1. PowerPoint Presentation Materials to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 20
STRUCTURAL GENOMICS
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2. 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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3. 1.83 million bp
~ 1,743 genes
Figure A complete map of the genome of the bacterium Haemophilus influenzae
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4. Genome sequences of other organisms are examined later
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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5. 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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6. 1. Cytogenetic mapping 2. Linkage mapping 3. Physical 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
Distances computed in map units (or centiMorgans)
3. Physical mapping
Relies on DNA cloning techniques
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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7. 20-7 Figure 20.2 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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8. Cytogenic Mapping Cytogenic mapping relies on microscopy
It is commonly used with eukaryotes which have much larger chromosomes
Eukaryotic chromosomes can be distinguished by
Size
Centromeric locations
Banding patterns
Chromosomes are treated with particular dyes
The banding pattern that results is used for mapping
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9. Cytogenic mapping relies on light microscopy
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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10. 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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11. 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 The technique of fluorescence in situ hybridization (FISH)
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12. 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
Remember that the probe will only bind a specific sequence
The results of the FISH experiment are then compared to Giemsa-stained chromosomes
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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13. Fig. 20.4

14. 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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15. 20-14

16. 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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17. 20-16 Figure 20.5 Restriction fragment length polymorphisms (RFLPs)
Arrows indicate sites cut by a restriction enzyme
Restriction site only found in individual 1
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 Restriction fragment length polymorphisms (RFLPs)
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18. EcoRI sites PRESENT on both chromosomes
EcoRI sites ABSENT from both chromosomes
Figure An RFLP analysis of chromosomal DNA from three different individuals
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19. EcoRI site found only on one chromosome
The three individuals share many DNA fragments that are identical in size
Polymorphic bands are indicated at the arrows
Indeed, if these segments are found in 99% of individuals in the population, they are termed monomorphic
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20. 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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21. Same three individuals as those of Figure 20.6
RFLPs are always inherited in a codominant manner
A heterozygote (individual 3) will have two bands of different lengths
A homozygote (individuals 1 and 2) will display only one band
Figure Southern blot hybridization of a specific RFLP
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22. 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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23. Figure 20.8
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24. 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
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25. 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
lod score = log10
Probability of a certain degree of linkage
Probability of independent assortment
For example, if the lod score is 3
3 is the log10 of 1000
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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26. 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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27. A few known genes are shown in red
The right side describes the map distances in map units
The left side describes the locations of RFLP markers
Figure 20.9
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28. 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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29. Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the -globin gene
Normal  -globin allele
Sickle-cell mutant -globin allele
175 bp
201 bp
Large fragment
DdeI
Ddel
376 bp
DdeI restriction sites in normal and sickle-cell alleles of
-globin gene.
Electrophoresis of restriction fragments from normal and sickle-cell alleles.
Normal allele
Sickle-cell allele
201 bp 175 bp
(a)
(b)
In this case the actual disease causing mutation also mutates a restriction site
b-globin coding sequence used as probe

30. 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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31. 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 b-globin gene
The normal allele (HbA) results in the formation of hemoglobin A
The mutant allele (HbS) results in the formation of hemoglobin S

32. Figure 20.10
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33. Three different RFLP were found among 73 individuals
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
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34. RFLPs associated with the normal b-globin gene
The observations are consistent with this diagram
RFLPs associated with the normal b-globin gene
RFLPs closely linked to the normal b-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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35. Genetic Mapping Using 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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36. 20-35 Figure 20.11 Identifying a microsatellite using PCR primers
The PCR primers specifically recognize sequences on chromosome 2
Add PCR primers
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 Identifying a microsatellite using PCR primers
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37. 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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38. Figure 20.12 Identifying pattern of microsatellites in a human pedigree
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39. A key difference between RFLPs and microsatellites
RFLPs use restriction enzymes and Southern blots
Rather difficult
Microsatellites use PCR
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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40. 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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41. 20-43 Figure 20.14 Chromosome sorting
Chromosomes are stained with two fluorescent dyes:
Hoescht 33258
Binds to AT-rich DNA
Chromomycin A3
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
This device can separate chromosomes at the amazing rate of 1,000 to 2,000 per second
Figure 20.14
Chromosome sorting
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42. 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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43. The numbers denote the order of the members of the contig
Clone individual pieces into vectors
The numbers denote the order of the members of the contig
Figure The construction of a contig
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44. 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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45. 20-47 Figure 20.15 The use of genetic markers to align a contig
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 The use of genetic markers to align a contig
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46. 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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47. 20-49 Figure 20.17 The use of YAC vectors in DNA cloning
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 The use of YAC vectors in DNA cloning
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48. Most commonly, a type of cloning vector called a cosmid is used
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
Therefore, libraries with smaller insert sizes are needed
Most commonly, a type of cloning vector called a cosmid is used
It is a hybrid between a plasmid vector and phage l
It can accept DNA fragments tens of thousands of bp long
Figure 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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49. Figure 20.18
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50. 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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51. 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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52. The number of steps required to reach the gene of interest depends on the distance between the start and end points
Figure 20.19
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53. 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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54. 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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55. 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
Cloning and characterization of genes
2. Medicine
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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56. 20-58
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