Introduction

Early Sequencing
Early sequencing was performed with tRNA through a
technique developed by Richard Holley, who published the first
structure of a tRNA in 1964. This involved breaking down RNA
molecules, then puzzling the pieces back together. However,
this was extremely time consuming, and due to its large size,
such methods could not readily be used for DNA sequencing
(Sanger 1988).
Frederick Sanger developed improved methods that allowed
sequencing of some DNA up to 50 nucleotides in length.
However, he realized the potential of copying DNA instead of
degrading it (Sanger 1980).
In 1975 Sanger developed the plus and minus method. This
included the principal of chain termination during
polymerization, and was used to sequence an entire genome
(almost) that of the X 174 bacteriophage. Despite this
result, Sanger was not satisfied and kept searching for better
methodology (Sanger 1980).
Sagners major breakthrough, which would become the basis
for subsequent techniques, came at a meeting in Germany
where Klaus Geider gave him a sample of ddTTP, which would
terminate chain polymerization upon incorporation. This would
be called the di-deoxy method.

dNTP

Addition of ddNTPs will terminate chain elongation. Location of ddNTP

insertion within a nucleotide chain can be determined using gel separation.
(image adapted from Sanger 1980)

Sanger used this to finish the remainder of the X 174 genome

in 1978.
These improvements and Maxim and Gilberts
chemical method of
led to a large attraction towards
sequencing (Sanger 1988).
Using the same methods as Sanger did in 1977, it would have
taken more than 1000,000 years to complete the human
genome. Three innovations came about that greatly expedited
the sequencing process: Shot-gun sequencing, PCR, and the
automation of sequencing. These developments led to the
publication of the first bacterial genome, H. influenzae Rd
KW20, in 1995 by Robert Fleischmann (Binnewies 2006). From
early sequencing of tRNA to the publication of a bacterial
genome took 30 years, but the groundwork was laid out for
future sequencing.

Human Genome Project

Erwin Chargaff could not have imagined the rapid technological
advancement in computers and robotics when he made the
often quoted statement by critics of the human genome project;
Even the smallest functional DNA varieties seen, those
occurring in certain small phages, must contain something like
5,000 nucleotides in a row. We may, therefore, leave the task of
reading the complete nucleotide sequence of a DNA to the 21st
century, which will, however, have other worries.
Progress was deemed too slow such that a decade to
publication of the 1st draft of the human genome, Bart Barrell
considered the HGP premature the outstanding advances
made then notwithstanding.
The magnitude of progress made in rapid DNA sequencing has
been quite phenomenal.
The prohibitive cost of sequencing a human genome can be
reduced through Novel detection assays, miniaturization in
instrumentation, microfluidic separation techniques, and
increase in number of assays per run.
Novel detection assays are mostly modifications the Sanger
sequencing assay, but non-Sanger methods such as
pyrosequencing and several modification of it hold promise of
dramatically reducing the cost of genome sequencing.

Current and Developing Techniques

Sequencing By Hybridization (SBH)
The array contains all possible
oligonucleotide sequences of a given
length.
DNA of unknown sequence is incubated
with the array.
The target hybridizes to the array
Oliver
wherever there is complementation to a
portion of the target.
Limitations
Hybridization of oligos are detected by
Difficult to reconstruct long
fluorescence.
sequences.
The probes are organized by overlaps
Very large libraries are
with one another to reconstruct the target
required.
sequence.
The normal approach to SBH
is also sensitive to errors.
Latest Improvement and Advantages
Universal bases are used instead of
normal oligonucleotides.
By acting as spacers the universal
bases make consecutive probes less
dependent on one another.
These are less sensitive to errors.
Does not require larger libraries.

SBS involves detection of the identity of each nucleotide immediately

after its incorporation into a growing strand of DNA in a polymerase
reaction. The SBS includes "fluorescent in situ sequencing" (FISSEQ)
and the pyrosequencing method.

(Seo 2005)

A different fluorophore is linked to each of the four bases through a

photocleavable linker.
DNA polymerase incorporates complementary a single-nucleotide
analogue.
Unique fluorescence emission detected depends upon the nt.
incorporated.
Fluorophore is subsequently removed photochemically. The 3-OH
group is chemically regenerated and the cycle proceeds.
Advantages
Allows parallel sequencing.
Use of photons requires no additional chemical reagents.
Clean products with no need of subsequent purification.

Emulsify beads and PCR

reagents in water-in-oil
microreactors

avidin-biotin
purification of
A/B fragment

Clonal amplification
occurs inside
microreactors

Break microreactor
enrich for DNA
positive beads

4 bases (TACG)
cycled 42 times
Chemiluminescent
signal generation
Signal processing
to determine base
sequence and
quality score

Well diameter: 44m

200,000 reads obtained in
parallel
A single cloned amplified
sstDNA bead is deposited
per well

Non-Sanger nonfluorescence technique that

quantitatively measures released PPi
Pyrogram corresponds to complementary base

Source: Biotage

Applications and advantages

SNP analysis
Ideal for rapidly mutating organisms
Quantifications provide additional data
Limitations
Short sequence reads
Homopolymer repeat problems

Lab-on-a-chip concept: integration of all sequencing steps, including PCR

amplification, sample purification and capillary electrophoresis using the Sanger
sequencing method.
Integration of Separation using 384
channels, accurately sequencing
560 bases with 99% accuracy.
High throughput and decreased
(6x) analysis time.
Reduced reagent and sample vol.
Potential for low cost commercial
product.

Cyclic Reversible Terminator (CRT)

(Blazej, et al. 2006)

Sequencing by CRT consists of three steps; incorporation, imaging and

deprotection. The reversible terminator must be cleaved efficiently with
photocleaving groups like 2-nitrobenzyl group.

Disadvantage
Failure in cleaving dye moiety.

Advances and declining costs for sequencing technology will yield

accessible genotype- phenotypic information to the scientific community. Rising
issues within the scientific community and the public include identifiably of
individuals, intellectual property vs. individual property rights to genomic
sequences, requirements to share research results, and targeting research
towards/away from certain races and cultures based on cost benefits.

Conclusion

Determines the order of nonconsecutive nucleotide additions.

Cy3-labeled-UTP is incorporated into the primer strand, donor dye. Subsequent
incorporation of a complementary Cy5-labeled-UTP or Cy5-labeled-dCTP
substrate results ins a spFRET signal.
Photobleaching of Cy5 dye addition of natural nucleotides dATP and dGTP
addition of Cy5-labeled dNTP.

Comparative Genome Sequencing

Test DNA is hybridized with reference DNA to identify regions of genomic
differences.
Genomic different regions are sequenced to identify SNPs.
Advantages
Fast, accurate sequencing of the regions of interest.

With quicker, faster, transportable, low-cost sequencing, applications include:

Individual sequencing leading to personalized medicine- gene therapy.
Rapid identification and characterization of pathogens.
Profiling tumor subtypes for diagnosis and prognosis.
Hypothesis testing for genotype/phenotype relationships.
Understanding B- and T- cell receptor diversity to allow antibody selection.

Ethical Issues

(Mitra.)

Limitations
Low readout length.
Error prone.

Limitations
Require longer read lengths.
Rate limiting step: sample
preparation.

Applications

Polony (polymerase colony) is amplified

product from single DNA molecule in
acrylamide gel.
Sequencing done by the incorporation
of cleavable fluorescent labeled
nucleotide.
Advantage
Scalability is easy by using 1m
magnetic beads.

Advantages
High degree of parallelization.
Sparing use of reagents.

Sequencing Reaction Miniaturization

Multiplexing Reactions

Informatics

Microfluidic Separation Platforms

sstDNA library
with adaptors

Single-Pair FRET (spFRET)

Cycle continues

Automation

Data acquisition

Pyrosequencing

Selection
(isolate
AB
fragment
only)

Polony Technology

Sequencing-By-Synthesis (SBS)

Robotics

Software control

Single-nucleotide addition (SNA)

Nebulization of
genome

Advantages
Avoids gel electrophoresis, functions in highly parallel fashion, high throughput,
speed and accuracy.

(Harvard Nanopore Group)

Microfluidics

Throughput

A method for multifluorescence

discrimination of nucleotides
separated by CE.
Four laser- four dye system
excites near absorption
maximum, uniformly intense
emission signal.
Elimination of cross-talk between
dye channels.
High fluorescent signal is
collected; it is easier to resolve
(Lewis, et al. 2005)
the correct sequence.
Less processing of fluorescent
data is required.
Future Implications
Potential for a transportable and compact DNA sequencing system.
Higher sensitivity, quicker analysis, and lower cost: shortened preparation
time, reduced sample and reagent volumes, and less data processing.

Ligation

Nanopore Sequencing
Utilizes a nanoscale device that translocates polymer molecules in
sequential monomer order through a very small volume of space.
Includes a detector that directly converts characteristic features of the
translocating polymer into an electrical signal. Transduction and
recognition occur in real time, on a molecule-by-molecule basis. It can
probe thousands of different molecules in a few minutes.
It can probe very long lengths of DNA.

Sensitivity

Pulsed Multi-line Excitation (PME)

Flow diagram of SBS developed by 454 Life Sciences

Anneal sstDNA to
an excess of DNA
capture beads

Efficiency

Integration of
Technologies

Arun Ammayappan, Ernest Nyannor, Jason Sinclair, Senthilkumar Palaniyandi and Sandi Kirsch

$1000

Speed

Nanoscaling

Comparative Genome Sequencing

How do the newest-latest DNA sequencing technologies work and what applications become
possible with much cheaper sequencing?

With the advancements in sequencing and its marriage with computer

science, the face of biology has been altered. Biology will merge with computer
science, mathematics, and physics as never before (Yao 2002), adding the
advancements made. However, lets not forget about a meeting in Germany 40
years ago when one man told another that he had some ddTTP.

References
Bart Barrell, 1991. DNA sequencing: present limitation and prospects for the future. FASEB J: 5: 40-45
Robert G. Blazej et al., 2006. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA
sequencing. PNAS: 103(19): 7240-7245
Biotage. http://www.pyrosequencing.com/DynPage.aspx?id=8726&mn1=1366
Morris W. Foster and Richard R. Sharp, 2006. Ethical issues in medical-sequencing research: implications of
genotype-phenotype studies for individuals and populations. Hum. Mol. Gen.: 15(R1): R45-R49
E.K.Lewis,et al., 2005. Color-blind fluorescence detection for four-color DNA sequencing. PNAS:102(15):534651
Oliver. http://www.chem.brown.edu/faculty/oliver/slide1.htm
Mitra. http://cbcg.lbl.gov/Genome9/Talks/mitra.pdf
M. L. Metzker, 2005. Emerging technologies in DNA sequencing. Genome Res.:15(12):1767-76
NimbleGen. http://www.nimblegen.com/products/cgr/index.html
Tae Seok Seo et al., 2005. Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent
nucleotides. PNAS: 102(17): 5926-5931
454 Life Sciences. http://www.454.com/enabling-technology/the-process.asp
Caitlin Smith. 2005. Genomics: Getting down to details. Nature 435, 991-994
Harvard Nanopore Group. http://www.mcb.harvard.edu/branton/projects-NanoporeSequencing.htm
Tim T. Binnewies et al., 2006. Ten years of bacterial genome sequencing: comparative-genomics-based
discoveries. Func. Integ. Genomics 6:165-85
Frederick Sagner, 1980. Determination of Nucleotide Sequences in DNA. Nobel Lectures, Chemistry 1971-80
Frederick Sanger. Sequences, Sequences, Sequences. Annu. Rev. Biochem. 57:1-28, 1988
Toru Yao, 2002. Bioinformatics for the genomic sciences and towards systems biology. Progress in Biophysics
and Molecular Biology 80:23-42