This document discusses advancements in Sanger sequencing using microchip-based platforms. It describes how microfluidics can integrate and miniaturize sample preparation, amplification, purification, electrophoretic separation, and detection steps. This allows processing of multiple samples simultaneously while reducing time, costs, and sample/reagent usage. The document also highlights developments in polymeric matrices used within microfluidic devices to achieve long read lengths over short distances, such as poly(N,N-dimethylacrylamide) networks and hydrophobically modified polyacrylamides. These materials enhance DNA separation performance critical for applications like genome sequencing.

1. MICROCHIP BASED
SANGER
SEQUENCING OF DNA
Monika U
18BCHA12

2. INTRODUCTION
For decades, the method of choice for the determination of
DNA sequences has been the Sanger reaction, followed by
size-based electrophoretic separation of single stranded DNA
ladders. The development of capillary array electrophoresis
(CAE) to replace the more traditional slab gels certainly led
to dramatically increased DNA sequencing throughput, but
sequencing human genomes by this technology was still far too
expensive. While many new technologies for the determination
of DNA sequences are under development, at this time, Sanger
sequencing is the only technology that can provide truly long
reads (i.e., the highly accurate sequence of more than 600
contiguous DNA bases). Current development of more
advanced Sanger-based electrophoretic sequencers involve the
miniaturization of the process onto a microfluidic chip
platform

3. MICROFLUIDICS
Microfluidics has the potential to greatly reduce costs in
each step of the sequencing process from sample
preparation to analysis. These devices can produce more
defined, narrower sample injection zones, which increases
DNA separation efficiency and thus decreases the total
distance and time required to obtain high resolution data.
Microfluidic devices can also be designed and fabricated so
that every step from sample preparation to separation can be
performed on one integrated device, and so that up to 96
samples can be processed simultaneously. Miniaturizing and
combining multiple processes onto a microfluidic device
offers obvious advantages; however, the engineering of
multiple chemical processing steps onto a small device has
created new challenges and problems not encountered in
comparative macroscale benchtop systems

4. INTEGRATED MICROFLUIDIC DEVICES
FOR GENOMIC ANALYSIS
➢ The development of a single microfluidic device
capable of pre-PCR DNA purification, amplification
via thermal cycling, post-Sanger reaction purification,
electrophoretic separation, and finally DNA detection
will play a significant role in the pursuit of rapid,
inexpensive genomic sequence determination.
➢ Combining these steps onto a single microfluidic
platform promises to greatly reduce the amount of
expensive reagents and materials needed for
sequencing and shorten the overall analysis time

5. ➢ The Mathies group at the University of California at
Berkeley have focused on developing prototypes of
microfluidic systems capable of achieving this goal
through a combination of glass fabrication techniques,
microfluidic valving in PDMS (PolyDimethylSiloxane),
hydrodynamic pumping, and electrophoresis.

6. ➢ The integrated devices developed by the Mathies
group have been tested with DNA samples that have
already been purified from their raw state (i.e., whole
blood, serum, etc.).
➢ Using resistive heaters integrated into the chip device
itself, the DNA sequencing ladder is synthesized via the
Sanger cycle sequencing reaction with a predetermined set
of reagents.
➢ To achieve high-resolution separations, DNA must
be separated from the extraneous Sanger reaction
components before it can be analyzed.
➢ The Mathies lab device uses acrylamide-based
copolymers with single-stranded oligonucleotides
randomly attached to the polymer backbone.

7. ➢ By electrophoresis, the DNA sample through the
polymer, single stranded DNA with a complementary
sequence to the immobilized capture
oligonucleotides selectively hybridizes while the
unwanted molecules (salt, dNTPs, and ddNTPs) are
electrophoresed away.
➢ By raising the temperature of the device above the
melting point of the captured DNA, the desired Sanger
fragments can then be released into another channel for
electrophoretic separation and detection via laser-
induced fluorescence (LIF) with four emission channels
(colors) to detect each DNA base, as required for DNA
sequencing.

8. ➢ In recent advancements, this system has been modified
to allow in-line injection of the sample, where the
DNA is captured in the same channel in which the
electrophoretic DNA separation will occur.
➢ This eliminates the need for excess DNA sample, much
of which is often wasted during the standard cross
injection in microchip systems, and hence is a step
toward significantly reducing sample and reagent
requirements by exploiting microfluidics.
➢ The Mathies group has also developed microchannel
devices with up to 96 sequencing lanes running in
parallel, which in principle are capable of sequencing
over 100,000 bases per hour when full automation can
be achieved.

9. ➢ The combination of these two technologies displays the
potential of microfluidics to prepare and analyze
numerous samples in extremely short periods of time
compared to conventional CAE systems.

10. IMPROVED POLYMER NETWORKS FOR
SANGER SEQUENCING ON
MICROFLUIDICS
❖ Poly ( N,N-dimethylacrylamide)
Networks for DNA Sequencing
➢ Developing lab-on-a-chip systems has been a major
focus of the DNA sequencing community that is
developing Sanger technology, and some very
significant advances have been achieved; however,
in general, much less attention has been paid to
DNA separation networks and polymeric channel
coatings utilized in these devices, compared to the
development of the devices themselves.

11. ➢ Typically, the same materials that were
successfully utilized in CAE systems have been
used in microfluidic devices ; however, just as
cross-linked polyacrylamide or agarose
networks, which performed extremely well in
slab gels, did not transfer well to capillary
systems, CAE specific polymer solutions also
need to be re-engineered for the new microchip
platforms.
➢ To achieve DNA sequencing read lengths of
600–700 bases, which are necessary for current
DNA sequence alignment algorithms to process
repeat-rich genomes, highly entangled solutions
of hydrophilic, high-molar mass polymers are
needed.

12. ➢ Highly hydrophilic polymer coatings for internal
microfluidic channel surfaces are also necessary to
reduce electroosmotic flow and bioanalyte
adsorption, which otherwise greatly reduce the
read length and resolution obtained in these
devices.
➢ Poly(N,N-dimethylacrylamide) (pDMA) used in
conjunction with poly (N-hydroxyethylacrylamide)
(pHEA) as a separation matrix and wall coating,
respectively, has been reported to provide chip-
based read lengths in excess of 600 bases in only 6.5
min in a 7.5-cm long glass channel.
➢ These results are 2–3 times faster than comparable
read lengths obtained in other microfluidic chips
[6, 7, 10, 35] and 10–20 times faster than a typical
CAE system, which requires 1–2 hrs.

13. ➢ The combination of differently sized polymers allows
for an increase in sequencing read length because
higher total polymer concentration, the most
important factor in separating small DNA
fragments, results in a smaller average mesh size,
while an increase in the polymer entanglement
strength, which is achieved with the higher
molecular weight polymers, favors optimal
separations of larger DNA fragments.
➢ Mixed molar mass pDMA matrices provide
average read lengths that are 10% longer than
matrices formulated with a single average molar
mass.

14. ➢ Interestingly, a commercially available linear
polyacrylamide (LPA) solution from Amersham
was tested under the same conditions as the
mixed molar mass pDMA and produced less
than 300 bases of good-quality data.
➢ This is a surprising result since this commercial
LPA matrix can often deliver read lengths in
excess of 700 bases in a CAE instrument.
➢ The increase in sequencing performance in
pDMA is attributed to a hybrid separation
mechanism that has been observed via single-
molecule fluorescent DNA imaging.

15. ➢ Theory on DNA electrophoresis through gels and
entangled polymer solutions postulates that DNA moves
through entangled polymer networks either in an
equilibrium coiled conformation (so-called Ogston
sieving) or by unwinding and snaking through the mesh
by a mechanism related to polymer reptation.
➢ Dilute polymer solutions, however, can separate DNA by
a mechanism known as transient entanglement coupling
(TEC) in which DNA entangles with loose polymer
chains in solution and transiently drags them through
solution in a U-shaped conformation.
➢ The pDMA matrix discussed above is thought to allow
DNA to move through the matrix by a hybrid mechanism
using a combination of reptation and TEC (for DNA
molecules too large to move through the polymer mesh
without unwinding).

16. ➢ The pDMA chains form an entangled network in
solution that promotes reptation, but the chain
entanglements are weak enough to allow the DNA to
pull polymer chains from the network, resulting in local
network disruption, so that they move through the matrix
in a U-shaped conformation similar to what is observed
in the TEC mechanism.
➢ While these are not DNA sequencing fragments, they do
show that the two mechanisms can coexist in one matrix,
under relevant field strengths. The reduction in band
broadening and increased read lengths achieved with the
mixed molar mass pDMA solutions on glass
microfluidic chips results in a polymer separation matrix
that is capable of providing long DNA sequencing read
lengths, very rapidly, in short separation distances.

17. ❖ Hydrophobically Modified Polyacrylamides for
DNA Sequencing
➢ Another advancement in the development of DNA
sequencing matrices for microfluidic chips has been
made by modifying LPA with hydrophobic N,N-
dialkylacrylamides to create a hydrophobically
modified block copolymer, which is used in
conjunction with a channel coating.
➢ To synthesize a reproducible block structure, free-
radical micellar polymerization is used, in which a
surfactant such as sodium dodecyl sulfate (SDS) is
added to the polymerization reaction above its critical
micellar concentration.

18. ➢ The SDS molecules form micelles with hydrophobic
cores where the hydrophobic monomer can be
incorporated and then effectively integrated into the
polymer backbone during the polymerization process.
➢ The ability to rapidly load and unload a DNA separation
matrix in a microfluidic chip is necessary to analyze
multiple DNA samples quickly.
➢ Hydrophobic block copolymers typically have much
higher viscosities than their homopolymer counterparts;
however, due to the extremely small amount of
hydrophobe needed to achieve an increase in read length,
these specially designed copolymers have very similar
viscosities and channel loading times to their
homopolymer counterparts.

19. ➢ The result is a polymer solution that provides very
high-resolution separations over a short distance,
which can also be loaded into a microfluidic device
in a reasonable amount of time.

20. CONCLUSION
With the advances in both integrated microfluidic
devices and high-performance polymeric materials, we
hope that the reader has a clearer picture of where Sanger-
based sequencing technologies are heading in the future.
The immense potential of integration of these devices and
the importance of long read lengths for some projects in
genomic sequencing signify that the Sanger approach and
DNA electrophoresis will continue to be a powerful tool
for future genomic technology, especially for DNA
sequencing projects aimed at decoding and correctly
assembling complex, repeat-rich genomes.

21. THANK YOU..