanical Engineering
January 19, 2007
Certified by
Todd Thorsen
Assistant Professor of Mechanical Engineering
Thesis Supervisor
Accepted by
Lallit Anand
Chairman, Department Committee on Graduate Students
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2
Development of a Microfluidic Platform for Integrated DNA
Sequencing Protocols.
by
Mayank Kumar
Submitted to the Department of Mechanical Engineering
on January 19, 2007 in Partial Fulfillment of the Requirements for the
Degree of Master of Science in Mechanical Engineering
Abstract
This thesis describes the design and development of a microfluidic platform to reduce
costs and improve the quality of in the DNA sequencing methodology currently
implemented at the Broad Institute in Cambridge, Massachusetts.
The Sequencing Center at the Broad Institute currently generates an average of 130
million bases per day with an average read length of 800. This is enabled by the
successful preparation and detection of over 97,000 unique samples. Most of the cost per
sample is tied up in expensive proprietary reagents utilized in the various reactions
comprising the preparation process. Through the application of microfluidics, the
possibility of drastically scaling down the amount of proprietary reagents is explored.
Stamp-sized elastomeric polydimethylsiloxane (PDMS) microfluidic devices were
developed and microfluidic sample manipulation techniques were standardized. Using
these devices and techniques, an attempt was made to adapt the various components of
the sequencing process to the microfluidic platform.
Work within the scope of this thesis is focused on the adaptation of the commercial
sequencing protocols, which are labor intensive, consume costly reagents and serve as
limitations for high-throughput parallelization of the process. The first is the
amplification reaction. By scaling down the process from a plate-based format to an
integrated microfluidic device, amplification reagent consumption was reduced by two
orders of magnitude while maintaining the quality and length of the sequencing reads
(with the subsequent sequencing reaction run off chip). As a follow up project, an attempt
was made to scale down the Sequencing Reaction, which, in spite of limitations,
suggested a good path toward the eventual development of an integrated microfluidic
device for the preparation of running the complete sequencing reaction protocol on-chip.
Thesis Supervisor: Todd Thorsen
Title: Assistant Professor of Mechanical Engineering
3
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4
Acknowledgements
First and foremost, I thank Professor Todd Thorsen for reposing faith in me and
entrusting me with the responsibility of working on this project. I am very grateful for
being able to use the resources of his lab at MIT. He was encouraging at my successes
and equally understanding at my failures. None of the work here in this thesis would have
been possible without his extremely friendly and affable nature. I feel very fortunate to
have had his guidance and creative suggestions at so many critical junctures.
I thank the Broad Institute for providing me with the most state-of-the-art resources in
Bioengineering research. It was a great privilege to have worked on the most advanced
instrumentation and methods. The most valuable, however, was the help and advise of a
great team of researchers in the Production Sequencing Development section of the
Broad Institute where I was employed. I have no words to express my gratitude to Joe
Graham who was my research supervisor at the Broad Institute. None of this would have
been possible without the calming assurance provided by his insightful suggestions, care
and understanding. I heartily feel that his affectionate behavior was always like an elder
brother than a supervisor.
In addition, I thank Andrew Barry, Sheila Fisher and Susan Faro at the Broad Institute.
Andrew is the most cheerful guy I have ever met. A shining beam of enthusiasm, he was
ever ready to help me regardless of the complication of the task at hand. I am grateful to
Sheila for providing me with her invaluable advise in the myriads of times of crisis and
for helping me with the bioengineering techniques which were completely foreign to me
to begin with. Susan's readiness to help coupled with her motherly care and affection
provided me with the most affable work environment possible. I will also like to thank
my labmates at the Thorsen Lab. I am grateful to J.P. Urbansky for helping me out with
the nitty-gritties of microfluidics on numerous occasions. His prior experience in the
issues I encountered made my work so much easier.
I will also like to thank Leslie Regan, Joan Kravit and the entire graduate office for
helping me out since the very beginning of my stay at MIT. They made MIT feel like a
second home to me. For the same reason, I thank my friends Siddarth, Piyush, Kaustuv,
Ajit and many others who made me feel homely and cared for.
I thank my family for always providing great strength to me in my hardest times. No
words come close to the amount of love I have received from my parents since times of
which I don't even have a conscious remembrance. The love from my mother, father and
sister is the most valuable thing of my life and whenever I think of that, the day-to-day
problems and challenges suddenly become very small and easily manageable.
Last but not the least, I thank that Source, the Ultimate Reality from and in which my
personality and its talents and capacities have sprung. I know that my abilities are mine
by accident and not by choice. I am grateful for this life and its enriching experiences.
Moreover, I am grateful to all my Spiritual Masters for this very understanding.
5
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6
Contents
1.
Introduction ........................................................................................................
9
2. D evelopm ent of Basic M icrofluidics ............................................................
23
3. Amplification ..................................................................................................
37
4.
Sequencing Reaction ......................................................................................
63
5.
Introduction ........................................................................................................
77
APPENDICES
1.
Fabrication ....................................................................................................
81
2.
LABVIEW Controls and Instrumentation .....................................................
85
3.
Amplification Trials ..................................................................
89
7
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8
Chapter 1
Introduction
The purpose of this thesis is to propose cost reduction and quality improvement
techniques in the DNA sequencing methodologies at the Broad Institute through the
adoption of a microfluidic platform.
The DNA Sequence
To set the goals and inspiration of this work, we need to first introduce the concept of the
DNA sequence. The deoxyribonucleic acid (DNA) is a double-stranded molecular chain
in the nuclei of our cells that carries the genetic information that is subsequently
translated into proteins that not only engage in complex regulatory pathways at the
cellular level, but form higher order structures like tissue and organs. Its shape is a double
helix (Figure 1.1), consisting of a string of simple units called nucleotides, which are held
together by a backbone made of sugars and phosphate groups.
9
The backbone consists of four types of molecules called bases and it is the sequence of
these bases that encodes information. The four bases as shown in Figure 1.1 in different
colors are Adenine (A), Cytosine (C), Guanine (G) and Thymine (T).
NWO
LI
K
I
~
Cytosine
\0
Ca
phosp ate
oW
backboneN
N,2
T*end
.0
5Wend
Figure 1.1 DNA Structure details
Hence, the DNA sequence is a sequence of these letters A, T, G and C, which are
representative of the bases in the DNA chain. DNA sequencing is the process of figuring
out the sequence of these bases through chemical reactions and the use of appropriate
technology.
Goals and Inspiration
State of the art in DNA sequencing technology at centers like the Broad Institute allows
the sequencing of up to 1000 continuous bases per run. The average mammalian genome
10
is six orders of magnitude larger than this read length.
This is the mitigating
circumstance, which has led to the current technique employed for large genome
sequencing. This technique consists of shattering the genome of interest (with or without
some level of preliminary sorting) into small cloneable segments, sequencing the ends of
these segments, and utilizing an assembly algorithm coupled with a large amount of
processing power to reassemble the genome based on overlaps in this sequence.
The Sequencing Center of the Broad Institute currently generates an average of 130
million bases of sequence every day. This is enabled by the successful preparation and
detection of over 97,000 unique samples.
Each of these preparations carries an
associated cost which is the sum of all reagents, consumables, labor, and detection time.
The value of a high throughput facility is in the ability to quickly amortize fixed costs for
start-up such as automation, process tracking software, initial R&D, etc.
When addressing process improvement, an obvious target is the reduction of consumable
costs (assuming no increase in detection capacity). For the sequencing process, most of
the cost per sample is tied up in expensive proprietary reagents utilized in the various
reactions comprising the preparation process. The most straightforward way to reduce
the costs associated with these reagents is to reduce the amount of reagent used per
reaction or eliminating steps all together.
The purpose of this project is to propose cost reduction and quality improvement by
directly cutting down the reaction volumes by at least two orders of magnitude through
the adoption of nanoliter sample preparation platforms.
At the Broad Institute currently, the limit on sequencing is detection. The production
process in place can easily, in a 40-hour week, generate enough samples to keep detection
machines running 24 hours a day, 7 days a week. In such a scenario, it does not make
much sense to concentrate efforts on decreasing process time for sample preparation.
However, introduction of microfluidics will easily serve to reduce the time and
complexity of the process as well.
11
Currently, the sequential series of steps involved in sample preparation, amplification and
sequencing reaction take place in discrete robotic stations. In addition, there is the tedious
manual transfer of reaction plates from one station to another. These are certainly
avoidable issues in labor efficiency. With the development of a suitable microfluidic
platform, electronic automated controls can make it possible for the above mentioned
series of steps to be performed in a single device sequentially without the need for
manual interference. Hence, apart from reduction in reagent consumption, microfluidics
is extremely useful in simplifying the entire macroscopic scheme of operations.
The quest to miniaturize lab processes has been ongoing in the biotechnology industry for
years, reflected in the evolution from 96 to 384 and then to 1536 well plates.
When
operating with reactions at the pL level, environmental factors begin to play a larger role
and the need to control evaporation, condensation, and contamination becomes a
necessity. In order to overcome with this issue, scientists are relying more and more on
microfluidic technology. The utilization of this type of technology is already pervasive in
the industry and can be seen in such applications as HPLC, Micro Electro Mechanical
Systems (MEMS), Lab-on-a-Chip, and Capillary Electrophoresis (CE).
A strong
argument can be made, in fact, that it was the advent of CE for DNA sequencing that
enabled the Human Genome Project to be finished years ahead of schedule at a reduced
cost due to the increased ease and throughput of this technology.
Numerous research groups have worked significantly towards miniaturizing the process
of DNA sequencing. Much work has been done on taking the electrophoretic separation
and detection part of the sequencing process to the microfluidic realm, for example by
Mathies group. Also, we have seen great progress in making the DNA amplification
technology of Polymerase Chain Reaction (PCR) possible on nanoliter and even picoliter
scale. This is significant since PCR is still used at some places as a part of the sample
preparation protocols. But we are yet to witness systematic efforts to sequentially map
and transfer the whole scheme of routine sample preparation techniques to the
microfluidic realm.
12
For example, at the Broad Institute, the sample preparation roughly consists of bacterial
cell culture, followed by Rolling Circle Amplification (RCA), Sequencing Reaction and
purification. Similar processes are in place at other major sequencing centers including
the Joint Genome Institute (JGI) in California. Still we have not seen any cases of RCA
being implemented at nanoliter scale to extend the possibility of its integration with the
other sample preparation processes at a microfluidic scale. Of course, complete
miniaturization of the process demands answering many issues including modifying the
dispensing mechanisms, bacterial transformation and library construction methods and
also probably the detection mechanism.
In this thesis, the efforts made at bringing down RCA and subsequently the Sequencing
Reaction to the nanoliter scale will be outlined. Chapter II discusses the development of
fundamental microfluidic fabrication and functional aspects, which will be required for
the prospective nanoliter sample preparation platform. Chapter III includes extensive
experimentation on the DNA amplification reaction on the nanoscale leading to a set of
optimized conditions and protocols on reagent consumption and template preparation.
Chapter IV similarly discusses the efforts made on making a nanoliter Sequencing
Reaction on a PDMS microfluidic format possible. But first we discuss here the protocols
already in use in the DNA Sequencing line at the Broad institute.
Library Construction
The process by which these DNA samples are prepared for sequencing begins with the
construction of a library. This is schematically shown in Figure 1.2. The first step is
shattering the genome of interest into short fragments, generally about 1000 base pairs in
length, using restriction enzymes shown as yellow beads in Figure 1.2. Then the same
enzyme is used to cleave the plasmid of interest at a similar site. The plasmid is a circular
piece of double stranded DNA generally about 2000 to 5000 base pairs long. The small
DNA fragment is then ligated into the vector DNA or the plasmid.
13
The remarkable property of plasmids is that the bacterial cells do not differentiate them
as foreign for purposes of replication. Moreover, the plasmids multiply within the host
cells in the absence of cellular multiplication. The vector DNA is then inserted into
bacterial cells (transformation).
The bacterial cells are then spread onto agar plates
where, provided they were successful transformed with an inserted vector, they propagate
into colonies of thousands of identical cells, each containing an exact copy of the inserted
DNA.
These colonies are then detected through standard imaging techniques and
transferred into sample plates via automated picking machines for purification and
sequencing. This is a labor intensive and time-consuming process, but it is necessary to
separate and amplify the DNA being interrogated into unique clones for sequencing.
EIZ~
Chromosomal DNA
Transformation
Insert Target DNA
Plasmid
Plasmids Multiply
Host Cells Multiply
Figure 1.2 Recombinant DNA Technology
Sample Purification and Sequence Set-Up
14
Host Cell
Colonies Grow
Once isolated, the bacteria are subjected to a series of purification steps to isolate and
amplify the inserted DNA of interest followed by a Sanger sequencing reaction to
generate detectable product. The amplification takes using TempliPhi 10000 (Amersham
Biosciences), which makes use of the Rolling Circle Amplification technique discussed
in detail in Chapter 1II. Currently, the whole process is accomplished as illustrated in
Figure 1.3, with samples migrating through a heavily automated process in 384 well
plates and undergoing pL volume reactions.
Inoctdat Ion
Transfer &lycerol
and Add Denaturing
Buffer
DenaT ure
Denature at
95C for
5min.
The rmocycling
S eq. Set -Up
Add TPh i
Add Tphi
from Ch iled
Resevoir
ETOH Precp.
&4WE~
4W
4Wli
4W
4W
TrnfeWo
Transfer
to
& Add
See .
Mi
Plates
960"C
5c5
oydes
'C40
Add Ethanol
and
Centrifuge
A mplify/
Heat Kill
bIut in
Amplify at
3
I 6hr
6*C/]yn
D Iuto with
3rUL wator
EDTA Elut ion
4W
4W
Add EDTA
bete Cion
W
4Wk
Load on ABI
3730
Detector
Figure 1.3 Complete Sequencing Process at the Broad Institute
The current process steps are:
1.
.5ptL of the isolated bacterial culture from one well of a 384 well glycerol plate
(200pL max. volume) is combined with 2pL of denaturing buffer in a new 384
well conical bottom plate (35 ptL max. volume).
2. The plate is then sealed and run through a conveyor oven which raises the sample
temperature to 90'C for 3 minutes.
3. The samples are then cooled to room temperature, the seals are removed and 3piL
of TempliPhi© at 2'C is added.
15
4. The plates are then resealed and placed in a chilling incubator which heats the
samples to 30'C for 16 hours then raises the temperature to 65'C for 1 minute
before cooling the samples to 10'C, which they remain at until removed and
placed in a refrigerator to await sequence set-up.
5. The plates are then diluted in a two-step process the first step of which adds
14.5RL of water, mixes, aspirates 15RL and deposits that to waste. The second
step of the dilution adds another 15pL of water to the remaining 5pL of dilute
sample and mixes.
6.
1.3pL of the dilute sample then is transferred into two wells of a new 384 conical
bottom plate (35ptL max. volume).
.75ptL sequencing mix consisting of Big
DyeC reaction mix and primers specific to either the 'forward' or 'reverse' site of
the vector then gets added to each of these wells.
7. These new plates are then sealed and placed on thermocyclers where they undergo
25 cycles of 96'C for 20 seconds to 50'C for 15 seconds to 60"C for 4 minutes.
Once the cycling is complete the plates are stored in a refrigerator.
8. The seals are removed and 14 tL of Ethanol is added to each well. The plates are
resealed and centrifuged at 2000 rpms for 2 minutes. The ethanol is then removed
by inverting and tapping the plates. The plates are then stored in a warm room.
9. The plates have 10pL of EDTA added to each well, are spun, sealed, and loaded
on a 3730 for detection.
16
DNA Amplification
Prior to the Sequencing Reaction, it is necessary to amplify the DNA of interest. This
amplification serves two purposes:
1.
When isolating the DNA from bacterial cells, selectively amplifying the DNA
allows it to become the overwhelming component of the reaction mixture thereby
eliminating the need to purify out any bacterial genomic DNA, proteins, or other
agents that may interfere with the sequencing reaction later on.
2. Amplification of the DNA of interest allows for a uniform amount of input into
the sequencing reaction. The amplification step is achieved through the use of
TempliPhiC which utilizes the Phi29 enzyme for rolling circle amplification
(Figure 7), explained in much greater detail in Chapter III.
Figure 1.4 Rolling Circle Amplification
One thing to take note of with regards to the amplification reaction is the dilution step.
This is essentially a 1: 615 dilution of the amplified product prior to input into the
sequencing reaction. Simply reducing the volumes associated with this reaction should
make it possible to eliminate this dilution step all together, a feasible adaptation for
microfluidic devices.
Prior reagent minimization efforts have been conducted by S. Fisher et al. [1], which
reduce the volume of TempliPhi reagent used down to 100nL while keeping the overall
reaction volume and template ratio the same with promising results. As reflected in
17
Figure 1.5, acceptable quality sequence can be obtained from template generated from
only 400 nL of TempliPhi without making any other adjustments. This shows the large
dynamic range of the reaction and suggests that further optimization is achievable by
lowering the overall reaction volume. It is interesting to see how this volume actually
scales down under real microfluidic experimentation in Chapter III.
50
45
40
35
30
20
15
10
5
0
0
CP
(0
C')
U')
2
rto
0
r0
a)
0a
C6
CC)
a)
-
a)
CO0
19
03
.-.
..
CD
0-
CO
LO
Volume TPhi (nL)
Figure 1.5 Tphi Volume Reduction
Sequencing Reaction
The scope of this proposal does not include an alteration of the instrumentation currently
utilized for detection. Consequently, efforts were carried out to work backwards through
18
LO
CV
0
CO
a
the process using the minimum input material required by the ABI 3730xl sequencer to
determine the extent to which the process can be scaled down.
It has been shown by J. Meldrim[2] that the current process yields sequencing product in
excess of what is required for detection. The data generated shows that a significant
quantity of acceptable quality sequence (Phred 20 standard) can be obtained using the
product generated by the current 2pL sequencing reaction dilute down to only 2% of its
original concentration (Figure 1.6).
Theoretically, this would mean that a 40nL
sequencing reaction would yield sufficient product for detection. This possibility is
systematically explored in Chapter IV.
800
700600500
-
__-_
__--__
-_
I
CD
-o 400-
S300200100r-
I u-I
c-4 o
C)
C0
000
0
0
0
F Lo
0
LO
-
6
C:)
Dilution
Figure 1.6 Phred 20 for dilution series performed on a single sample
This analysis is not that straight forward; however, for one thing, the data also showed a
significant decline in the detection intensity for the dilute samples (Figure 1.6). This
means the reliability of generating consistent reads would be challenged by the optical
sensitivity of the detectors. This may be due to the sample loading interface to the
detectors, which currently requires a minimum elution volume for electrokinetic
19
injection. Effectively injecting the largest amount of sample possible, yielding the
greatest intensity, would most likely require a modification to the injection interface to
reduce the necessary injection volume. Also, reaction kinetics cannot be overlooked
when dealing with reactions at this volume.
The Dispensing Problem
The scale down as shown in Figures 1.5, 1.6 is possible only with specialized dispensing
devices. However, with the current dispensing machines in use at the operating stations at
the Broad Institute, the Equator and Multimek, it is not possible to go down to the
nanoliter scale and utilize the complete dynamic range of the reactions. Hence, even after
knowing that the different reactions involved in DNA sequencing can be scaled down to
the nanoliter scale, it is not possible with the existing dispensing instrumentation to
utilize this potential advantage. A methodology for nanoliter dispensing is required
which, as described in Chapter II, can be easily afforded in a microfluidic device.
The Possibilities
Denature
)ilution
Volume uL
/olume uL
0.5
0.63%
2
2.50%
Tphi
3
3.75%
Water
74.5
93.13%
Template
0.5 20.00%
Denaturing Buffer 2
80.00%
).5
Big Dye*
Big Dye Buffer*
H20 for Big Dye*
Primer*
otal Volume
80
2.5
*Based on 1/64th BigDye concentration
Table 1.1 Preliminary Scale-down possibilities
20
Table 1.1 shows an initial estimate of the possibilities afforded by switching to a
microfluidic platform. These calculations are basically linear and are roughly based on an
elementary study of how the reads scale down when the amount of sequencing product is
reduced in a 384 well plate reaction. Beginning with the proposed sequencing reaction
volume of 40nL (2% of the current volume) and extrapolating out the required volumes
for the rest of the process keeping the ratios the same and eliminating the dilution, the
new 'microfluidic' process would consist of the volumes reflected in Table 1.1.
If
successful this would equate to a 98% reduction in Big Dye volume and a 99.94%
reduction in TempliPhi volume.
Picking Colonies
Amplification and Heat Kill
Denaturation
TempliPhi Addition
Big Dye Addition
Thermocycling
Figure 1.7 Operating stations for various processes with robotic automation
Hence, resorting to a microfluidic platform has great potential to cut down reagent costs.
Furthermore, the whole sequence of operation can be greatly simplified with great
advantages in labor efficiency. As shown in Figure 1.7, the current process takes place
sequentially at several robotic stations requiring manual labor at several places. With
microfluidics it is possible to being all these steps potentially to a single point of
operation. This will greatly simplify process controls.
21
References
[1] Fisher, S., et al., Experiment conducted by the Technology & Development group of
the Broad Institute, 2003.
[2] Meldrim, J., Limit of Detection, Technology & Development at the Broad Institute
Presentation, 2004
22
Chapter 2
Development of Basic Microfluidics
Preliminary Device Design
The PDMS
microfluidic
lithography. [Appendix I].
devices
were
fabricated through the technique
of soft
In this chapter, we focus on the design of microfluidic
circuitry in terms of the geometry of the flow channels and also on the protocols for some
basic operations required to manipulate fluidic reagents on the nanoliter scale. The layout
of devices is designed in Adobe Illustrator.
These devices are dual-layer devices having a flow layer for fluid flow and a control
layer incorporating valves for controlling the flow. The fabrication and control using
Labview of these devices is explained in Appendices I and II.
The first design as shown in Figure 2.1 was an ambitious one. This design aims to
accomplish all the three prospective stages of sample preparation including amplification,
sequencing reaction and purification within a single reaction chamber, which happens to
23
be the peristaltic rotary mixer. Moreover, it was proposed that the accurate metering of all
the reagents involved would also be accomplished in the same design.
-+
Sample Volume at
Prior to injection = 50 nI.
> Total
Cross Injection for metering definite
amount of Template, Templi-phi and
Big Dye, Primer.
-- >
Dilution water forces these
-+
Template : 0.4 ni, Templi-phi: 2.5 nI
Big Dye: 19 nl , Water: 28 ni
fixed amount of reagents in the
rotary mixer.
--
i12
Beads
Valves
+ Alcohol
Template
Templi-phi
Waste out
First Dilution
Force Template +* Templi-phi
Amplified
Template
4F
--
Big Dye, Primer
I
1
Figure 2.1 Microfluidic Layout for Design
1
24
Second Dilution
Force Big Dye, Primer
Peristaltic Pump
Figure 2.1 makes the proposition quite clear. To the left side of the design, we would
have the template (0.4 nl) and templi-phi (2.5 nl) accurately metered. They would then be
forced by dilution water into the bottom left portion of the rotary mixer as shown in
Figure 2.1. The reactants would be securely 'valved-off from both sides and would
occupy merely 1/4 h of the 50 nI reaction chamber.
The mixing takes place by diffusion. Taking the diffusivity of average biomolecules in
water as parameter, it was found that 7 minutes would be taken for complete diffusion
mixing between the template and the T-phi. Considering that the amplification reaction
itself consumes 16 hours of time in the macro-scale reaction, the diffusion time scale is
really negligible to the reaction time scale.
After the amplification reaction is over, the requisite amount of big dye (19 nl) is metered
in the bottom portion on the design (Figure 2.1) and forced in into the remaining
3 /4 th
portion of the rotary mixer by dilution water (18.5 nl). The volumes of reactants can be
controlled using the cross-injection metering scheme. Figure 2. 3 illustrates the metering
process in detail. The accuracy of metering really depends on the accuracy of the whole
soft lithography process, which is discussed later in this thesis.
The two closed valves on both sides now only separate the adjacent volumes of amplified
template and diluted Big Dye. Both these valves also form two of the three valves
constituting the peristaltic pump. Three valves occurring in series on a channel when
activated in a suitable pattern constitute a pump. Now, pumping is turned on by the
mechanism as outlined by Unger et. al.[l]. Peristalsis is typically actuated by the pattern
101, 100, 110, 010, 011, 001, where 0 and 1 indicate "valve open" and "valve closed,"
respectively. The Labview control required for this operation is systematically discussed
in Appendix 2.
The mixing would take quite bit of time here. The radius of the circular mixer here is 4
mm and peristaltic pumping at the operating valve pressure of 10-15 psi is barely
25
sufficient to support mixing in such a large volume. This deficiency is improved upon in
the later designs.
The microfluidic device is subsequently placed on a flat-plate thermocycler for the
sequencing reaction to take place. After these proposed sequence of steps, the DNA
sample still remains to undergo purification in order to remove the excess ddNTPs from
the Big Dye mix. Another inlet purges out 1/5th of the sequence reaction products, as
shown in Figure 2.1, and replaces with magnetic DNA affinity beads. The circular
chamber is again sealed and mixing done.
Subsequently, the DNA is sticking on to the beads. The chip is then placed onto a
magnetic 96 well plate counterpart leading to the beads being immobilized in-situ. Water
is flushed through the circular chamber cleaning away the impurities. The chamber is
sealed again and then 1/5th of the products flushed out by an appropriate concentration of
alcohol. The alcohol displaces the DNA strands from the magnetic beads. The sequencing
reaction products can be taken out of the chip and electrokinetically injected into the
AB3730 detectors.
Microfluidic Metering and Pumping
This first design as proposed was quite ambitious. It was desirable to the break up the
individual DNA reactions involved and study them individually. But before that, the first
practical stage of the project was to test the microfluidic devices for basic operations such
as valve opening/closing, microfluidic metering and pumping.
The first endeavor in this regard is depicted in Figure 2.2.
26
Figure 2.2 Microfluidic Device based on Design 1
The device shown above in Figure 2.2 was designed to meter the requisite amounts of
templi-phi and the DNA template for amplification. This is the same design as proposed
in Figure 2.1. Figure 2.2 is indeed Figure 2.1 rotated counterclockwise by 90 degrees!
The channel width here is 100 ptm and the height is 10 pim. The reactants metered in the
metering region (as in Figure 2.1) would be forced into the circular peristaltic mixer for
mixing followed by amplification. The metering region is zoomed-in and shown in
Figure 2.3.
The valves 1,2,3,4 were used to meter in the template. The volume 1-4 as shown in the
figure accommodates approximately 0.4 nI of Template. The valves 4,5,6,7 were used to
meter templi-phi. The volume 4-7 as shown accommodates about 2.5 nl of TempliPhi.
27
Figure 2.3 Metering Region in Design
1
The aforementioned volumes were not arrived at arbitrarily. As analyzed by Joe Graham
(Broad Institute) in Table 1.1, on a microfluidic platform it is indeed unnecessary to first
do a highly concentrated DNA T-phi reaction and then take a miniscule portion of it
discarding the rest. In the 384 well plate reaction, this strategy becomes necessary
because the amplification reaction does not proceed well until there is a minimal
concentration of reactants. It was shown in Table 1.1 that discarding the 2 stage dilutions,
we can afford to manage with only 8 nl of template and about 50 nl of TempliPhi.
28
Figure 2.4. Cross-Injection across valves 5,6 bounded by valves 4,7
Further, it was observed that such a reaction still produces excess sequencing product.
An analysis by J. Meldrim[2] showed that only 2% of the sequencing product may be
enough to produce decent reads on the 3730 detector. We decided run the reaction with
5% of the volumes suggested in the scale-down of Table 1. Thus the volumes of
Template and Templi-phi required scale down to 0.4 nl and 2.4 nl respectively. This
linear scale-down is a heavy approximation and suitable experimentation is needed to
find out if this is applicable.
Here the methodology of cross-injection is defined. Lets take the case of valves 4-7 for
metering Templi-phi shown in green in Figure 2.4. Template, as shown in orange, has
already been metered. Valves 4,7 should be closed prior to the operation. Now, valves 5,6
are open and Templi-phi is forced via valve 5 through valve 6.
29
-
iiiimi
''I
I I I
m.iiii...
11111111
111
I Iii
-.
-
11T
- -
- - -
-
-
Peristaltic
Pump
Variable Metering
Region
Figure 2.5 Microfluidic Layout for Design 2.
30
This situation is exactly shown in Figure 2.4. As soon as the fluid comes out of the outlet
(this operation can be suitably 'timed' via Labview controls), valve 6 is closed. Valve 5 is
still open so that fluid completely fills the recesses of the channel and forces out the air
through the ends of the valved-off regions. After it is observed through the microscope
that there are no more vacant spaces in the channel, valve 5 can be closed off as well.
Figure 2.6 Microfluidic Device based on Design 2 - Metering valves are numbered
Now valve 4 separating Template and Templi-phi is opened and the mixture is forced
into the circular chamber through dilution water. This amplification mix occupies 1/ 5 th of
the circular chamber.
It was noted, however, that such a design as in Figures 2.1-2.4 does not allow one to vary
the volume of Templi-phi between successive experiments. To see the variation of
31
amplified product with the amount of Templi-phi used, a new chip would have to be
designed with a different channel length between valves 4,7. It was quite easy, however,
to vary the amount of input DNA template in the reaction since input DNA is available in
a variety of concentrations. But Templi-phi comes in a standard concentration and so we
decided to design a chip wherein variable volumes of Templi-phi could be metered.
The corresponding graphic design 2 is shown in Figure 2.5 and Figure 2.6. Here it is
possible to meter variable volumes of Templi-phi in increments of 0.5 nl. The smallest
volume available for Templi-phi metering is 0.5 nl between valves 3,4 with inlet and
outlet bounded by valves 1,2 respectively. The next larger volume is 1 nl between valves
3,5 followed by a volume of 1.5 nl between valves 3,6 and so on.
Figure 2.7 Metering accurate amount of Templi-phi
32
Once Templi-phi has been metered as needed, it can be forced into the rotary mixer
chamber by pushing the diluted Template into the channel. This operation is illustrated in
Figure 2.7 and Figure 2.8. In figure 2.7, 1.5 nl of Templi-phi, in green, has been metered
between valves 3 and 6. In Figure 2.8, Templi-phi is forced ahead into the rotary mixer
by the appropriately diluted template, in yellow, into the rotary mixer. The templi-phi,
template mix has been forced to fill only a portion of the rotary mixer in Figure 2.8. This
is done just as a demonstration of the valve and metering controls. In a practical operating
situation the mixture will fill the complete rotary mixture. The template will have to be
appropriately diluted so that the required amount of template DNA is available for
amplification reaction.
Figure 2.8 Metered templi-phi forced into the rotary mixer by the template
Another feature of this design is that the size of the rotary mixer is significantly reduced
as compared to the design 1. Here the total volume within the annular chamber is 10 nl as
33
o;.. - - -_--- ------
-
-
_-I-----
_
opposed to 50 nl earlier on. The radius of the annular chamber is 0.8 mm as compared to
4 mm in design 1. The width and height of the channels is 100 ptm and 20 pim
respectively. The lesser diameter of the annular chamber allows much faster peristaltic
pumping.
As the valving and cross-injection aspects of microfluidics had already been investigated
in design 1, the primary application of design 2 was to test peristaltic pumping as
demonstrated by Unger et. al.[1].
Complete mixing of the reactants in this design is
completed in less than a minute. Figure 2.9 shows the scenario after the mixing of three
colored dyes - orange, yellow and green.
Figure 2.9 Dye color at the conclusion of mixing
34
References
[1] Unger MA, Chou HP, Thorsen T, Scherer A, Quake SR, "Monolithic Microfabricated
Valves and Pumps by Multilayer Soft Lithography", Science 288: 113-116 (2000)
[2] Meldrim, J., Limit of Detection, Technology & Development at the Broad Institute
Presentation, 2004
35
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36
Chapter 3
Amplification
In order to systematically work towards the integrated project goals the final task was
divided into individually manageable sub-tasks. As Templi-phi amplification was the first
step in the sample preparation protocols, initial efforts focused on the development of a
microfluidic chip to automate the amplification process. Chip design 2 was used as the
foundation for this work.
Amplification Mechanism of TempliPhi
The amplification technique used at the Broad Institute is called the Rolling Circle
Amplification (RCA). This differs from the conventional Polymerase Chain Reaction
(PCR) amplification method, which cycles through a temperature range for each base
extension, in that it is carried out isothermally (300 C). This greatly simplifies the
packaging in a microfluidic environment as thermocycling is no longer required.
37
RCA eliminates the requirement for extended bacterial growth prior to sequencing and
saves laboratory personnel hands-on time by eliminating the centrifugation and transfer
steps required by older preparatory methods. Additionally, costly purification filters and
columns are not necessary, as amplified product can be added directly to a sequencing
reaction. Starting material can be any circular template from a colony, culture, glycerol
stock or plaque.
TempliPhi 10,000
TempliPhi 100/500
4
2.5
C
2
y..0.5
y..
0.
0
0
4
8
12
0
16
4
8
12
16
20
24
Time (hours)
Time (hours)
Figure 3.1 Templi-phi kinetics for the Standard Manufacturer kits
Before RCA, the conventional method for sequencing template production was bacterial
culture - a labor intensive multi-step procedure that takes up to 24 h and produces
templates of varying quality and quantity [1]. This deficiency is overcome in RCA. The
biggest advantage of RCA is really that the amplification product can be directly
sequenced, eliminating the need for costly commercial purification columns and plates.
Templi-phi is available generally in two varieties, TempliPhi 500 and TempliPhi 10000 both have been used for development of on-chip RCA in this project. TempliPhi 500
provides the various components of the reagent in separate bottles. This makes it easier to
change the proportions of components like primers, enzyme and ddNTPs in the reaction,
which, in turn allows the flexibility of finding out the optimal reactant proportions on the
38
nanoliter scale. TempliPhi 10000 kit comes with all the components mixed together in
proportions presumably optimized on the microliter scale.
As Figure 3.1 from the manufacturer's catalogues demonstrates, both of the product kits
(Templi 500 and Templi-phi 10000) can achieve sufficient amplification within 4-6
hours. However, at the Broad Institute, the amplification routinely is done using only the
TempliPhi 10000 kit and the process is designed to be completed within 16 hours. The
idea in this section will be to find out how this kinetics scales down when the reaction is
performed at nanoliter scale in the microfluidic environment.
Rolling circle amplification (RCA) is a technique used to amplify circularized DNA with
a sequence specific primer and a strand displacing DNA polymerase, producing a
repeating linear construct. The polymerase, TempliPhi from the bacteriophage Phi29,
processively creates a copy of the circular plasmid DNA template utilizing random
hexamer primers. RCA was discovered owing to the observation that replicating phage
DNA in infected bacteria was larger than in phage particles because of the formation of
concatamers of genomic phage DNA. Since phage DNA is circular, tandem repeats are
produced by rolling circle replication (RCR), in which the phage DNA polymerase
replicates and subsequently displaces the newly made strand.
The amplification mechanism is shown in detail in the following figures.
39
The reaction begins with denatured single stranded circular DNA
Phi29 DNA polymerase binds
the primers
Polymerase replication
Polymerase replication
C)
Most DNA polymerases will
stop here, unable to displace
the newly replicated downstream I
40
Phi29 DNA polymerase is unique,
it begins strand displacement with no loss of replication speed at all
Strand displacement
The newly displa ced strands are single stranded.
There is no need for thermal cycling to create
single stranded DNA (as with PCR), and this
new DNA is a tem plate for replication
41
More random primers
bind the new displaced strands
and ar
xtended by polymera se
Eventually all of the nucleotides are depleted and the reaction stops. By this time the
enzyme will have made 4-5 pg of double stranded, tandemly repeated copies of the circle.
This DNA does not need to purified,
and can be added directly to a sequencing reaction
Figure 3.2 Mechanism of Rolling Circle Amplification
Random hexamers bind to the denatured circular template allowing Phi29 DNA
polymerase to initiate multiple amplification events (Figure 3.2). The inherent stranddisplacement activity of the enzyme displaces the 5'-ends of downstream strands. As
DNA
synthesis and displacement continue, the enzyme produces single-stranded,
complementary
concatamers
of the circular template (Figure 3.2).
Priming and
polymerization directed by the displaced strands produces double-stranded DNA.
Nucleotides in the TempliPhi premix fuel the reaction to produce as much as 5 pig of
DNA in the pl scale reaction in a 384 well plate. Amersham Biosciences catalogues claim
that this is possible from as little as 1 pg of template in 4 hours. In this chapter, we
investigate if such a limit is practically achievable on a microfluidic platform and also if
42
this can be exceeded and DNA amplification completed from much lower starting
template amounts.
The rate of the strand synthesis by Phi29 DNA polymerase is claimed to be
approximately 50 nucleotides/second, due in part to the enzyme's high processivity.
Phi29 DNA polymerase is able to incorporate greater than 70,000 nucleotides during a
single binding event without the aid of accessory proteins.
The microfluidic experiments outlined in this thesis are designed to test the lower limits
of input template that can be used as claimed by Amersham Biosciences. A parametric
study was carried out in the microdevices, varying input template and Templi-Phi
polymerase concentrations as well as the total reaction time. In the process of protocol
development, the microlfuidic device underwent several design modifications to control
issues like sample evaporations and biofouling of the microchannel walls.
Fluorescence Measurement Methodology
The first issue towards the above mentioned goals was to find out an effective way of
quantifying the on-chip Templi-phi reaction. An early option investigated was to take the
amplified DNA out of the chip and determine the extent of amplification by gelelectrophoresis. However, the quantity produced on chip was below the threshold of
standard quantification by gel-electrophoresis.
Preliminary calculations from Table 1.1 indicated that 0.6 ng of amplified DNA product
may be required to feed into the subsequent DNA sequencing reaction. Off-chip
quantification of the amplification product, even if feasible, introduces the possilibty of
sample loss or contamination that compromises the sequencing reaction. To realize the
true utility of a microfluidic platform, it is desirable to perform as many operations as
43
possible on the chip itself. Hence, the idea of using DNA quantification fluorescent dyes
was employed. There were several candidates like Pico Green, SYBR Green, Oligreen,
Ethidium Bromide, Hoechst 33258 etc. Since 80% of the product generated by RCA is
double-stranded, single-stranded detection dyes like Oligreen cannot be used. The dye
should really be dsDNA specific and ideally show no fluorescent enhancement in the
presence of ssDNA. Further, we need sensitivity of detection to small quantities of DNA
at the scale of -1
ng since we need to calibrate the extent of DNA amplification with
respect to our input parameters (input DNA
template Templi-Phi concentrations,
amplification time).
It was found that PicoGreen was best suited to the application at hand. PicoGreen is an
intercalating
agent, and
is inserted between the individual
DNA
strands.
The
concentration of intercalated PicoGreen is linear with respect to the number of base pairs
of DNA, and the fluorescence emission of the dye can be used to obtain quantitative
measurements of DNA concentration. The fluorescent enhancement of this dye is >1000
fold upon binding to dsDNA with excitation around 495 nm and emission around 520
nm.
Unlike Hoechst 33258 and some other dues, PicoGreen fluorescence intensity is the same
upon binding to poly(dA).poly(dT) and poly(dG).poly(dC)
important for providing a dependable measure of DNA
homopolymers. This is
concentration. The linear
concentration range for DNA quantitation extends over four orders of magnitude - 25
pg/ml to 1 ptg/ml - with a single dye concentration [2].
However, in our case, it is expected that the post-amplification concentrations in the
microfluidic chamber might be of the order of 100 ptg/ml. This would not only lie outside
the linear range of PicoGreen but also out of the routinely tested range of most of the
dyes. It was interesting to find out whether PicoGreen would provide a sufficient
variation to set up a calibration curve in this high-end range. A non-linear calibration
44
curve was indeed found out and it provided good variation over the pertinent range of
quantitation.
Firstly, a set of titration experiments was performed to calibrate fluorescence emission in
the microfluidic channels. Certain issues had to be handled well here. There is always
some variability in the microfabrication procedure varying the height of microfluidic
channels in spite of using the same exposure mask in photolithography. To ensure that
the set of channels used for calibration and the set of channels used for successive real
experiments had the same height dimensions, a profilometer was used to measure
channel-channel
variations between different devices. Final devices selected for
experimentation has height variations of less than one micron vs. an average channel
height of 20 microns.
The fluorescent images were captured from a fluorescence microscope (Nikon TE -2000,
20X Objective). Figure 3.3 shows a snapshot of 10 nM concentration of DNA in the
channel. The fluorescent values were obtained using ImageQuant software. It should be
noted here that the fluorescence observed depends not only on the channel depth but also
the exposure settings of the microscope. For Fig.3.3, the settings were Gain = 16 and
Capture Time = 31 ims. It was necessary to adopt the same exposure settings in real
experimentation as in calibration.
In order to increase the amount of final product obtained, the height of the channels in
design 2 was increased to 20 [tm (from 10 ptm in Design 1). This corresponds to a volume
of 10 nl in the peristaltic mixer as mentioned earlier. A final amplified product of 1 ng
corresponds to 55 nM of DNA in a 10 n1 reaction. Hence we would expect much brighter
fluorescence compared to Figure 3.3, which is only 5 nM, if the Templi-phi reaction
proceeds successfully.
45
Figure 3.3 PicoGreen Fluorescence image for IOnM of pUC19 DNA in the microchannel.
It was subsequently decided that PicoGreen would not be used as a part of the Templi-phi
reaction mix as the dye intercalates between the grooves of the DNA and hence might
hamper the activity of the Phi 29 DNA polymerase. Hence, PicoGreen was added only
after the amplification reaction was over.
Referring to Figure 3.4, once the reaction was completed, valves 2,3 were opened while
keeping valves 1,4 closed. PicoGreen was forced through valves 1,4 flushing some of the
amplification product outside the reaction chamber. The region 1-4 however comprises
only 1/7th of the reaction volume and the remaining 6 /7th of the reaction product is still
available to mix with PicoGreen reagent. Valves 1,4 are now closed and valves 2,3
opened with peristaltic mixing turned on.
46
It should be noted that valves 1,4 serve the dual purpose of acting as gates to allow only
a specific amount of amplified product to be replaced with PicoGreen and also as two of
the three valves required for peristaltic pumping.
Commercially available 1-X PicoGreen reagent is very concentrated and is generally
diluted before using for DNA quantitation. Here 1/10-X PcioGreen is used. This strength
is arrived at by linearly scaling the amount of dye required to detect DNA on the order of
5 ng.
I
I
M
PicoGreen Out
13
I1
2
-r
t
PicoGreen In
Figure 3.4 Valves 1,4 act as gates to allow only a specific amount of amplified product to be replaced with
PicoGreen reagent. They are also 2 of the 3 valves required for peristaltic pumping!
47
Amplification Trials
Appendix III shows all amplification reaction trials in tabular form.
The first trials of Templi-phi reaction on chip were performed using design 2 (Figure 2.5)
which had a reaction volume of 10 nl. Relying on preliminary scale-down calculations
from Table 1.1 and some guess work, the first reaction mix tried was 2 nl 10000 Templiphi + 8 nl 1 nM template with a reaction time of 30 min.
The amount of Templi-phi was scaled down by three orders of magnitude from the 3 pl
used in the 384 well plate reactions. According to a linear scaling analysis, the final
amplified product was predicted to be 1-2 ng down by three orders of magnitude from the
2-2.5 ptg produced in the 384 well plate reactions.
The time required for reaction was also scaled down from 4-16 hours to 30 min. It was
argued that the time needed for the reaction to finish was really the time for the enzyme
to incorporate all the nucleotides. Since the amount of nucleotides and template has been
scaled down by three orders of magnitude, the time should also scale down drastically.
Accordingly the time was scaled down by one order of magnitude, this being an initial
guess only. A lesser reaction time could have been assumed but a conservative estimate
was preferred in the initial set of experiments. The template used was pUC 19, which is
generally provided as a control in Templi-phi reaction kits.
Thirty minutes after the reactants were mixed on-chip, the product was tested within
PicoGreen in the way outlined in the preceding section. However, no change in
fluorescence was observed, suggesting that the reaction was not taking place.
The background fluorescence measurement for comparison was taken by mixing the
reactants with PicoGreen on chip before the onset of the proposed reaction. In this way,
48
any effects of background fluorescence and also of the initially present double stranded
template were accounted for.
Subsequently, the possible failure scenarios were examined. The amount of starting
template used was 36 pg and it could be argued that it was too low for any significant
amplification to take place. However, in the catalogues of Amersham Biosciences for
TempliPhi, it was reported that robust amplification was observed with as low as 1 pg of
starting plasmid DNA [3].
Another pertinent idea was that the binding of hexamers, present in TempliPhi mix, with
the template is thermodynamically more favorable if the template is single stranded rather
than double stranded. The template is indeed denatured in the 384 well plate reactions to
allow the vector DNA to escape form the E. Coli host cells without lysing the cells,
producing single stranded DNA in the process. Hence, it was decided to denature the
template off-chip before proceeding with the on-chip reaction.
Further, a careful revision of enzyme kinetics tells us that the time required for reaction to
complete may not scale down as drastically as previously expected.
dS
-- ccS..
.............................................................
dt
AS
A t 0c --
S
........................................................................
(1)
(2 )
where S is the concentration of the substrate at any time. The above relation indicates that
the time taken depends on the fractional change in concentration of the substrate
concentration. Accordingly, even when the amount of substrate and enzyme is scaled
down tremendously, the reaction time may not decrease at all since the fractional change
in concentration of the substrate required to complete a portion of the reaction still
remains the same in the scaled-down reaction as it was in the original reaction. Keeping
49
this analysis in mind, it was also decided to run the experiments for longer periods of
time identical to the 384 well plate reactions.
Hence, several reactions were tried where the template was denatured prior to the
reaction and with times varying from 2 hours to 16 hours. However, as the reaction time
went into hours from minutes, it was observed that the reaction mix evaporated almost
completely even at room temperature. In spite of the valves remaining closed throughout
the reaction, the reaction mix evaporated invariably within an hour from the onset of the
reaction.
It was recognized then that PDMS is gas-permeable and so it easily allows water vapor to
escape through the pores in the polymer matrix. Several procedures have been used by
researchers to address this issue. The channels can be coated with suitable chemicals to
inhibit evaporation. However, here one has to careful in choosing the chemical regarding
its affinity with DNA and enzymes. A three-layer PDMS device can be constructed
instead of a two-layer device wherein the topmost layer has a network of water jackets.
Using water jackets above the reaction channels provides a saturated environment in the
polymer pores and so evaporation is curbed.
However, in the present device it is much easier to incorporate these water jackets in the
control layer, which lies about 20 pm above the flow layer. Figure 3.5 shows the water
jackets and other control layer channels in red around the black reaction chamber. With
the use of these water jackets, evaporation was curbed for reaction times up to 4 hours
50
Water Jackets to counter Evaporation
Figure 3.5. Spiral Water Jackets around the Circular Reaction Chamber to provide a Saturated Environment
With the modified design as shown in Figure 3.5, longer reactions were performed.
Appendix III shows all the amplification reaction trials in tabular form. Still only 2-3 fold
amplification was observed.
Further troubleshooting led to the hypothesis that the enzyme availability was limited in
the microfluidic devices due to partitioning of enzyme into the PDMS or adhesion to the
PDMS microchannel walls. To check how much the PDMS surface can impede the action
of the enzyme, several reactions were performed in the 384 well plate format with some
small pieces of PDMS submerged in the reaction mix. However, there was no
considerable decrease in the amount of amplified product and subsequent generated
sequence.
In spite of negligible effect of incorporating PDMS pieces into the 384 well plate
reaction, nothing can be inferred about the corresponding behavior at the micro-scale as
the surface area to volume ratio increases tremendously in microfluidic devices. Hence,
the possibility of the enzyme being the limiting species in the reaction had to be explored.
51
One possibility could be that the amount of enzyme scaled down by three orders of
magnitude for the micro-scale reaction was just not significant enough to catalyze the
reaction. Another more feasible scenario was that the PDMS channel surfaces may lead
to biofouling and hence the leave very little enzyme available in solution to participate in
the reaction.
Hence, a decision was made to use TempliPhi 500 kit instead of the TempliPhi 10000 kit.
This was because the TempliPhi 500 kit provides the enzyme, dNPTs and the
hexamers/reaction buffer separately as opposed to the TempliPhi 10000 kit, which
bunches the three together in unknown concentrations. Hence, we would have the
flexibility to play with the amounts the enzyme and other constituents.
In the next set of reactions, the Phi-29 enzyme was used in excess as compared to the
manufacturer's specifications. Also, the template was denatured off-chip and preannealed with the reaction buffer mixture containing the random hexamers. However, no
amplification was observed.
52
Number of Spiral Water Jackets is
Number of Spiral Water Jackets is
increased to enable longer Reactions.
Metering is no longer performed on-chip.
Figure 3.6 Microfluidic Layout around the Circular Mixer for Design 3.
As the amplification protocols failed on the chip in spite of the troubleshooting efforts,
one final hypothesis was explored. Given the complexity of the amplification process,
utilizing multiple substrates with reaction kinetics that are not first order, perhaps the
scalability of the reaction collapses as critically small reaction volumes owing to a
combination of the aforementioned problems (i.e. substrate or enzyme concentrations) or
subtle changes in the buffer composition due to evaporation or osmotic equilibration. To
test this hypothesis, we carried out a final set of experiments with scaled-up reaction
volumes on-chip.
The total reaction volume in Design 3 is 40 nl. The width of flow channels is 200 pm and
the height is 20 pm. Further the radius of the annular chamber is increased to 3.2 mm
from 1.6 mm in Design 2. Complete mixing is still observed to happen within 90 seconds
in this design. Further, the number of spiral jackets is greatly increased as compared to
53
Figure 3.5. These water jackets make possible reaction times up to 24 hours and longer
without any significant evaporation.
Figure 3.7 Successful amplification gives a strong fluorescence signal - Row 9, Appendix III
The reaction mix for this 40 nl reaction can be seen in row 9 in Appendix III. It
comprises of 24 nl TempliPhi 10000 and 15.5 nl TempliPhi 500 (6 nl dNTPs, 5.5 nl
reaction buffer and 4 nl enzyme). For the first time, a strong fluorescence signal was
obtained from the PicoGreen indicating a successful reaction. From a starting template
amount of 45 pg and a reaction time of 16 hours, a strong amplification of 80 fold was
observed leading to an amplified product of 3.5 ng. The corresponding fluorescence
image is shown in Figure 3.7.
The amplified product from the above reaction was purged off the chip, diluted to 1.3 g1
and mixed with 0.75 p1 of Big Dye mix to yield 2.05 pl of sequencing reaction mix. This
volume composition is the standard one used in 384 well plate reactions. The sample,
upon processing in the Broad Institute Sequencing line, yielded a read length of 300. The
54
result was very encouraging considering that a routine 384 well plate reaction gives an
average read length of 800.
Following this success, it was decided to work further on optimizing the reaction
parameters. The TempliPhi 10000 is more efficient that the 500 kit and so it made sense
to push the amount of TempliPhi 10000 mix versus the amount of TempliPhi 500 mix in
the reaction. Also, it was required to minimize the amount of enzyme used from the 500
kit since the enzyme is the most expensive component of the reaction mix.
Upon subsequent tests, it was observed that reducing the amount of dNTPs and the
random hexamers from the TempliPhi 500 kit did not significantly affect the outcome of
the reaction (reaction 10 from Appendix III). However, reducing the amount of enzyme
significantly affected the extent of completion of the amplification. It can be observed
from reactions 11,12 in Appendix III that when no extra enzyme from 500 kit is used, the
amplification drops to only 13 fold.
The results suggest that the enzyme is the limiting species in the reaction. With lower
surface-to-volume rations in Design 3, enzyme partitioning into the PDMS or adhesion to
the microchannel surface would be reduced. With lower partitioning, more available
TempliPhi enzyme would be present to drive the amplification reaction.
Experiments were also performed in order to observe the minimum amount of template,
which could undergo amplification and also yield a decent read length. Reactions 13-16
in Appendix III demonstrate that amplification is possible from as little as 112.g ag of
starting template consistently yielding 1.5-3 ng of amplified DNA. In other words,
1.6x10 7 fold amplification is possible from as little as 40 copies of pUC 19 vector.
However, reactions 15,16 failed to produce any read lengths from the sequencing line at
the Broad Institute. It can be inferred that some non-specific amplification is taking place
in reactions 15,16. It is not possible to infer the extent of non-specificity involved from
this data.
55
Reaction 14 produced an average of 2.5 ng amplified DNA from 45 fg of starting
template, equivalent to 16000 starting copies of pUC 19 producing 60000 fold
amplification. The amount of starting template used here for successful amplification is
lower than any documented limit of rolling circle amplification performed on any
platform. This is a significant result also because it also produced a 60,000 fold
amplification, exceeding
that achieved in the 384 well plate format at Broad.
Furthermore, reaction 14 yielded specific product showing an average of 900 reads on the
ABI 3730 detector. Some tests provided read lengths as high as 1000. This result is very
interesting, considering the average read length of 800 in the 384 well plate format.
Moreover, plugging diluted samples of this reaction product into the Broad Sequencing
line demonstrated that as little as 1 ng of amplification product could undergo a
successful sequencing reaction on the thermocycler leading to a good read length from
the ABI 3730 detectors. This result is also significant since the Broad Institute
sequencing line routinely uses ~50 ng of amplified DNA. In Chapter I, it was predicted
that only 400 nl of TemplPhi might be needed to produce a sequence of acceptable
quality. In the current series of experiments, it is actually demonstrated that robust
sequencing is indeed possible from as little as 1 ng of amplified DNA, which comes from
only 15 nl of TempliPhi (Figure 3.9) as opposed to 400 nl. Hence, this reduction is
TempliPhi volume as made possible by the current microfluidic platform is expectedly
sharp and promising.
56
Amplification Product Vs. Time
3
2.5
z
1.5
E
1
0.5
0
0
1
2
4
3
5
6
7
Time (h)
Figure 3.8 Templi-phi kinetics on the Microfluidic Chip
Reactions 16,18 from Appendix III show that the amplification can be completed in just
five hours. Reaction 20 shows that the reaction does not reach completion in three hours.
A series of experiments performed on cell culture as template investigated the minimum
reaction time as shown in Figure 3.8. It was indeed confirmed that five hours is the
optimal time for the TempliPhi reaction. This is still less that the 16 hours required in the
384 well plate format at the Broad.
A notable point here is that the amplification reaction becomes non-specific with starting
template amounts less than 45 fg. It can be inferred that non-specific amplification targets
become much more significant compared to specific pUC 19 targets when the amount of
template goes below 45 fg. Hence, in order to amplify on smaller copy numbers of pUC
19, we need to provide a reaction mix, which has much lesser impurities. Indeed,
TempliPhi manuals indicate that non-specific amplification is easily observed with very
low amounts of starting template [3].
57
Another interesting observation pertains to the variation of amplified DNA produced with
the amount of TempliPhi 10000 provided. It was also observed earlier in reactions 1-7
that negligible amplification is observed in a reaction volume of 10. However,
significantly high amplification is observed for a TempliPhi volume of 24-32 nl as in
reactions 9-22. It can be easily inferred that the variation of amplified product with
TempliPhi reagent is highly non-linear. This relationship is investigated and shown in
Figure 17. The amplification substantially increases after a volume of 10 nl of TempliPhi
10000 reagent is exceeded.
One possible reason for this non-linearity is that some biofoiling is happening inspite of
using BSA. Since the surface area to volume ratio is much high for a device with a
reaction volume 10 nL as compared to a device with a reaction volume of 40 nL, we can
expect that much more biomolecules would be leaving the solution for their affinity with
PDMS in a 10 nL volume than a 40 nL volume. Another mechanism of biofouling is
biomolecules actually partitioning into the PDMS structure. This possibility is explored
in detail in Chapter IV.
Amplified Product VS TempliPhi 10000
5
4.5
4
3.5
-
3
z
2.5
A?
S2
E
< 1.5
1
0.5
0
0
5
10
20
15
TempliPhi 10000 (n)
Figure 3.9 Plot of amplified product versus TempliPhi reagent used.
58
25
30
35
Tests on more concentrated TempliPhi reactions are difficult to do because the TempliPhi
manufacturers provide standard reagents and do not disclose the constituents of the
reaction mix. Reactions were tried with Phi 29 enzyme from different a source in varying
concentrations
and also with dNTPs from different a manufacturer
in varying
concentrations. However, as shown in reaction 17, dNTPs added from a different
manufacturer provide very low amplification probably because the TempliPhi reaction
mix also contains various salts and other constituents necessary for the rolling circle
amplification to proceed. Hence working on more concentrated TempliPhi reactions is
indeed a separate project in itself and was not pursued fully because of time constraints.
The amplification reaction was also tried starting with a portion of a colony from
overnight cell culture. Approximately
1 / 4 0 0 0 th
of a colony (-250 cells) went into the
amplification reaction on chip, equivalent to 350 fg of starting DNA template. Reaction
21 in Appendix III shows this reaction, which equivalently used around 50000 plasmid
copies. This reaction provided read lengths of 750 on an average upon passing through
the Broad Institute Sequencing line.
To prepare starting template for reaction 21, one colony from overnight cell culture was
taken in 5 [l of TE buffer. 3.5 [tl of this mixture was mixed with 3.5 tl of sample buffer
from TempliPhi 500 kit. This mixture containing the template as well as the random
hexamers was denatured in a three-minute cycle to allow hexamers to anneal with the
single stranded template. This annealed buffer mix was added to enzyme, dNPTs and
TempliPhi 10000 kit reagents and loaded onto the chip.
Upon discovering that the amplification reaction is working well on microfluidic
platform with both pUC 19 vector and cell culture as starting template, it was decided to
work on strategies for reducing enzyme consumption. Up to this point, extra enzyme
from 500 kit was being used. Reducing this amount would be very useful as the enzyme
is the most expensive component of the reagent kit.
59
To begin with, it was assumed that PDMS surface had an affinity for the Phi 29 enzyme
and so by incorporating a proper coating it was possible to greatly reduce enzyme
consumption. The first coating tried was BSA and it showed great results. Reaction 22
demonstrated that using 4-6 nI of BSA made it unnecessary to use any extra enzyme.
Using BSA, reaction 22 produced 4-4.5 ng of amplified DNA on an average, which was
the greatest amount of product attained in the present microfluidic platform. This was
probably because with no reagent from TempliPhi 500 kit being used, all TempliPhi
reagent came from the TempliPhi 10000 kit which is far more efficient as compared to
the 500 kit, as evident from the manufacturer's catalogues.
In conclusion, it was established in this section how amplification reaction can be adapted
to the microfluidic platform. Optimal operational conditions starting from both plasmids
and cells were confirmed and reported. It was shown that approximately 4 ng of
amplified DNA can be obtained on chip which is enough to produce quality reads on the
ABI 3730. This opens the path to bringing down the complete sequencing process to the
microfluidic format.
References
[1] Michael J. Reagin, Theresa L. Giesler, Alia L. Merla, Jeanine M. Resetar-Gerke,
Kinga M.
Kapolka, J. Anthony Mamone,
"TempliPhi:
A
Sequencing
Template
Preparation Procedure That Eliminates Overnight Cultures and DNA Purification",
Journal of Biomolecular Techniques, 14:143-148, 2003
60
[2] Singer V. L., Jones L. J., Yue S. T., Haugland R. P., "Characterization of PicoGreen
reagent and development of a fluorescence-based solution assay for double-stranded
DNA quantitation", Anal. Biochem., 1997 Jul 1;249(2):228-38
[3] TempliPhi 100/500 Amplification Manual, Amersham Biosciences.
61
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62
Chapter 4
Sequencing Reaction
Integration with Amplification reaction
After standardizing and optimizing the amplification reaction, the next step was to work
on bringing the sequencing reaction down to the nanoliter scale. As discussed in Chapter
I, the amplification reaction is followed by a couple of dilution steps and then the
sequencing reaction at the Broad Institute. In a microfluidic format, since we no longer
need any dilutions, we can make the sequencing reaction work using the amplification
product on chip as the input template.
We have already seen in chapter III that the amplification reaction on chip produces an
average of 3 ng of DNA whereas roughly 1 ng is needed for the sequencing reaction on
the 384 well plate format. Hence, even after flushing some of the product off the chip to
make room for the sequencing reaction reactants, primarily the Big Dye, we should have
enough amplified DNA on chip to make a successful sequencing reaction.
63
With the above conviction, a chip was designed as shown in Figure 4.1, which could
perform the amplification and sequencing reaction sequentially. This chip was designed
to perform four such reactions in a parallel fashion. The four circular reaction chambers
would ideally align over the four corresponding adjacent wells of a 96-well plate. The
output product of the sequencing reaction can be used for a purification procedure on
chip or can directly be flushed upwards through the chip into an exit well complimentary
to the ABI 3730 suction needles. However, this means that compared to a 384 well plate
format, we are providing only
1 /4th
of the reaction wells required due to design
constraints. However, the design here only preliminary and by no means optimal and the
flow, control channels can be re-designed in a spatially optimized way so as to provide
384 output reaction wells in conformation with the ABI 3730 detectors.
With regard to the pumping and valving scheme for microfluidic manipulation, this
design is very similar to the chip design used throughout Chapter III for DNA
amplification. The scheme detailed for purging amplification product out to make room
for PicoGreen detection will be used here to make room for Big Dye, M13 primer and
other sequencing reaction constituents.
64
+n
+
14-
.1-
-IFigure 4.1 Proposed Microfluidic Layout for sequential amplification and sequencing reactions.
Having established that the amplification reaction works well on chip, initial on-chip
sequencing reaction experiments utilized amplification material produced off-chip. To
test the sequencing reaction from a reaction mix prepared off-chip, we utilized Design 4
used throughout Chapter III for amplification.
65
Biology of the Sequencing Reaction
Before describing the various trials done to test sequencing reaction on-chip, it is useful
to review the existing methodology on the 384 well plate format. The sequencing reaction
is very similar to the Polymerase Chain Reaction (PCR) used for the amplification of
DNA. PCR amplifies the input template by using a pair of primers flanking the DNA
segment of interest. Apart from the primers and polymerase, we have dNTPs also present
in large concentration, which are really the building blocks of DNA as detailed in
Chapter I. PCR consists of three thermocycling steps which are repeated for about 30
cycles. Typically, the first step at 94 0C is the denaturation step followed by primer
0
annealing at 550C and extension at 72 C. After 30 cycles we have exponentially
amplified product, which is double stranded.
The polymerase enzyme used in the sequencing reaction hence is very similar to the Taq
polymerase used in PCR. There are two main differences between PCR and the
sequencing reaction. Whereas in PCR we use two primers, only one primer is required is
the sequencing reaction. The primer is determined by the plasmid used for cloning itself
and in our case it is the M13 primer that is used. Another difference is the use of a small
of the concentration of dNTPs) of extra extension molecules called ddNTPs
in addition to dNTPs. The comparison between a dNTP and ddNTP molecule is shown in
amount (1%
Figure 4.2.
0
WHO-qwOCH
BMs
H(
H
deoxynuclootde (dNTP)
dkleoxyoucleottde (ddlP)
Figure 4.2 Comparing ddNTP and dNTP molecules
66
The ddNTP molecule differs from a dNTP in the sense that one of the OH groups is
replaced by an H group. Just as we have four types of dNTPs in solution, we also have
four types of ddNTPs I solution - A, T, G, C. Furthermore, each type of ddNTP is labeled
with a uniquely colored fluorescent dye.
The comparatively rare event of incorporation of a ddNTP in place of a dNTP terminates
the extension reaction. Thus at the end of sequencing reaction, the last molecule in almost
every strand is a labeled ddNTP where the color of the fluorescent dye really determines
which base it corresponds to among A, T, G and C. The comparison between the PCR
and the sequencing reaction is shown schematically in Figure 4.3a,b and Figure 4.4a,b.
Whereas in PCR the final product is double-stranded, we have single-stranded product at
the end of sequencing reaction. This is because we use only one primer in sequencing
reaction for the reason that each of the single strands in sequencing reaction terminate in
a different colored dye and we want each of these strands to travel and be detected
separately on the gel as shown in Figure 4.5.
Such a detecting mechanism is available on the commercial ABI 3730 detectors used in
the Broad Institute. The sequencing reaction product has strands of all the possible
intermediate lengths and hence migrates to different positions on the gel where the
corresponding base at that length can be detected by the specific color of the fluorescent
dye.
67
I
I.
30 - 40 cycles of 3 steps:
Step 1 :
I minut 94 'C
Step 2 : anel
45 seconds 54 'C
5P~
34
5I
ft rward and reverse
primersxten
Step 3 : extenso"A
34
2 minutes 72 'C
only dNTP's
Figure 3a. PCR steps
-
4di cycle
41h cycle
aTnted gene
Sh
CYCyl
--
=
2nd IyII
------------- W35th cycle
ist cycle
template DNA
<-<
22
4 copies
2
- 16111
=
8 copies
16 coIll
Figure 3b. PCR exponential amplification pattern
68
32 copic
2
=68
bIlion copies
30 cycles of 3 steps:
Step I:
I minut 94 'C
Step 2: anneal
15 seconds 50 *C
33'
I primer !
11111111111113'
5
31
sigure
Step 3 : extenw
4 minutes 60 *C
mixture of dNTP's
and ddNTP's
I
4a. Sequencing Reaction steps
Ist cycle
6 template strands
6 complementary strands
2nd cycle
12 cwnnpImentury strands
1'_______________________________
\
30 cycles: 180 complkemntary
-*
strands
mixture of strnnds with different
length which end an a fluorescently
labeled ddNTP
PCR product
-Primer fts
only on
one strand
-On incorporation of a fluotescently labelled ddNTP (complerenmry
with the base on the template) the elongation stops
Figure 4b. Sequencing Reaction linear amplification pattern
69
U"W"
X0
/eector
t - I hour
t = 2 hours
Figure 4.5 Gel Electrophoresis of Sequencing Reaction Product
Sequencing Reaction Trials
With an overview of the biological mechanism behind the sequencing reaction, the
microfluidic experiments can be described in propoer context. As previously mentioned,
initial on-chip sequencing reaction efforts utilized the amplification chip (Design 4). The
standard reaction mix for sequencing reaction is 1.3 p.L input template plus 0.75 pL Big
Dye mix. Typically, the Big Dye mix consists of 0.125 p.L of Big Dye, 0.008 tL of M13
primer and the rest is buffer and dilution water. The Big Dye is a proprietary reagent
consisting of unknown quantities of polymerase, dNTPs and ddNTPs. Hence, we do have
the freedom to play with the amount of Big Dye, input template, buffer and primer
concentrations in our reaction mix on the chip.
It was decided to initially proceed with the standard reaction mix as used in the 384 well
plate format. The 2 pL plate reaction volume was reduced to 40 nL on-chip. BSA (? v/v)
70
was added to reducing biofouling, as per the amplification experiments. However, the
standard reaction mix with BSA did not yield any sequence on the 3730 detectors.
From a troubleshooting perspective, we looked at the relative quantity of amplified
template in the plate and on-chip reactions. Approximately 1.5 ng of DNA was being
used as input template on-chip vs. concentrations as low as 1 ng of DNA for the 384 well
plate format. Through independent experiments on the 384 well plate format, it was
confirmed that using high relative input DNA concentrations was detrimental to the
reaction. Hence, it was decided to use lower amounts of input template on chip. Still no
sequencing product was observed. This suggested that other failure scenarios need to be
explored as well.
There was also a possibility that thermocycling was not effective. A flat-plate
thermocycler was being used with the glass-slide in contact with thermocycler plate.
Approximate heat transfer calculation revealed that the time constant for heat transfer
through the thin glass slide would be insignificant compared to the time scale of
thermocycling itself. Using a thermocouple, it was indeed observed that the temperature
variation in the channels during thermocycling very closely mimicked the programmed
temperature variation of the thermocycler. Notwithstanding, the thermocycling times and
ramp rates were varied to look for unforeseen variables. Especially, the time of the
extension step was increased to allow for complete reaction. However no encouraging
results were observed.
A tentative guess was that biofouling was still occurring by some mechanism yet
unknown. In order to test this hypothesis, standard sequencing reaction mix was flushed
through the chip and then put on the 384 well plate sequencing reaction followed by
clean-up and detection. It was observed that only 150 reads of low quality were generated
as opposed to the normal of 800 reads. On the contrary, if the sequencing reaction was
first done on the 384 well plates and then the product flushed through the chip, it gave
good read lengths on the 3730 detectors.
71
Hence, it could be easily argued that adhesion of some critical components of the reaction
was happening in the process of flushing the sequencing reaction mix in the chip. The
adhesion was so rapid that the concentration of the critical species went down in a matter
of seconds as the reaction mix was flushed through the chip. Since the sequencing
reaction product itself was not affected as it was flushed through the chip, it could be
argued that it is not the long DNA strands, which have an affinity for PDMS but some
other smaller molecule.
As a result, it was decided to increase the reaction volume from 40 nL to 200 nL. Blazej
et. al., 2006 [1] report successful sequencing reaction on a glass device at a reaction
volume of 250 nL. Hence, 200 nL volume should definitely yield some product if the
biofouling is kept in check.
Several passivation techniques apart from BSA were tried to reduce the adhesion of
biomolecules to PDMS. Bo et. al. [2] report a material n-dodecyl-P-D-maltoside (DDM)
which is excellent in passivating the PDMS surface against protein absorption. A static as
well as dynamic coating of DDM was tried as reported in the above paper but still
sequencing reaction was not successful. Subsequently, the silanization of the PDMS
channels was also tried with PEG-grafting but with no success.
It was then decided to find out which component exactly present in limiting
concentrations in the solution due to biofouling. PCR was tried on 200 nL device and was
found to be successful with routine reaction mix and thermocycling parameters. As
explained earlier in this chapter and depicted in Figures 4.3 and Figure 4.4, the PCR
reaction mix differs from the sequencing reaction mix mainly in the sense that
sequencing reaction mix consists of ddNTPs whereas PCR reaction mix does not. The
other differences such as slight variation in the structure of polymerase and use of two
primers in the PCR as opposed to only one in the sequencing reaction are not pertinent
here. Hence, it can be deduced that it is the ddNTP molecules which are present in
72
limiting amounts and which are being removed from solution due to their affinity with
PDMS.
In hindsight, this is very possible scenario. While testing the amplification reaction
product on chip using PicoGreen, it was observed that the PicoGreen dye has a great
affinity for PDMS and it remains sticking to PDMS inspite of repeated washes with DI
water. The ddNTP molecule being dye-labeled can be expected to have a similar affinity
to PDMS, which cannot perhaps be prevented by the passivation techniques tried in this
project. BSA coating, however, does serve to present the enzyme from sticking onto the
PDMS walls as demonstrated in Chapter III.
Figure 4.6 Fluorescence shows the extent of partitioning of small dye molecules into PDMS structure.
Later it was discovered that surface adhesion is not the only mechanism responsible for
biofouling. It is in fact possible for small molecules like ddNTPs to partition from the
solution into the PDMS structure as reported by Toepke et. al. [3] in 2006 (Figure 4.6).
This partition can be strong in some cases that the actual concentration in the PDMS
channel can be as low as 100 times less than the intended concentration. Small molecules
are much more susceptible to this effect and the ddNTP being a small molecule is most
probably being partitioned from the solution through this mechanism itself.
73
An argument can be made that dNTP is also a molecule very similar to ddNTP and still it
is visibly not present in limiting amounts as proved by the success of PCR on PDMS in
the presence of BSA as passivating agent. However, it should be noted that in case
surface adhesion is solely responsible for biofouling then there is a strong possibility that
the adhesion of ddNTP is happening only because it is small dye-labeled molecule and
there is reason to believe that PDMS has an affinity towards small hydrophobic dyes as
proved by its affinity towards PicoGreen. This might possibly the reason as to why dNTP
is not the limiting species.
If the dominant mechanism of biofouling is actual partitioning of the smaller molecules
into the PDMS structure, then there is no reason why the dNTP molecule should not also
similarly leave the solution and partition into PDMS like the ddNTP molecule. In this
case, it should be noted that the concentration of dNTP is much higher as compared to the
ddNTPs and perhaps PCR can still proceed with a handy fraction of dNTPs partitioning
into PDMS. However ddNTPs are only present in miniscule amounts in the sequencing
reaction mix and only intended to be reaction terminators. Hence, sequencing reaction
cannot proceed successfully if a significant portion of the ddNTPs partition into PDMS.
This being said, further research is still required to figure out the exact or dominant
mechanism by which ddNTPs are rendered limiting in the reaction mix. Also an efficient
passivating scheme needs to be figured out to allow the sequencing reaction to happen on
a PDMS microfluidic platform.
74
References
[1] Robert G. Blazej, Palani Kumaresan, Richard A. Mathies, "Microfabricated
bioprocessor for integrated nanoliter-scale Sanger DNA sequencing", PNAS, Vol. 103,
No. 19, May 9, 2006.
[2] Bo Huang, Hongkai Wu, Samuel Kim, Richard N. Zare, " Coating of
poly(dimethylsiloxane) with n-dodecyl-b-D-maltoside to minimize nonspecific protein
adsorption", Lab on a Chip, 5, 1005-1007, September 2005.
[3] Michael W. Toepke, David J. Beebe, "PDMS absorption of small molecules and
consequences in microfluidic application", Lab on a chip, 2006, 6, 1484-1486.
75
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76
Chapter 5
Conclusions and Future Work
In this project, the possibility of bringing down the DNA sequencing sample preparation
protocols to the nanoliter scale was explored. The purpose was to bring about cost
reduction and quality improvement by directly cutting down the reaction volumes by at
least two orders of magnitude.
Through the adoption of microfluidic techniques outlined in Chapter II, manipulation of
nanoliter quantities of reagents was made possible. Further, in Chapter III it was observed
that it is indeed possible to bring down the Rolling Circle Amplification (RCA) technique
down to the nanoliter scale. Experiments were done to standardize and optimize the scale
down as tabulated in Appendix III.
The results obtained in Chapter III are critical since RCA is an integral part of the sample
preparation procedures in major sequencing centers like the Broad Institute and the Join
Genome Institute. Prior to this work, we have not seen any cases of RCA being
implemented at nanoliter scale to explore the possibility of its integration with the rest of
the sample preparation processes at the microfluidic scale. Here, we have definitively
77
shown that RCA is totally amenable to operation at the nanoliter scale on a PDMS
platform. This was amply demonstrated by the excellent read-lengths obtained from the
RCA samples from chip. This paves the way for further integration at nanoliter scale.
In Chapter IV, the efforts directed towards scaling down the sequencing reaction were
outlined. In spite of various trials, success at scaling down the sequencing reaction was
not achieved. Through several subsequent trials, it was inferred that the ddNTPs were the
limiting species in solution owing to their partitioning away from solution. The
attenuation in the concentration of ddNTPs is expected to be severe based on research
carried out by independent groups.
There are several directions to move ahead from here onwards. The foremost requirement
is to make the sequencing reaction work on the microfluidic platoform. As stated in
Chapter IV, this requires the development of an effective passivation technique to curb
the partitioning of ddNTPs from the solution onto the PDMS walls or into the PDMS
structure itself Several passivation have already been explored in this project and further
research needs to be done yet.
Perhaps modifying the PDMS structure might help. Modifying the chemical composition
of the polymer itself might help in making it impervious to the ddNTP molecules. The
more common technique is to coat the walls of the channels with a suitable chemical so
that the ddNTPs are repelled from them.
It has been confirmed to the foremost extent possible at this stage that treating this single
problem will allow the sequencing reaction to run successfully at the microfluidic
platform. As mentioned in Chapter IV, a couple of recent research articles have already
demonstrated
the possibility of a nanoliter sequencing reaction
microfluidic device.
78
on an all-glass
In addition, we need to work on the starting template material used for sample
preparation. In this project, sequencing procedures were implemented starting only from
plasmid vectors or from cell culture. However, we need to explore the possibility of
starting from a single plasmid or a single cell. If a technique allowing us to sequence
starting from a single plasmid is developed, then there will be no need to undergo tedious
cell culture protocols saving us a lot of labor and making the process much more
efficient.
If the possibility of starting from a single plasmid is actualized and the nanoliter
sequencing reaction standardized on the PDMS platform, then the idea of a nanoliter
integrated sample preparation procedure can easily be materialized leading to a
revolution in DNA sequencing and also giving rise to a myriad of bio-medical
possibilities.
79
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80
Appendix I
Fabrication
The microfluidic devices used in this project were two layer PDMS devices fabricated
through the technique of soft lithography. This comprises of two basic steps: i) making a
master mold on silicon substrate and ii) using the mold as a stamp to create PDMS
devices via coating followed by curing of PDMS on the mold.
First we explain the development of the mold. The molds were developed at the
Microsystems Technology Laboratory at MIT. The process is schematically depicted
below in Figure 1.
A silicon wafer is taken and first cleaned by acetone, methanol and isopropanol in that
order. Then there is a pre-bake at 130 0C for 10 minutes. Then the wafer is mounted on a
spin coater and a couple of drops of HMDS are added at the center of the wafer. The
coater is then ramped for 10-15 s to around 2000 rpm where HMDS is around to spread
first and then evaporate. HMDS promotes the adhesion of photoresist to the wafer. Then
the photoresist is added to the center of the wafer. The spin coater is ramped for about 10
s followed by steady rotation at 1400 rpm for 50 s. The photoresist used here is AZ
81
P4620 and an rpm of 1400 accomplishes a thickness of 10 prm of photoresist. Then there
is a post-bake in 90 0 C oven for 10 min.
Silicon wafer
Pre-Bake @ 130 0C followed by AZ P4620 spin-coat
Post Bake
@ 90 0C followed by UV
expose
Develop in AZ 440
Spin Coat PDMS
Cure in 800C oven and peel off
Figure 1. Microfabrication Steps
The photoresist thickness used in this project was either 10 pm or 20 pm. If a thickness of
20 ptm is desired then another layer of AZ P4620 is coated after the post bake. After
coating the second layer another post-bake is done for 10 min.
82
Followed by the post-bake, we do UV exposure on the MJB3-BroadBand mask aligner.
For a thickness of 10 pm, we expose for a total of 20 s in two batches of 10 s each. For a
thickness of 40 im, we do four exposures of 10 s each. It is not preferable to expose at all
once since it leads to a lot of bubble effervescence while exposure leading to non-uniform
layer thickness.
Once we have the molds for both the flow layer and the control layer, we can go ahead to
fabricate the PDMS microfluidic device using the molds as the master. As shown in
Figure 1, the mold is first coated with PDMS. Depending on whether the flow layer or the
control layer is fabricated the process differs. For the control layer, I part of PDMS
polymer is mixed with 5 parts of the curing agent and then degassed. The mixture is then
poured onto the mold for a thickness of around 500 ptm and then left for curing into a
80 0C oven for 20 minutes. The control layer is then peeled off the mold and kept for
aligning over the flow layer.
For the flow layer, PDMS is spin coated on the mold. PDMS is mixed with the curing
agent at a ratio of 20:1. Coating at an rpm of 3000 yields a thickness of 25-30 ptm, which
is suitable for a flow layer mold of 20 gm photoresist thickness. The mold is then left for
curing in the 80 0C oven for 20 minutes. After curing, the control layer is aligned over the
flow layer mold under the microscope. The mold is then left for curing in the 80 0C oven
for 20 minutes.
After pulling out the mold from the oven, the flow layer has bonded with the control
layer. Now when the device is peeled off the mold, the flow layer comes off with the
control layer. The alignment of the two layers is shown schematically in Figure 2.
Cover glass slides are also spin coated with PDMS in the meantime at an RPM of 4000
yielding a thickness of 10 pm. The glass slides are also left for curing in the 80 0C oven
for 20 minutes. After curing, the flow layer bonded with control layer is simply places
onto the glass slide. The device is again left for curing for 20 minutes after which we
83
obtain a two-layer PDMS device. Here the lower flow layer is bonded to the PDMS layer
on the glass slide, giving rise to secure flow channels.
mold
flat
substrate
Figure 2. Flow and Control Layers: Peeling and Alignment
84
Appendix II
LABVIEW Controls and
Instrumentation
The instrumentation used in this project has been picked up from what has been in use at
the Thorsen Lab at MIT. The connecting tubing, lee valves, electronic drive board,
LABVIEW card and cables are a few of the equipments that were used in order to work
towards the objectives of this work.
The pneumatic controls used for operating valves were controlled through LABVIEW
software. A schematic of the front panel used for most part of this project is shown in
Figure 1. The block diagram is not shown here for the sake of clarity. This LABVIEW
code can be used to operate 16 valves at once. This number of valves was sufficient for
all the various test designs employed in this project.
Also, this design can also allow us to operate the peristaltic pump in parallel with the
operation of the valves. There is a separate switch, which allows us to turn on/off the
85
pumping as and when needed during device operation. All the microfluidic operations as
described in Chapter II can be easily performed using this code.
Figure 2 shows a part of the experimental set up where the microfluidic device with the
tubing connections is placed on the flat-bed thermocycler for the sequencing reaction.
86
Figure 2. Microfluidic Chip on the Flat-bed Thermocycler
87
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88
Appendix III
Amplification Trials
Chamber
Volumne=10nl
Experi
ment
0.
Expr0
0
.2 E
C)0
z
0E
E
E
_ >
>
a. X
00 (L
0)0
~~
.0
aEE
CL
z
0
0~
.2~
=
2
>
C
=
CP
41
Q0-
0
-
=a
E
M 0
)*
Notes
w>_
o
0
1
36 pg 1.3 E7 10000
2n
2 Hr
23
.33x 2n
N
N
N
3nM No Amplification
2
36 pg 1.3 E7 10000
2ni
8 Hr
23
.33x 2ni
N
N
N
3nM No Amplification
3
36 pg 1.3 E7 10000
5nl
16 Hr
23
.33x 2n
N
Y
N
7nM 2-3 fold Amp
4
45 pg 1.6 E7 10000
5n
16 Hr
23
0.33
2nl
Y
Y
N
7nM 2-3foldAp
5
45 pg 1.6 E7
500
3.68 3.68 0.14 18Hr
ni ni ni
23
0.33 2n1
X
Y
Y
N
6nM 2 fold Amp: Standard
composition of 500 kit used
6
36 pg 1.3 E7
500
2.7nl3.2nl2.inl 18Hr
30
0.33
X 2nl
Y
N
Y
Stick
89
______Ratios
in 500 kit altered
500
7
36 pg 11.3 E7
8
500 +
45 pg 1.6 E7 10000
2.7nl3.2nl2.lnl 18Hr
7nl
1.5ni
ann
eal
1nl
30
0.33 2nl
x
0.33
2nl
3-4 fold Amp: Ratios in 500
kit altered
Y
N
Y
9nM
Y
N
Y
10000 kit (More efficient) +
stick extra enzyme,dNTPS from
500 kit
18Hr
30
6 nl 5.5 4 nl 16Hr
ni
30
0.33 4.5nl
Y
N
Y
3.5 ng 80-fold Amp; Sequence
generated of 300 reads
30
0.33
x
Y
N
Y
2.8 ng
.n
Chamber Volume = 40 ni
9
45 pg 1.6 E7 500+ 24 ni
10000
10
45 pg 1.6 E7 5000+0
34 nl
100ni
0
1
4 nl 16 Hr
X
4
ni
_0-fold
11
45 pg 1.6 E7
0000 100ni
38 nl
0
0
16 Hr
30
0.33
x
4
ni
Y
N
Y
Amp
0.5 ng
13-fold Amp
12
13
45 pg 1.6 E7 500+
10000
0.45
Pg
38 nl
1.6 E5 500+ 24 nl
10000
0
1.5
nI
6 nl 5n
0
16 Hr
4 nl 16Hr
30
30
0.33
x
4.5
nl
Y
0.33 4.5ni Y
N
N
Y
Y
0.5
ng
Same test as above; 13-fold
Amp - Enzyme is limiting!!
2 ng
4500-fold Amp
14
15
45 fg 1.6 E4 5000+ 22 n
220n0
2.25 fg 800 500+ 22 ni
copies 10000
6 n
5.
ni
6 nl
16Hr
6 nl 5.5 6 nI 16Hr
nI
30
30
0.33 4.5ni
Y
0.33 4.5nl
Y
X
X
N
Y
2.5 ng
60K-fold Amp, Sequence
generated of 1000 reads
N
Y
3 ng
1.33 E6-fold Amp, No
sequence
16
112.5
40
500+
ag copies 10000
22nl
6 nl 5.5 6 nl 16 Hr
ni
90
30
0.33
x
4.5
ni
Y
N
Y
1.5 ng
1.3 E7- fold Amp, No
Sequence
17
18
19
20
21
45 pg 1.6 E7 5000+0
2.25
fg
Cells
Cells
Cells
800
1 E4
1 E4
5 E4
22 n1m
6 n 5
30nl
4 nl 1.5 4nl
ni
5Hr
500+ 30 nl
10000
3 ni 1.5 5nl
nl
16 Hr
5000+
10000 30 ni
3 ni 1.5 5nl
ni
3 Hr
500+
10000 30 nl
3 nl 1.5
nI
5000+
10000
6 ni 16 Hr
30
0.33 45
X
nI
Y
N
Y
0.3 ng
30
0.33
4.5
ni
Y
N
Y
2.8 ng
0.33 4.5
x
nl
Y
0.33 4.5
x
ni
Y
0.33
x 4.5
nI
Y
x
nl 10 Hr
30
30
30
Little Amp - Rxn buffer works
better than additional dNTPs
Amp can be completed in 5
Hours
N
Y
3 ng
Generated Sequence does
not match
N
Y
0.7 ng
Amplification is not complete
in 3 hours
N
Y
3 ng
Sequence of 750 reads
22
45 fg 1.6 E4 10000 32 nl
0
0
0
5 Hr
91
30
0.33 4.5
x
ni
Y
N
Y
4 ng BSA used - extra enzyme
from 500 kit not required