Transcript Brooker Chapter 20

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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 20
STRUCTURAL GENOMICS
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INTRODUCTION

The term genome refers to the total genetic
composition of an organism

The term genomics refers to the molecular analysis
of the entire genome of a species

Genome analysis consists of two main phases



Mapping
Sequencing
In 1995, researchers led by Craig Venter and
Hamilton Smith obtained the first complete DNA
sequence of an organism

The bacterium Haemophilus influenzae
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20-2
1.83 million bp
~ 1,743 genes
Figure 20.1
A complete map of the genome of the bacterium Haemophilus influenzae
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20-3

In 1996, the genome of the first eukaryote was completed
by a worldwide consortium led by Andre Goffeau in Belgium


Saccharomyces cerevisiae (baker’s yeast)
The genome contains 16 linear chromosomes

~ 12 million bp containing ~ 6,200 genes

Genome sequences of other organisms are examined later

Structural genomics begins with the mapping of the genome
and progresses ultimately to its complete sequencing

Functional genomics examines how the interactions of
genes produces the traits of an organism

Proteomics is the study of all the proteins encoded by the
genome and their interactions
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20.1 CYTOGENETIC AND
LINKAGE MAPPING

There are three common ways to determine the
organization of DNA regions

1. Cytogenetic mapping

Also called cytological mapping

2. Linkage mapping

3. Physical mapping
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
1. Cytogenetic mapping



Relies on microscopy
Genes are mapped relative to band locations
2. Linkage mapping


Relies on genetic crosses
Genes are mapped relative to each other


3. Physical mapping


Relies on DNA cloning techniques
Genes are mapped relative to each other


Distances computed in map units (or centiMorgans)
Distances computed in number of base pairs
Figure 20.2 compares these three types of maps for two
genes in Drosophila melanogaster


sc  scute, an abnormality in bristle formation
w  white eye
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20-6
Obtained from
analysis of polytene
chromosomes
Note: Correlations
between the three
maps often vary from
species to species
and from one region
of the chromosome to
another
Figure 20.2
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20-7
Cytogenic Mapping

Cytogenic mapping relies on microscopy


Eukaryotic chromosomes can be distinguished by




It is commonly used with eukaryotes which have much
larger chromosomes
Size
Centromeric locations
Banding patterns
Chromosomes are treated with particular dyes

The banding pattern that results is used for mapping
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20-8

Cytogenic mapping tries to determine the location
of a particular gene relative to a banding pattern


It is often used as a first step in the localization of genes
in plants and animals
Cytogenic mapping relies on light microscopy

Therefore, it has a fairly crude limit of resolution


In most species, it is accurate within limits of ~ 5 million bp
The resolution is much better in species that have
polytene chromosomes

Such as Drosophila melanogaster
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20-9
In situ Hybridization

In situ hybridization can locate the position of a gene
at a particular site within an intact chromosome



It is used to map the locations of genes or other DNA
sequences within large eukaryotic chromosomes
Researchers use a probe to detect the “target” DNA
The most common method uses fluorescently
labeled DNA probes

This is referred to as fluorescence in situ hybridization
(FISH)
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20-10
Cells are treated with agents that make them swell
and are then fixed on slides
DNA probe has been
chemically modified to
allow the fluorescent
label to bind to it
Figure 20.3 The technique of fluorescence in situ hybridization (FISH)
20-11

To detect the light emitted by a fluorescent probe, a
fluorescence microscope is used

The fluorescent probe will be seen as a colorfully glowing
region against a nonglowing background


The results of the FISH experiment are then
compared to Giemsa-stained chromosomes


Remember that the probe will only bind a specific sequence
Thus, the location of a probe can be mapped relative to the
G banding pattern
Figure 20.4 illustrates the results of a FISH
experiment involving six different probes
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20-12
Fig. 20.4
Linkage Mapping

Linkage mapping relies on the frequency of
recombinant offspring to map genes


Geneticists have realized that regions of DNA, which need
not encode genes, can be used as genetic markers
A molecular marker is a DNA segment that is found
at a specific site and can be uniquely recognized

As with alleles, the characteristics of molecular markers
may vary from individual to individual

Therefore, the distance between linked molecular markers can be
determined from the outcome of crosses
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20-14
Restriction Fragment Length
Polymorphisms (RFLPs)

Restriction enzymes recognize specific DNA
sequences and cleave the DNA at those sequences

Along a very long chromosome, a particular
restriction enzyme will recognize many sites

These are randomly distributed along the chromosome

When comparing two individuals, a given restriction
enzyme may produce certain fragments that differ in length
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20-15
Restriction site only
found in individual 1
Arrows indicate
sites cut by a
restriction enzyme
Thus, there is a polymorphism in the
population with regard to the length
of a particular DNA fragment
This variation can arise as a result
of deletions, duplications,
mutations, etc.
Figure 20.5 Restriction fragment length polymorphisms (RFLPs)
20-16
EcoRI sites
PRESENT on both
chromosomes
EcoRI sites
ABSENT from both
chromosomes
Figure 20.6
An RFLP analysis of chromosomal DNA from three different individuals
20-17
EcoRI site found
only on one
chromosome
The three individuals share
many DNA fragments that
are identical in size
Indeed, if these segments
are found in 99% of
individuals in the
population, they are termed
monomorphic
Polymorphic
bands are
indicated at
the arrows
20-18

In actual RFLP analysis, DNA samples
containing all chromosomal DNA would be
isolated


EcoRI digestion would yield so many fragments
that the results would be very difficult to analyze
To circumvent this problem, Southern blotting
is used to identify RFLPs
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20-19
RFLPs are always inherited
in a codominant manner
Same three
individuals
as those of
Figure 20.6
A heterozygote (individual
3) will have two bands of
different lengths
A homozygote (individuals
1 and 2) will display only
one band
Figure 20.7 Southern blot hybridization of a specific RFLP
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20-20
The Distance Between Two Linked
RFLPs Can Be Determined

We can map the distance between two RFLPs by
making crosses and analyzing the offspring

However, we look at bands on a gel rather than
phenotypic characteristics
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20-21
Figure 20.8
20-22
If the RFLPs are not linked,
a 1:1:1:1 ratio of all four
types would be expected in
the offspring
(due to independent
assortment)
If the RFLPs were linked, a
higher percentage of
parentals would be
expected
In fact, there are
more parental
offspring
Therefore
the RFLPs
are linked
Figure 20.8
20-23

The likelihood of linkage between two RFLPs is determined
by the lod (logarithm of odds) score method

A statistical test developed by Newton Morton in 1955

Computer programs analyze pooled data from a large number of
pedigrees or crosses involving many RFLPs
They determine probabilities that are used to calculate the lod score



Probability of a certain degree of linkage
Probability of independent assortment
For example, if the lod score is 3
 3 is the log
10 of 1000


lod score = log10
Therefore, there is a 1000-fold greater probability that the markers are
linked than assorting independently
A lod score of + 3 or higher is traditionally considered as evidence of
linkage
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20-24
RFLP Maps

RFLP linkage analysis can be conducted on many
different RFLPs to determine their relative locations
in the genome

A genetic map composed of many RFLP markers is
called an RFLP map


RFLP maps are used to locate genes along particular
chromosomes
Figure 20.9 shows a simplified RFLP map of the five
chromosomes of the plant Arabidopsis thaliana
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20-25
A few known
genes are
shown in red
The left side
describes the
locations of
RFLP markers
Figure 20.9
The right side
describes the
map distances
in map units
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20-26
Experiment 20A: RFLP Analysis
and Disease-Causing Alleles

RFLP analysis can be used to determine if a
person is heterozygous for a disease-causing
allele
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20-27
Using restriction fragment analysis to distinguish the
normal and sickle-cell alleles of the -globin gene
Normal  -globin allele
201 bp
175 bp
In this case the actual disease
causing mutation also mutates
a restriction site
DdeI
DdeI
Large fragment
DdeI
DdeI
Sickle-cell mutant -globin allele
Large fragment
376 bp
Ddel
Ddel
Ddel
(a) DdeI restriction sites in normal and sickle-cell alleles of
-globin gene.
Normal Sickle-cell
allele
allele
-globin coding sequence
used as probe
Large
fragment
376 bp
201 bp
175 bp
(b) Electrophoresis of restriction fragments from
normal and sickle-cell alleles.

The assumption is that a disease-causing
allele had its origin in a single individual,
known as a founder



The founder lived many generations ago
Since that time the allele has spread throughout
the human population
A 2nd assumption is that the founder is likely to
have had a polymorphic marker near the
mutant allele

Therefore, the two will be linked for many
generations
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20-28
Using RFLPs as Genetic
Markers

In 1978, Yuet Kan and Andree Dozy
confirmed that RFLP markers can be
used to predict heterozygosity

Their experiment focused on the -globin
gene


The normal allele (HbA) results in the formation
of hemoglobin A
The mutant allele (HbS) results in the formation
of hemoglobin S
Figure 20.10
20-30
Interpreting the Data
Three different RFLP were found among
73 individuals
The occurrence of these RFLPs was
not at random, with regard to the
HbA and HbS alleles
For example, the 13 Kb RFLP was usually
found in persons who were known to
have at least once copy of the HbS allele
20-32

The observations are consistent with this diagram
RFLPs associated
with the normal
-globin gene
RFLPs closely
linked to the normal
-globin gene

This type of information can be used as a predictive tool


An individual found to be heterozygous, 7.6/13, is fairly likely to be
heterozygous (HbAHbS), and thus a carrier of the mutant allele
An individual found to be homozygous, 7.6/7.6, is fairly likely to be
homozygous for the normal HbA allele
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20-33
Genetic Mapping Using Microsatellites

Microsatellites




Short, simple sequences
Abundantly dispersed throughout a species’
genome
Variable in length among different individuals
The most common human microsatellite is the
sequence (CA)n , where n may range from 5 to
more than 50

(CA)n is found about every 10,000 bases in the
genome
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20-34
Add PCR
primers
The PCR
primers
specifically
recognize
sequences on
chromosome 2
The amplified region is called a
sequence-tagged site (STS)
The two STS copies in this case
are different in length
Therefore, their microsatellites
have different numbers
of CA repeats
Figure 20.11 Identifying a microsatellite
using PCR primers
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20-35
Genetic Mapping Using Microsatellites

The inheritance pattern of microsatellites can be
studied


Indeed, PCR amplification of particular microsatellites
provides an important strategy for analysis of pedigrees
This idea is shown in Figure 20.12

Prior to this analysis, a unique segment of DNA containing
a microsatellite has been identified

PCR amplification (Figure 20.11) provides a mechanism to
test for this microsatellite in a family of five
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20-36
Figure 20.12 Identifying pattern of microsatellites in a human pedigree
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20-37

A key difference between RFLPs and microsatellites

RFLPs use restriction enzymes and Southern blots


Microsatellites use PCR


Rather difficult
Relatively easy
A newer kind of molecular marker combines the
above two approaches


The markers are termed amplified restriction fragment
length polymorphisms (AFLPs)
To identify AFLPs, chromosomal DNA is digested with one
to two restriction enzymes

Specific fragments are then amplified via PCR
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20-38
20.2 PHYSICAL MAPPING

Physical mapping requires the cloning of many
pieces of chromosomal DNA

The cloned DNA fragments are then characterized by




1. Size
2. Genes they contain
3. Relative locations along a chromosome
In recent years, physical mapping studies have led to
the DNA sequencing of entire genomes
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20-39

Chromosomes are stained
with two fluorescent dyes:

Hoescht 33258


Chromomycin A3


Binds to AT-rich DNA
Binds GC-rich DNA
Thus, each chromosome
will have a distinct level
of fluorescence
Excites the
fluorescent dyes
Chromosome is
given a negative
charge if the
detector indicates it
is the desired
chromosome
Stream of
chromosomes
separated into
individual droplets

Thus, a chromosome can
be separated from a
mixture of chromosomes

Figure 20.14
This device can separate
chromosomes at the
amazing rate of 1,000 to
2,000 per second
Chromosome sorting
20-43
Physical Maps of Chromosome
are Constructed from Contigs

A sample of chromosomal DNA can be digested into
many smaller pieces with restriction enzymes


The fragments can then be cloned into vectors to create a
chromosome-specific library
The next step is to organize the chromosome pieces
according to their exact location on a chromosome


To do this, researchers need a series of clones that contain
overlapping pieces of chromosomal DNA
Such a collection of clones, is known as a contig

It contains a contiguous region of a chromosome that is found as
overlapping regions within a group of vectors
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20-44
Clone individual pieces
into vectors
The numbers denote the order of the
members of the contig
Figure 20.15 The construction of a contig
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20-45

Different experimental strategies can be used to
align the members of a contig




Southern blotting
Use of molecular markers (such as STSs)
Analysis of restriction enzyme digests
An ultimate goal of physical mapping is to obtain a
complete contig for each chromosome in a genome

Geneticists can also correlate cloned DNA in a contig with
markers obtained from linkage or cytological methods

In Figure 20.16, two members of a contig carry genes
already mapped to be ~ 1.5 mu apart on chromosome 11
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20-46

Genes A and B had been mapped previously to specific regions of
chromosome 11



Gene A was found in the insert of clone #2
Gene B was found in the insert of clone # 7
So Genes A and B can be used as genetic markers (i.e., reference points)
to align the members of the contig
Figure 20.15 The use of genetic markers to align a contig
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20-47
YACs and BACs

To create contigs of eukaryotic genomes, the cloning
vectors have to accept large chromosomal fragments


In general, most plasmid and viral vectors cannot accept
inserts that are larger than a few tens of thousands bp
However, other cloning vectors can!

Yeast artificial chromosomes (YACs)


Inserts can be several hundred thousand to 2 million bp long
Bacterial artificial chromosomes (BACs)

Inserts can be up to 500,000 bp long
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20-48
EcoRI is found at
low concentrations
Each arm has a different
selectable marker
Therefore, it is possible
to select for yeast cells
with YACs that have
both arms
Figure 20.17 The use of YAC vectors in DNA cloning
20-49

YACs and BACs are commonly the first step in
creating a rough physical map of the genome

However, their large insert sizes make them difficult to use
in gene cloning and sequencing experiments


Most commonly, a type of cloning vector called a
cosmid is used



Therefore, libraries with smaller insert sizes are needed
It is a hybrid between a plasmid vector and phage l
It can accept DNA fragments tens of thousands of bp long
Figure 20.18 compares the cytogenic, linkage and
physical maps of chromosome 16

This is a very simplified map

A more detailed map takes over 10 pages of your textbook to print!
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20-50
Figure 20.18
20-51
Positional Cloning Can Be Achieved
Using Chromosome Walking

Positional cloning is a strategy to clone a gene
based on its mapped position along a chromosome

This approach has been successful in the cloning of many
human genes, especially those that are disease-causing


Examples: Cystic fibrosis, Huntington disease
A common method used in positional cloning is
chromosome walking

A gene’s position relative to a marker must be known

This provides a starting point to molecularly “walk” toward the gene
of interest
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20-52

Figure 20.19 considers a chromosome walk in order
to locate a gene we will call gene A



Genetic crosses had earlier shown that gene A is
approximately 1 mu away from another gene, gene B
A cloned DNA fragment of gene B is used as the starting
point to walk to gene A
The chromosome walk consists of a series of
subcloning and library screening

In subcloning a small piece of one clone is inserted into
another vector
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20-53
The number of steps
required to reach the gene of
interest depends on the
distance between the start
and end points
Figure 20.19
20-54
The Human Genome Project

In 1988, the NIH established an Office of Human
Genome Research, with James Watson as director

The human genome project officially began on
October 1, 1990


It has been the largest internationally coordinated
undertaking in the history of biological research
From the outset the goals of the human genome
project are the following:
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20-55

1. To obtain a genetic linkage map of the human genome

2. To obtain a physical map of the human genome

3. To obtain the DNA sequence of the entire human genome

4. To develop technology for the management of human
genome information

5. To analyze the genomes of other model organisms

6. To develop programs focused on understanding and
addressing the ethical, legal, and social implications of
the results obtained from the Human Genome Project

7. To develop technological advances in genetic
methodologies
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20-56
Many Genomes Have Been Sequenced

In just a couple of decades, our ability to map and
sequence genomes has improved dramatically

Motivation behind genome sequencing projects
come from a variety of sources

1. Basic research


2. Medicine


Cloning and characterization of genes
Identification of genes that (when mutant) play a role in disease
3. Agriculture

Development of new strains of organisms with improved traits
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20-57
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