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Since the discovery of the chemical nature of DNA in the 1950s that, it is
written in a simple four-letter code of nucleotides, and is the hereditary material in
all living organisms, sequencing, or "reading" the genetic code has become of
increasing interest to scientists. RNA sequencing was one of the earliest forms of
nucleotide sequencing. The major landmark of RNA sequencing is the sequence of
the first complete gene and the complete genome of Bacteriophage MS2,
identified and published by Walter Fiers. Prior to the mid-1970s no method
existed by which DNA could be directly sequenced. Knowledge about gene and
genome organization was based upon studies of prokaryotic organisms and the
primary means of obtaining DNA sequence was so-called reverse genetics in
which the amino acid sequence of the gene product of interest is back-translated
into a nucleotide sequence based upon the appropriate codons. Given the
degeneracy of the genetic code, this process can be tricky at best. In the mid-
1970s two methods were developed for directly sequencing DNA. These were the
Maxam-Gilbert chemical cleavage method and the Sanger chain-termination
method. Prior to the development of rapid DNA sequencing methods in the early
1970s by Frederick Sanger in England and Walter Gilbert and Allan Maxam at
Harvard, a number of laborious methods were used. For instance, in 1973, Gilbert
and Maxam reported the sequence of 24 basepairs using a method known as
wandering-spot analysis. The chain-termination method developed by Sanger and
coworkers in 1975 soon became the method of choice, owing to its relative ease
and reliability. Technical variations of chain-termination sequencing include
tagging with nucleotides containing radioactive phosphorus for radiolabelling, or
using a primer labeled at the 5 end with a fluorescent dye. Several changes took
place in these technologies owing to the high demand for low-cost sequencing and
it has driven the development of high-throughput sequencing technologies that
parallelize the sequencing process, producing thousands or millions of sequences
at once. High-throughput sequencing technologies are intended to lower the cost
of DNA sequencing beyond what is possible with standard dye-terminator
methods.
Simple point mutations can cause altered protein shape and function.
DNA
A nucleic acid, that carries the genetic information in the bodys cells. made up of
four similar chemicals called bases and abbreviated A, T, C, and G that are
repeated over and over in pairs.
DNA sequencing
Gene
A gene is a distinct portion of a cells DNA that codes for a type of protein or for
an RNA chain.
Gene sequencing
Genome sequencing:
Breaking the whole genome into small pieces, sequencing the pieces and then
reassembling them in proper order to arrive at the sequence of the whole genome.
Genomics:
1980 Frederick Sanger and Walter Gilbert receive the Nobel Prize in
Chemistry
1995 Craig Venter, Hamilton Smith, and colleagues at The Institute for
Genomic Research (TIGR) publish the first complete genome of a free-
living organism, the bacterium Haemophilus influenzae by shot gun
method.
1995 Richard Mathies et al.. publish fluorescence energy transfer dye-based
sequencing.
1996 Pal Nyren and his student Mostafa Ronaghi at the Royal Institute of
Technology in Stockholm publish their method of pyrosequencing.
1998 Phil Green and Brent Ewing of the University of Washington publish
phred for sequencer data analysis.
1999 Completion of sequencing of the chromosome 22
2000 completion of rough draft of human genome.
The newly synthesized and labeled DNA fragments are heat denatured, and
separated by size (with a resolution of just one nucleotide) by gel electrophoresis
on a denaturing polyacrylamide-urea gel with each of the four reactions run in one
of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by
autoradiography or UV light, and the DNA sequence can be directly read off the
X-ray film or gel image. A dark band in a lane indicates a DNA fragment that is
the result of chain termination after incorporation of a dideoxynucleotide (ddATP,
ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among
the four lanes are then used to read (from bottom to top) the DNA sequence.
A G T C
A
T
A
G
C
G
T
A
G
C
G
T
A
G
C
G
T
A
G
C
T
A
G
C
G
A
T
T
A
A
T
T
A
Advantages:
Works with ssDNA and dsDNA and thus eliminates the need for M13
phage
Requires only small amounts of template
Can be set up in microtitre plates or microtubes
Can use internal labeling with [-32P], [-33P],or [35S]or with 5- end
labeled primer
Can be adapted for rapid screening
High-throughput sequencing
The high demand for low-cost sequencing has driven the development of
high-throughput sequencing technologies that parallelize the sequencing process,
producing thousands or millions of sequences at once. The dye-terminator
sequencing method, along with automated high-throughput DNA sequence
analyzers, is now being used for the vast majority of sequencing projects.
Dye-terminator sequencing
The most dramatic advance in sequencing and the one that carried DNA
sequencing into a high throughput environment was the introduction of automated
sequencing using fluorescence-labeled dideoxy-terminators. In 1986, Leroy Hood
and colleagues reported on a DNA sequencing method in which the radioactive
labels, autoradiography, and manual base calling were all replaced by fluorescent
labels, laser induced fluorescence detection, and computerized base calling. In
their method, the primer was labeled with one of four different fluorescent dyes
and each was placed in a separate sequencing reaction with one of the four
dideoxynucleotides plus all four deoxynucleotides. Once the reactions were
complete, the four reactions were pooled and run together in one lane of a
polyacrylamide sequencing gel. A four-color laser induced fluorescence detector
scanned the gel as the reaction fragments migrated past. The fluorescence
signature of each fragment was then sent to a computer where the software was
trained to perform base calling. This method was commercialized in 1987 by
Applied Biosystems. Automated DNA-sequencing instruments (DNA sequencers)
can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a
day. A number of commercial and non-commercial software packages can trim
low-quality DNA traces automatically. These programs score the quality of each
peak and remove low-quality base peaks (generally located at the ends of the
sequence). Best estimates of error rates for base calling with slab gel based
sequencing is PHRED and for capillary sequencing is Life Trace.
chromatogram
Capillary electrophoresis:
In the early 1990s Harold swerdlow and colleagues reported the use of
capillaries to obtain DNA sequences. Capillaries are small, a 50m inner diameter,
and they dissipate heat very efficiently due to their high surface area to volume
ratios. This means that a capillary based system can be run with much higher
voltages thus dramatically lowering the run times. Most importantly, capillary
systems can be automated, a major limitation in gelbased systems (dye
terminater sequencing is only semi automated that too in case of base calling).
Capillaries could be flushed out after a run and replaced for the next run without
having to touch the capillary (Gupta P K 2009). DNA sequencing reactions can be
carried out in a single reaction tube and be prepared for loading once the reaction
reagents had been filtered out. Load the sequencing reaction into the capillary,
apply a constant electrical current through the capillary, and have the resolved
fragments migrate past an optical window where a laser would excite the dye
terminator, a detector would collect the fluorescence emission wavelengths, and
software would interpret the emission wavelengths as nucleotides.
CAPILLARIES
DETECTORS
OUTPUT
SIGNAL
Alternative sequencing methods: (primrose and Twyman 2003)
Pyrosequencing:
Sequencing by ligation
Sequencing by hybridization
References:
Gupta P K 2009 Cell and Molecular biology. 3rd edition Rastogi publications.
Hardiman G 2008 ultra-high-throughput sequencing, microarray based genomic
selection and pharmacogenomics. Phamacogenomics 9 (1): 5-9.
http://www.integratedDNAtechnologies.com
http://www.appliedbiosystems.com
http://www.biostudio.com
http://www.biologyanimations.com
Primrose S B and Twyman 2003 principles of genome analysis and genomics. 3 rd
edition Blackwell publishing co.
Lagerquist J 2010 nanopore based sequence specific detection of duplex DNA for
genomic profiling. Nano letters April 2010 (on line journal).
Lizardi P M A new hybridization based technique offers advantage in sequencing
genomes. Nature biotechnology 26: 648-650.