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Genomics Notes
Introduction:
Genomics is an area within genetics that concerns the sequencing and analysis of an
organism’s genome
...
Experts in genomics strive to determine complete DNA sequences and
perform genetic mapping to help understand disease
...
The
fields of molecular biology and genetics are mainly concerned with the study of the role
and function of single genes, a major topic in today’s biomedical research
...

Genomics involves the study of all genes at the DNA, mRNA, and proteome level as well
as the cellular or tissue level
...
He
initiated the practice of sequencing and genome mapping as well as developing
bioinformatics and data storage in the 1970s and 1980s
...

The term genomics was first coined in 1986 by Tom Roderick, a geneticist at the
Jackson Laboratory in Maine, during a meeting about the mapping of the human genome
...
It
includes all of the DNA, not just the genes
...

For simple viruses, with a single nucleic acid molecule, the genome is obvious,
although of course for RNA viruses it is RNA rather than DNA
...
The genome of a species will be a representation of all the DNA (or RNA in
case of RNA viruses), to make up a typical genome for the individuals of that species
...
Thus the female and male genomes will differ if the sex chromosomes are
different
...

When the phrase “genome size” was first used to describe the total haploid DNA
content, there were strong objections, based on the concept that there is additional
information in the chromosomal proteins, and even in body structures, that is necessary to
propagate the lineage
...

The majority of the DNA of a genome is not in the genes themselves and their
known associated regulatory sequence
...
In most species, a
large fraction of the DNA is repeated sequences that cause genetic recombination and
unequal crossing over, resulting in genomic rearrangements, but their overall significance
is not understood
...
Striking evidence that the actual coding capacity is likely to be relatively
constant among plants is seen when comparing the genomes of Arabidopsis and maize
...

Similarly, maize and sorghum are closely related plants that both have 10 chromosomes,
but the maize genome is more than three times the size of that of sorghum
...
In fact, several of the genes in sorghum show the same chromosomal
arrangement as their counterparts in maize
...
This finding supports
the conclusion that the majority of nuclear DNA may play a supporting role in the
structure and organization of the genome but does not contribute directly to its proteincoding capacity
...
The DNA content of
haploid eukaryotic cells (C value) ranges from 107 to 1011 bp
...
Although it has been assumed that organism
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complexity correlates roughly with genome size – humans have larger genomes than
most insects, and insects have larger genomes than fungi- this correlation is by no means
universal
...

Interestingly, genomes of plant are represented throughout the size range
...
Rice,
maize and wheat fall in between these two, having genome sizes of 5 X 108, 6
...
6 X 1010 bp, respectively
...
We have no satisfactory
explanation yet for the C-value paradox, but in plant, at least, we know that genome size
can to some degree be attributed to repetitive DNA and duplicated genomes (Polyploidy)
...
Comparative genomics
compares the content and organization of genomes of different organisms
...

Functional genomics:
Functional genomics is a field of molecular biology that attempts to make use of
the vast wealth of data produced by genomic projects (such as genome sequencing
projects) to describe gene (and protein) functions and interactions
...
Functional genomics attempts to answer questions about the
function of DNA at the levels of genes, RNA transcripts, and protein products
...

A major branch of genomics is still concerned with sequencing the genomes of
various organisms, but the knowledge of full genomes has created the possibility for the
field of functional genomics, mainly concerned with patterns of gene expression during
various conditions
...

Methods for gene discovery
Need for gene discovery

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Necessity to produce higher yield
o

Food requirement of ever increasing population

o

Non availability of lands for cultivation
How to produce higher yields

o

Developing abiotic/biotic stress tolerant genotypes

Methods to develop high yielding stress tolerant genotypes
Exploration of germplasm to identify contrasting germplasm
Candidate gene/QTL identification
Use it in breeding programs/transformation
Strategies to identify candidate genes:
QTL mapping – needs development of a segregating population
segregating for specific traits
Expression Profiling
o

RT-PCR, Northern analysis

o

DD-RT-PCR, cDNA library screening, SAGE,

o

microarrays, proteomics

Methods:
1) High-throughput Marker Genotyping
species specific genome sequences and large EST collections aid in
extensive data mining for genetic markers
genome sequences provide direct access to large numbers of microsatellite
markers and detection of SNPs
this marker resource can be used to map and fine map QTL and to identify
quantitative trait genes and causative quantitative trait nucleotides
...
Fine mapping can
be done by EST based markers
...
Useful
in directing SNPs
...

3) Mass spectrometry
Mass spectrometry is a robust device for analyzing biomolecules and suited for
SNP typing and finger printing
4) Pyrosequencing
method based on primer extension on a PCR amplified template
based on the detection of pyrophosphate, a byproduct of DNA synthesis
which is converted to ATP, which stimulates luciferase activity causing the emission of a
chemi-luminescent signal
...
A holistic analysis of gene expression in cells/tissues is
desirable
...
Microarray based transcript analysis - here the printed DNA probes are
hybridized with labeled biological targets
...

2
...
Massively Parallel Signature Sequencing (MPSS)
MPSS generates millions of short sequence tags for counting mRNA
frequencies
...
thus suitable
for studying rare transcripts
...
Proteomics:
analyzing the proteome of a genome

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point of time
...


complete set of proteins in a given tissue, cell or biological fluid at a given
Variations can be at protein synthesis, protein secretion or protein

Structural genomics:
Structural genomics seeks to describe the 3-dimensional structure of every protein
encoded by a given genome
...
The principal difference between structural genomics and traditional
structural prediction is that structural genomics attempts to determine the structure of
every protein encoded by the genome, rather than focusing on one particular protein
...
Structural genomics involves taking a large number of approaches to
structure determination, including experimental methods using genomic sequences or
modeling-based approaches based on sequence or structural homology to a protein of
known structure or based on chemical and physical principles for a protein with no
homology to any known structure
...
This raises new
challenges in structural bioinformatics, i
...
determining protein function from its 3D
structure
...
The map improves with the scientific
progress and is perfect when the genomic DNA sequencing of the species has been
completed
...
These may be genetic markers (PCR products) or
the unique sequence-dependent pattern of DNA-cutting enzymes
...
The term "mapping" is
used in two different but related contexts
...
Genetic mapping uses classical
genetic techniques (e
...
pedigree analysis or breeding experiments) to determine
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sequence features within a genome
...

Physical Mapping:
In physical mapping, the DNA is cut by a restriction enzyme
...
The resulting pattern of DNA migration (i
...
,
its genetic fingerprint) is used to identify what stretch of DNA is in the clone
...
Now a good choice of clones can be made
to efficiently sequence the clones to determine the DNA sequence of the organism under
study (seed picking)
...
There are alternative ways to determine how DNA in a group of clones overlap
without completely sequencing the clones
...
This type of
mapping is more accurate than genetic maps
...

Physical mapping involves finding a contiguous series (or ‘contig’) of cloned
DNA fragments which contain overlapping portions of the genome
...
If at least some of the clones contain
markers that have been independently mapped by other means, then the position of the
entire contig in the genome is also known
...
(Complete restriction digestion
of the DNA would mean that no clones could be found that overlapped across the
restriction sites for that enzyme)
The fragments are usually cloned in bacterial hosts (normally Escherichia coli),
using bacteriophage, cosmid, plasmid or other vector systems
...
Even larger fragments (over 1 Mb) can be cloned in yeast
(Saccharomyces cerevisiae) using yeast artificial chromosome (YAC) vectors; however,
such clones are often unstable, undergoing deletions or internal rearrangements
...
In STS content mapping, the library is screened to identify all
clones that contain a specific STS marker
...
In either case, all clones containing a given STS marker are identified
...
If one clone contains two
different STSs, then those two STSs must be consecutive in the genome
...

A second common approach to physical mapping is by the use of restriction
fingerprinting
...
If two clones are found that have several fragment sizes in common, then
they must represent overlapping parts of the genome, with the shared fragments coming
from the region of overlap
...

Physical mapping is also vulnerable to a variety of clone artefacts
...
Clones that have suffered internal deletions or rearrangements can
also cause errors
...
Despite its limitations, however, physical mapping has
the unique advantage that, as well as producing a map giving the relative locations of the
clones (and of any STS markers they are known to contain), it also produces a tangible
resource – the clones themselves – representing known segments of the genome
...

Grid libraries:
Traditionally, library screening by hybridization involves taking a plaque lift or
colony blot, which generates a replica of the distribution of clones on an agar plate
...
Once a laborious process, gridding or arraying has been
considerably simplified through the use of robotics
...
Using traditional
libraries, positive clones are detected by autoradiography and the X-ray film must be
aligned with the original plates in order to identify the corresponding plaques
...
Since
identical sets of membranes can be easily prepared, duplicates can be distributed to other
laboratories for screening
...
Thus, one
library can serve a number of different users and all data can be centralized
...
Therefore, they are often prepared for high-value libraries with
wide applications, such as genomic libraries cloned in high-capacity P1, BAC or YAC
vectors and also for valuable cDNA libraries
...

Map resolution:
The first method for assembling short, sequenced fragments into a wholegenome sequence, called a map-based approach, requires the initial creation of detailed
genetic and physical maps of the genome, which provide known locations of genetic
markers (restriction sites, other genes, or known DNA sequences) at regularly spaced
intervals along each chromosome
...

This process leads to gaps in assigning the order of clones that have
sequenced
...

If it is having low number of gaps then it is termed as high resolution and if the sequence
contains more number of gaps, it is called as low-resolution physical mapping
...

The first methods for quickly sequencing DNA were developed between 1975 and 1977
...
The Sanger method quickly became the
standard procedure for sequencing any purified fragment of DNA
...
The fragment
to be sequenced is used as a template to make a series of new DNA molecules
...

The method relies on the use of a special substrate for DNA synthesis
...
In DNA synthesis, two phosphate groups on the 5’carbon atom of a dNTP are removed, and a phosphodiester bond is formed between the
remaining 5’-phosphate group of the dNTP and the 3’ OH group of the last nucleotide on
the growing DNA chain
...
The ddNTPs are identical with
dNTPs, except that they lack a 3’-OH group
...
When a ddNTP has been incorporated into a DNA chain, however, no more
nucleotides can be added, because there is no 3’-OH group to form a phosphodiester
bond with an incoming nucleotide
...
A single
DNA molecule cannot be sequenced; so any DNA fragment to be sequenced must first be
amplified by PCR or by cloning in bacteria
...
Each part is placed in a different tube, to which are added:
1
...
All four deoxyribonucleoside triphosphates (dCTP, dATP, dGTP, and dTTP), the
normal precursors of DNA synthesis;
3
...
DNA polymerase
...
Within each of the four tubes, the DNA
polymerase enzyme carries out DNA synthesis
...
Within this tube, each of the single strands of
target DNA serves as templates for the DNA synthesis
...
DNA polymerase elongates a new strand of DNA from
this primer, by using the target DNA strand as a template
...
Because there is more dATP than ddATP
in the reaction mixture, dATP is incorporated most often, allowing DNA synthesis to
continue
...
The incorporation of ddA into the new strand occurs randomly at different
positions in different copies, producing a set of DNA chains of different length, each
ending in a nucleotide with adenine
...


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In the tube that received ddCTP, all the chains terminate in a nucleotide with
cytosine; in the tube that received ddGTP, all the chains terminate in a nucleotide with
guanine; and, in the tube that received ddTTP, all the chains terminate in a nucleotide
with thymine
...

The contents of the four tubes are separated side by side on an acrylamide gel so
that DNA strands differing in length by only a single nucleotide can be distinguished
...
The shortest strands, which terminated at positions early in the DNA
sequence, migrate quickly and end up near the bottom of the gel; longer fragments, which
terminated late in the sequence, migrate more slowly and end up near the top of the gel
...
The
next band up is from the tube that contained ddATP; so the next nucleotide in the
sequence is adenine (A), and so forth
...

Keep in mind that the sequence obtained is not that of the target DNA but that of its
complement
...
The trick is to
insert a sequence that will be recognized by the primer into the target DNA
...
The target DNA is then isolated from the vector and will
contain universal sequencing primer sites at each end
...
The dideoxy
reaction is also used here, but the ddNTPs used in the reaction are labeled with a
fluorescent dye, and a different colored dye is used for each type of dideoxynucleotide
...

In this case, the four sequencing reactions can take place in the same test tube and can be
placed in the same well during electrophoresis, given that each ddNTP is distinctively
marked
...
The different-sized fragments produced by the sequencing
reaction separate within a tube and migrate past a laser beam and detector
...
Each colored dye emits
fluorescence of a characteristic wavelength, which is read by the optical scanner
...
First, manual sequencing can generate excellent data, but even in the best sequencing
laboratories poor autoradiographs are frequently produced that make sequence reading difficult
or impossible
...
Skilled DNA sequencers ignore bad sequencing tracks, but many
laboratories do not
...
The use of a single-gel track for all
four dideoxy reactions means that this problem is less acute in automated sequencing
...
It should be noted that long runs of the same
nucleotide or a high G+C content can cause compression of the bands on a gel, necessitating
manual reading of the data, even with an automated system
...
The second advantage of automated
DNA sequencers is that the output from them is in machine-readable form
...
A third advantage
derives from the new generation of sequencers that have been introduced recently
...
The key
feature of this system is that the equipment has been designed for use with robotics, thereby
minimizing hands-on time and increasing throughput
...


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Maxam-Gilbert procedure:

In this procedure single-stranded DNA is subjected to several chemical treatments that
cleave the DNA molecules and generate a family of short single stranded fragments
...
Several cleavage protocols are used,
each of which is base specific and provides the base sequence by the following rule
...
Thus, if a 36-base fragment results from the protocol that
identified guanine, then it is known that guanine is the 37th base in the original molecule
...
The fragment to be sequence is processed
in the following manner
...
The radioactive DNA is then denatured with
alkali and two single strands are purified usually by Electrophoretic separation
...
However, not only do the two determinations serve as a check on one another, but,
as will be seen shortly
...

The sequencing procedure is following
...
To one portion (I) is added dimethylsulfate, which methylates
purines, however, G is methylated five times more effectively than A
...
Since methylation occurs at random position, the particular
A/G that is methylated differs in each strand
...
Sample Ia is treated, a treatment that removes all methylated bases, leaving
the deoxyribose
...
This heating cleavage protocol generates a set of
fragments of varying size, and the differing number of nucleotides in each fragment is
determined by the different positions of the methylated G/A
...
Sample Ib is not heated but
instead with treated with dilute acid, which removes mainly methylated A (and some G)
...

Thus, in sample Ib fragments are produced whose size is determined mainly by the position of
the methylated A, but some G- these are called the A+G fragments
...

The two samples Ia (G only) and Ib(A+G) are now electrophresed in a 20%
polyacrylamide gel containing 8M urea, a denaturant that prevents hydrogen bonding and hence
keep the fragments single stranded
...
The single terminal 32P
atom, which was enzymatically added before the methylation reaction, is the sole source of
radioactivity
...

The position of A and G in the single strand is determined by the following rules: 1
...
2
...

So far, the analysis has used only sample I
...
This sample is also divided into two portions, II a and IIb, which
are reacted with hydrazine in either dilute buffer (sample IIa) or in 2 M NaCl (sample IIb)
...

Cleavage at the site of hydrazine reaction is accomplished by treatment with peperidine, which
breaks the sugar phosphate backbone at the 5’ side of each base that has reacted with hydrazine
...
Thus,
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after electrophoresis and autoradiography the positions of C and T are determined by the
following rules
...
If a fragment containing n nucleotides is present only in the (C+T) lane (sample IIa),
there is a T at position n+1 in the original molecule;
2
...

All the four samples (Ia, Ib, IIa and IIb) are usually electrophoresed simultaneously, so all
bands are seen in the single gel
...
The
shortest fragments are those that move the fastest and farthest
...

If only one single strand of the original double stranded DNA molecule were analyzed in the
way just described, the complete base sequence could not be obtained
...

First, if the cleavage is at position n+1 (counting from the 32P labeled terminus), the number of
bases in the fragment is n
...

Second, the mononucleotide that would identify the penultimate base cannot (for technical
reasons) be detected and unambiguously
...
Sequencing both
strands also has the advantage that the sequence determined from one strand confirms the
sequence of the other strand
...
To obtain the sequences of the molecule containing 1000s of nucleotides one
uses sets of overlapping fragments
...
The genomic features may include the DNA sequence, genes, gene
order, regulatory sequences, and other genomic structural landmarks
...

The major principle of comparative genomics is that common features of two organisms will
often be encoded within the DNA that is evolutionarily conserved between them
...
Based on these,
genome and molecular evolution are inferred and this may in turn be put in the context of, for
example, phenotypic evolution or population genetics
...
With the explosion in the number of genome project due to the advancements in DNA
sequencing technologies, particularly the next-generation sequencing methods in late 2000s, this
field has become more sophisticated, making it possible to deal with many genomes in a single
study
...

It has also showed the extreme diversity of the gene composition in different evolutionary
lineages
...
"Dynamics of Genome Rearrangement in
Bacterial Populations"
...


•

Sequence and analysis of chromosome 4 of the plant arabidopsis thaliana, K
...

Schuèller, R
...
Murphy, Nature, vol 402 ,16 december 1999
...


•

Hongyan Zhu, Hong-Kyu Choi, Douglas R
...
Shoemaker, Bridging
Model and Crop Legumes through Comparative Genomics, Comparative Genomics of
Legumes, Plant Physiology, April 2005
...
nature

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