Download Genomes 3/e

Terry Brown
Genomes
Third Edition
Chapter 3:
Mapping Genomes
Copyright © Garland Science 2007
Chapter Objectives
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Why mapping is important before sequencing of the
genome?
What are genetic and physical maps?
Learning the techniques to construct genetic map of a
genome
The role of linkage analysis in map construction
Learning the techniques to construct physical map of
a genome
Objectives of
Today’s Lecture
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Importance of genome mapping before sequencing of
the genome?
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Understanding the way genome are mapped
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Genetic Mapping
• Linkage Analysis
Importance of genome
mapping before sequencing of
the genome?
Mapping Genomes
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Genome sequencing methodology depends on
sequencing technology available.
Even the most sophisticated techniques available
now can sequence about 750bp in a single
experiment.
So we need to construct the sequence of long
DNA molecules from a series of shorter
sequences.
By breaking the molecule into fragments and then
determining the sequence of each one.
Then assemble the sequence by searching over
lap and then build up the master sequence.
This method is known as shotgun method.
Mapping Genomes
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The Shotgun method works very well with the
small prokaryotic genomes.
The complexity of analysis increases
disproportionately when large genomes are
analyzed.
• The number of possible over laps increases when
number of fragments increases like:
•
2n2-2n for 2 fragments we there would be 4 possible
over laps while for 4 fragments where would be 24!
• The problems with reparative sequence
Mapping Genomes
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So the shotgun sequencing technique can not be
applied solely in sequencing of larger eukaryotic
genomes.
Rather a genome map is generated first which provide
a road map for sequencing project and help in
assembly of genome sequence.
The genetic map provides gene position and other
distinctive features.
After having genome map the sequencing can proceed
in either of the two ways:
• Whole-genome shotgun method
• Clone contig method
Figure 3.3 Genomes 3 (© Garland Science 2007)
Understanding the way genome
are mapped
Genetic and Physical Maps
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The Genetic Map:
Based on the use of genetic technique to construct maps
showing the position of genes and other sequences features
on a genome.
Obtained by linkage analysis using cross-breeding
experiments and family histories (pedigrees)
Physical Maps:
Uses molecular biology techniques to examine DNA
molecules directly in order to construct map showing the
positions of sequence features, including gene.
Genetic and Physical Maps
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The Genetic Maping:
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Based on Markers (Land marks)
• Important genes
• Biochemical markers
• DNA markers
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Restriction Fragment Length Polymorphisms (RFLPs)
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Simple Sequence Length Polymorphisms (SSLPs)
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Single Nucleotide Polymorphisms (SNPs)
Linkage analysis of Markers (enable positioning of land
marks)
• Based on recombination frequencies of genes/markers
Genetic Maps
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The genetic map shows the genetic markers as land map shows
distinctive features on landscape like rivers, road and building.
Genes were the first genetic markers
The early map constructed during early twentieth century were of
genes having two alternative forms i.e. alleles with different
phenotypes.
These genes were those with can be recognized by eye i.e. pea
pod color, height of plant, shape of wing of fruit fly. Etc
The limitation of observable characters and to map those
organisms which have few visible characters like microbes, The
need of other markers were soon realized:
• Biochemical markers
• ABO blood groups
• HLA typing system
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(HLA-DRB1=290 alleles
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HLA-B= >400 alleles)
DNA Markers for Genetic Mapping
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Using gene as a marker is very useful but it has limitation.
Gene occupy very small portion/space of genome and are not
evenly distributed in the genome.
And also every gene not have allelic forms or can not
distinguishable easily.
Therefore the map based on gene is not detailed and
comprehensive.
So other features which were not a gene, were used as marker
and known as DNA markers
• Restriction fragment length polymorphisms (RFLPs)
• Simple sequence length polymorphisms (SSLPs)
• Single nucleotide polymorphisms (SNPs)
RFLP
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Based on recognized restriction sequence length EcoRI (46= 4096)
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Density : 105 RFLPs in a mammalian genome.
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Simple Sequence Length
Polymorphisms (SSLPs)
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SSLPS are array of repeat sequences that display length variations i.e.
different alleles containing different numbers of repeat units.
They can be multiallelic i.e. each SSLP can have a number of different
length variant.
They are Minisatellite or variable number of tandem repeats (VNTRs)
• Repeat unit is up to 25bp in length (not evenly distributed found at ends)
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Microsatellite or simple tandem repeats (STRs)
• Repeat unit are 13bp or less (10-30 copies 6bp) (5x105 micro >6bp in human)
Single Nucleotide Polymorphisms
(SNPs)
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The individual of a species have genome which differ at many
nucleotides positions. i.e. A in one person and G in other.
Some of these may give rise to RFLPs
There are about 4Millions SNPs in human genome (one SNP per 10kb of
eukaryotic genomes).
Theoretically each SNPs should have four alleles but most of SNPs are
biallelic ?????
The SNPs are scoured by
• oligonucleotide hybridization analysis
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DNA chips
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Solution hybridization techniques
Single Nucleotide Polymorphisms
(SNPs)
DNA microarrays and Chips
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DNA microarrays: Target sequences are spotted onto a glass or nylon
membrane of 18x18mm into 80x80=6400 spots
DNA chips: DNA target sequence are synthesized by photolithography
onto a wafer of glass or silicon with high density about 300,000 oligos
per cm2
Hybridization with an
oligonucleotide with a terminal
mismatch
Oligonucleotide ligation assay (OLA)
& Amplification refractory mutation
system (ARMS)
Linkage analysis is the basis of
genetic mapping
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Principles of Inheritance:
• Law of random segregation of alleles
• Law of independent segregation of pairs of alleles.
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Genes on the same chromosomes should
inherent together
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Linkage
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Partial linkage
Figure 3.14 Genomes 3 (© Garland Science 2007)
Figure 3.15 Genomes 3 (© Garland Science 2007)
Figure 3.16 Genomes 3 (© Garland Science 2007)
From partial linkage to genetic
mapping
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Morgan and his student Arthur Sturtevant proposed
the frequency of recombination is a measure of
distance between two genes.
This can be used to construct the order and map of
genes along the chromosome.
Limitations
• Recombination hot spot
• Double cross overs
Linkage analysis with different types
of organisms
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Linkage analysis with species such as fruit flies and mice, WHERE
PLANNED BREEDING EXPRERIMENTS ARE POSSIBLE
Linkage analysis with humans, WHERE PLANNED BREEDING
EXPRIMENTS ARE NOT POSSIBLE
Linkage analysis with bacteria, WHERE MEIOSIS DOES NOT OCCURE.
Linkage analysis with planned
breeding experiments
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First developed by Morgan and his colleague.
Based on recombination frequency calculation with the
breeding experiments. i.e. with fruit flies.
Possible for all eukaryotic systems including humans.
Ethical limitations narrows the scope of this technique for
humans???
Based on observable markers as well as DNA markers i.e.
RFLPs, SSLP and SNPs.
Use of DNA marker also makes possible the direct
observation of games i.e. sperms and ovum to calculate
recombination frequencies.
Linkage analysis by using human
pedigree
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With humans its difficult to plan
breeding experiments therefore for
finding the recombination
frequencies we need to base on the
genotypes of available marriages
and their off springs in the
pedigree.
The direct observation of sperms is
also possible but is difficult.
Example of pedigree analysis of
association of gene and a maker M
with four alleles.
Association of linkage is
established with LOD score
(Logarithm of the odds)
Genetic analysis in Bacteria
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Meiosis is not there in bacteria but they do exchange genetic material
which can recombine.
The recombination frequencies can be calculated for these
recombination if genetic difference (mutation) are associated with some
phenotypes.
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Genetic exchange occurs by:
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Conjugation
• Complete chromosome or part of it can be transferred by conjugation tube.
• Chromosomal DNA can be transferred by episome transfer integrated in
plasmid (up to 1MB)
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Transduction
• By bacteriophage (50kb)
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Transformation
• Direct from environment (less than 50kb)
Physical Mapping
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Genetic map alone is rarely sufficient for directing the
sequencing phase of genome project.
Resolution:
• The resolution of genetic map depends on the number of crossovers
that have been scored.
• This is easy with bacteria and small eukaryotes which can be grown
in huge number so many crossovers can be observed enabling the
construction of highly detailed genetic maps.
• E. coli genome sequencing project in 1990s, the genetic map
contained 1400 markers (average 1maker per 3.3 kb).
• Saccharomyces cerevisiae project (1150 makers 1 per 10kb).
• But with humans large number of progeny can not be obtained so
few cross over can be studies.
• The genetic map is not finely resolved i.e. genes several kilobases
apart may appear at same position on the genetic map
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Inaccuracy:
• Crossovers are not randome i.e. recombination hot spot etc
Physical Mapping
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We need to SUPPLEMENT and RECHECK the Genetic
map with other techniques such as Physical mapping .
Physical mapping can be done with:
• Restriction mapping
• Fluorescent in situ hybridization (FISH)
• Sequence tagged sites (STS) mapping
Restriction mapping
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We can utilize unique cutter for restriction
mapping.
We can map the restriction site in a DNA
up to 50 kb of size using
• double restriction
• and partial restriction.
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The resolution limit can be enhanced by
using special Gel electrophoresis
techniques i.e.
• Orthogonal field alteration gel
electrophoresis (OFAGE).
Direct observation of DNA molecules
for restriction sites
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We can directly observe the restriction site
on chromosomal DNA by:
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Gel Stretching
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Molecular combing
Fluorescent in situ hybridization
(FISH)
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FISH enables the position of a marker
on chromosome or extended DNA to be
directly visualized.
By hybridization with fluorescent
probe.
The FISH can also be applied to
genome clone library, enabling the
mapping of clones to genome map.
FISH can be done with Mechanically
stretch chromosomes
• Centrifugation based 20X stretching
(resolution 200-300 kb)
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Nonemetaphase chromosomes
• Interphase chromosomes (resolution 25kb)
• Fiber FISH (10kb) (Stretching of interphase
chromosome by gel stretching or
molecular combing)
Sequence tagged site mapping
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To generate a detailed physical map of a large genome we need high
resolution and high throughput techniques.
• Restriction mapping can not be applied on large genomes
• FISH provide detailed mapping but takes much time and require huge
experimentations.
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Presently the most powerful physical mapping technique of large genomes
is STS mapping.
STS is simply a short DNA sequence generally 100 to 500 bp and occurs
only once in the chromosome or genome.
STS mapping is performed by multiple STS or set of STS on
broken/fragmented chromosome/genome.
A collection of DNA fragments is madee by isolating a chromosome and
then breaking it into smaller pieces, so that in collection a single point can
be represented about five/six times.
Sequence tagged site mapping
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The mapping is performed by amplification of STS unique sequence using
PCR and looking for the presence of two different STS on the same fragment
from the collection.
The frequency of having two STS on the same fragment depends how close
they are to each other.
Closer the STS to each other higher the chance to find them together on the
more fragments.
Or frequency at which breaks occur between two markers.
Any unique DNA sequence can be
used as an STS
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The STS can be any sequence which:
• Have known sequence
• Should have unique position in chromosome/genome
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The most common STS are:
• Expressed sequence tags (ESTs) (taken from cDNA projects: limited to genes only)
• Simple sequence length polymorphism (SSLPs) (mini and micro satellite )
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Help in directly linking the genetic and physical map
• Random genomic sequences
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Fragment of DNA for STS mapping
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For STS mapping fragment of a chromosome or
genomic DNA are needed, which are known as
mapping reagents.
These can be produced in many ways:
Radiation hybrids
• The genome can fragmented by irradiation by X-ray
(3000-8000 rad) and then can be fused to make radiation
hybrids with hamster cells
• A single chromosome can be separated by technique like
flow cytometry and can be used to make radiation
hybrids
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A genomic clone library
• This can be directly mapped with STS and provide a
direct linked with STS mapping and then can be
sequenced.