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PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission.

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Presentation on theme: "PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission."— Presentation transcript:

1 PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 20 STRUCTURAL GENOMICS

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 20-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3 20-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 20.1 A complete map of the genome of the bacterium Haemophilus influenzae 1.83 million bp ~ 1,743 genes

4 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-4

5 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20.1 CYTOGENETIC AND LINKAGE MAPPING 20-5

6 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 Distances computed in map units (or centiMorgans) 3. Physical mapping Relies on DNA cloning techniques Genes are mapped relative to each other 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-6

7 20-7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 20.2 Note: Correlations between the three maps often vary from species to species and from one region of the chromosome to another Obtained from analysis of polytene chromosomes

8 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Cytogenic Mapping 20-8

9 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-9

10 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) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In situ Hybridization 20-10

11 20-11 Figure 20.3 The technique of fluorescence in situ hybridization (FISH) 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

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-12

13 Fig. 20.4

14 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Linkage Mapping 20-13

15 20-14

16 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Restriction Fragment Length Polymorphisms (RFLPs) 20-15

17 20-16 Figure 20.5 Restriction fragment length polymorphisms (RFLPs) 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 Arrows indicate sites cut by a restriction enzyme This variation can arise as a result of deletions, duplications, mutations, etc.

18 Figure 20.6 An RFLP analysis of chromosomal DNA from three different individuals EcoRI sites PRESENT on both chromosomes EcoRI sites ABSENT from both chromosomes

19 20-18 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 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-19

21 Figure 20.7 Southern blot hybridization of a specific RFLP 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

22 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Distance Between Two Linked RFLPs Can Be Determined 20-21

23 Figure

24 Figure If the RFLPs are not linked, a 1:1:1:1 ratio of all four types would be expected in the offspring If the RFLPs were linked, a higher percentage of parentals would be expected (due to independent assortment) In fact, there are more parental offspring Therefore the RFLPs are linked

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 = log 10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Probability of a certain degree of linkage Probability of independent assortment For example, if the lod score is 3 3 is the l og 10 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

26 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RFLP Maps 20-25

27 Figure The left side describes the locations of RFLP markers The right side describes the map distances in map units Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A few known genes are shown in red

28 RFLP analysis can be used to determine if a person is heterozygous for a disease-causing allele Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Experiment 20A: RFLP Analysis and Disease-Causing Alleles 20-27

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 bpLarge fragment DdeI Ddel 376 bp Large fragment 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 Large fragment 201 bp 175 bp 376 bp (a) (b) In this case the actual disease causing mutation also mutates a restriction site  -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 2 nd 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-28

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  -globin gene The normal allele (Hb A ) results in the formation of hemoglobin A The mutant allele (Hb S ) results in the formation of hemoglobin S

32 Figure

33 Interpreting the Data Three different RFLP were found among 73 individuals The occurrence of these RFLPs was not at random, with regard to the Hb A and Hb S alleles For example, the 13 Kb RFLP was usually found in persons who were known to have at least once copy of the Hb S allele 20-32

34 The observations are consistent with this diagram Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 (Hb A Hb S ), 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 Hb A allele

35 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genetic Mapping Using Microsatellites 20-34

36 20-35 Figure Identifying a microsatellite using PCR primers Add PCR primers The PCR primers specifically recognize sequences on chromosome 2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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

37 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 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genetic Mapping Using Microsatellites

38 20-37 Figure Identifying pattern of microsatellites in a human pedigree Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-38

40 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20.2 PHYSICAL MAPPING 20-39

41 20-43 Figure Chromosome sorting Chromosomes are stained with two fluorescent dyes: Hoescht 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 Stream of chromosomes separated into individual droplets Chromosome is given a negative charge if the detector indicates it is the desired chromosome 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

42 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Physical Maps of Chromosome are Constructed from Contigs

43 20-45 Figure The construction of a contig Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The numbers denote the order of the members of the contig Clone individual pieces into vectors

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-46

45 20-47 Figure The use of genetic markers to align a contig Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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

46 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display YACs and BACs

47 20-49 Figure 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

48 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 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! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-50

49 Figure

50 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Positional Cloning Can Be Achieved Using Chromosome Walking

51 Figure 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-53

52 20-54 Figure The number of steps required to reach the gene of interest depends on the distance between the start and end points

53 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: Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Human Genome Project

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-56

55 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Many Genomes Have Been Sequenced

56 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20-58


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