Introduction: 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-1970’s 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-1970’s 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
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Introduction:
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-1970’s 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-
1970’s 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.
Need for gene sequencing:
• Understanding a particular DNA sequence can shed light on a genetic
condition and offer hope for the eventual development of treatment.
• An alteration in a DNA sequence can lead to an altered or non functional
protein, and hence to a harmful effect in a plant or animal.
• Simple point mutations can cause altered protein shape and function.
Terminology related to sequencing:
DNAA nucleic acid, that carries the genetic information in the body’s 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
Determination of the order of the nucleotide bases - adenine, guanine, cytosine, and thymine in a molecule of DNA.
Gene
A gene is a distinct portion of a cell’s DNA that codes for a type of protein or for an RNA chain.
Gene sequencing
Gene sequencing is a process in which the individual base nucleotides in an organism's DNA are identified.
Complete copy of chromosomal and extra chromosomal gene insrtuctions.
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:
Sequencing of genomes, determination of the complete set of proteins encoded by an organism and functioning of genes and metabolic path ways in an organism.
Historical facts in DNA sequencing:
1953 Discovery of the structure of the DNA double helix.
1972 Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA.
1975 The first complete DNA genome to be sequenced is that of bacteriophage φX174
1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation". Frederick Sanger, independently, publishes "DNA sequencing by enzymatic synthesis".
1980 Frederick Sanger and Walter Gilbert receive the Nobel Prize in Chemistry
1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb.
1986 Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the first semi-automated DNA sequencing machine.
1987 Applied Biosystems markets first automated sequencing machine, the model ABI 370.
1990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (at 75 cents (US)/base).
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.
Different sequencing methods:
Chemical cleavage method:
In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA
sequencing method based on chemical modification of DNA and subsequent
cleavage at specific bases by taking advantage of two step catalytic process. It
involves piperidine and two chemicals that selectively attack purines and
pyrimidines. Purines will react with dimethyl sulfate and pyrimidines will react
with hydrazine in such a way as to break the glycoside bond between the ribose
sugar and the base displacing the base (Step 1). Piperidine will then catalyze
phosphodiester bond cleavage where the base has been displaced (Step 2). The use
of these selective reactions to DNA sequencing then involved creating a