Structural genomics
- 2. Structural GenomicsStructural Genomics Structural genomics is concerned with sequencing and understanding the content of genomes. The first steps in characterizing a genome is to prepare its maps: ◦ Genetic Maps ◦ Physical Maps These maps provide information about the: ◦ relative locations of genes, ◦ Molecular markers, and ◦ chromosome segments,
- 3. Genetic MapsGenetic Maps Genetic maps (also called linkage maps) provide a rough approximation of the locations of genes relative to the locations of other known genes. These maps are based on the genetic function of recombination Individuals heterozygous at two or more genetic loci are crossed, and the frequency of recombination between loci is determined by examining the progeny. Recombination frequency between two loci is 50%, then the loci are located on different chromosomes or are far apart on the same chromosome.
- 4. Recombination frequency <50%, the loci are linked. For linked genes, the rate of recombination is proportional to the physical distance between the loci. Distances on genetic maps are measured in percent recombination (centimorgans, cM) or map units.
- 5. Limitations in Genetic MapsLimitations in Genetic Maps 1st is resolution or detail. 3.4 billion base pairs of DNA and has a total genetic distance of about 4000 cM, an average of 850,000 bp/cM. Even if a marker occurred every centimorgan (which is unrealistic), the resolution in regard to the physical structure of the DNA would still be quite low. 2nd they do not always accurately correspond to physical distances between genes. Based on rates of crossing over, which vary; so the distances on a genetic map are only approximations of real physical distances along a chromosome.
- 6. Physical MapsPhysical Maps Based on the direct analysis of DNA, and they place genes in relation to distances measured in number of base pairs, kilobases, or megabases. A common type of physical map is one when a pieces of genomic DNA is cloned in bacteria or yeast. Physical maps generally have higher resolution and are more accurate than genetic maps. Physical maps are often used to order cloned DNA fragments.
- 7. A number of techniques exist for creating physical maps including: Restriction mapping, which determines the positions of restriction sites on DNA; Sequence-tagged site (STS) mapping, which locates the positions of short unique sequences of DNA on a chromosome; Fluorescent in situ hybridization (FISH), by which markers can be visually mapped to locations on chromosomes; DNA sequencing.
- 8. Genetic and physical maps may differ in relativeGenetic and physical maps may differ in relative distances and even in the position of genes on adistances and even in the position of genes on a chromosome.chromosome. • Compares the genetic map of chromosome III of yeast with a physical map determined by DNA sequencing. • There are some discrepancies between the distances and even among the positions of some genes. • In spite of these limitations, genetic maps have been critical to the development of physical maps and the sequencing of whole genomes. A physical map is analogous to a neighborhood map that shows the location of every house along a street, whereas a genetic map is analogous to a highway map that shows the locations of major towns and cities.
- 10. Restriction MappingRestriction Mapping Restriction mapping determines the relative positions of restriction sites on a piece of DNA. When a piece of DNA is cut with a restriction enzyme. The fragments are separated by gel electrophoresis. The number of restriction sites in the DNA and the distances between them can be determined by the number and positions of bands on the gel.
- 11. Example: We have sample of a linear 13,000-bp (13-kb) DNA fragment 1st sample is cut with the restriction enzyme EcoRI; Second sample of the same DNA is cut with BamHI; Third sample is cut with both EcoRI and BamHI (double digest). The resulting fragments are separated and sized by gel electrophoresis Determine the positions of the EcoRI and BamHI restriction sites on the original 13-kb fragment?
- 13. Most restriction mapping is done with several restriction enzymes, used alone and in various combinations, producing many restriction fragments. With long pieces of DNA (greater than 30 kb), computer programs are used to determine the restriction maps. Restriction mapping may be facilitated by tagging one end of a large DNA fragment with radioactivity or by identifying the end with the use of a probe.
- 14. DNA-Sequencing MethodsDNA-Sequencing Methods The most detailed physical maps are based on direct DNA sequence information. 1975 and 1977 Frederick Sanger and his colleagues created the dideoxy sequencing method based on the elongation of DNA; Allan Maxam andWalter Gilbert developed a second method based on the chemical degradation of DNA.
- 16. How the primers are constructed when we don’tHow the primers are constructed when we don’t know the sequence of DNA?know the sequence of DNA? • By cloning the target DNA in a vector that contains sequences recognized by a common primer (called universal sequencing primer sites) on either side of the site where the target DNA will be inserted. • The target DNA is then isolated from the vector and will contain universal sequencing primer sites at each end
- 17. Automated dideoxy methodAutomated dideoxy method
- 18. Sequencing an Entire GenomeSequencing an Entire Genome The ultimate goal of structural genomics is to determine the ordered nucleotide sequences of entire genomes of organisms. The main obstacle to this task is the immense size of most genomes. ◦ Bacterial genomes are usually at least several million base pairs long ◦ Eukaryotic genomes are billions of base pairs long and are distributed among dozens of chromosomes. ◦ For technical reasons, it is not possible to begin sequencing at one end of a chromosome and continue straight through to the other end; ◦ Only small fragments of DNA—usually from 500 to 700 nucleotides—can be sequenced at one time.
- 19. The DNA be broken into thousands or millions of smaller fragments that can then be sequenced. Again a problem is there: ◦ Putting these short sequences back together in the correct order. Two approaches are used to resolve this task. ◦ Map-based sequencing ◦ Whole-genome shotgun sequencing The genome of bacteriophage , consisting of 49,000 bp, was completed in 1982. In 1995, the first genome of a living organism (Haemophilus influenzae) was sequenced by Craig Venter and Claire Fraser of the Institute for Genomic Research (TIGR) and Hamilton Smith of Johns Hopkins University. By 1996, the genome the first eukaryotic organism (yeast) had been determined, followed by the genome of Eschericia coli (1997), Caenorhabditis elegans (1998), and Drosophila melanogaster (2000). The first draft of the human genome was completed in June 2000.
- 20. Map-based approachMap-based approach Map-based approach, requires the initial creation of detailed genetic and physical maps of the genome, which provide known locations of genetic markers at regularly spaced intervals along each chromosome. These markers can later be used to help align the short, sequenced fragments into their correct order. After the genetic and physical maps are available, chromosomes or large pieces of chromosomes are separated by: PFGE -large molecules of DNA or whole chromosomes are separated in a gel by periodically alternating the orientation of an electrical current. Flow cytometry: chromosomes are sorted optically by size
- 21. Each chromosome (or sometimes the entire genome) is then cut up by partial digestion with restriction enzymes. Thus partial digestion produces a set of large overlapping DNA fragments. Which are then cloned by using cosmids, yeast artificial chromosomes (YACs), or bacterial artificial chromosomes (BACs). Clones are screened with specific probe. A set of two or more overlapping DNA fragments that form a contiguous stretch of DNA is called a contig. This approach was used in 1993 to create a contig of the human Y chromosome consisting of 196 overlapping YAC clones
- 22. Each clone can be cut with a series of restriction enzymes, and the resulting fragments are then separated by gel electrophoresis. A computer program is then used to examine the restriction patterns of all the clones and look for areas of overlap. The overlap is then used to arrange the clones in order
- 23. Whole-genome shotgun sequencingWhole-genome shotgun sequencing small-insert clones are prepared directly from genomic DNA and sequenced. Powerful computer programs then assemble the entire genome by examining overlap among the small-insert clones.
- 24. Whole-genome shotgun sequencing utilizesWhole-genome shotgun sequencing utilizes sequence overlap to align sequenced fragments.sequence overlap to align sequenced fragments.
- 25. The Human Genome ProjectThe Human Genome Project The Human Genome Project is an effort to sequence the entire human genome. Begun in 1990, a rough draft of the sequence was completed by two competing teams: An international consortium of publicly supported investigators Private company Celera Genomics, both of which finished a rough draft of the genome sequence in 2000.
- 26. Data Supporting Structural GenomicsData Supporting Structural Genomics In addition to the DNA sequence of an entire genome, several other types of data are useful for genomic projects and have been the focus of sequencing efforts. Including: - SNPs - ESTs
- 27. Single-Nucleotide PolymorphismsSingle-Nucleotide Polymorphisms Are single-base-pair differences in DNA sequence between individual members of a species. Arising through mutation. Single-nucleotide polymorphisms are numerous and are present throughout genomes. In a comparison of the same chromosome from two different people, a SNP can be found approximately every 1000 bp. Because of their variability and widespread occurrence throughout the genome, SNPs are valuable as markers in linkage studies.
- 28. Expressed-SequenceTagsExpressed-SequenceTags Another type of data identified by sequencing projects consists of databases of expressed-sequence tags (ESTs). In most eukaryotic organisms, only a small percentage of the DNA actually encodes proteins; in humans, less than 2% of human DNA encodes the amino acids of proteins. If only protein-encoding genes are of interest, it is often more efficient to examine RNA than the entire DNA genomic sequence. RNA can be examined by using ESTs—markers associated with DNA sequences that are expressed as RNA. RNA Reverse transciptase cDNA Short stretches of cDNA fragments are then sequenced, and the sequence obtained (called a tag) provides a marker that identifies the DNA fragment. Expressed-sequence tags can be used to find active genes in a particular tissue or at a particular point in development.
- 29. Functional Genomics attempts to understand the function of information in genomes
- 30. Goals of functional genomicsGoals of functional genomics The goals of functional genomics include: Identifying all the RNA molecules transcribed from a genome (the transcriptome) All the proteins encoded by the genome (the proteome). Functional genomics exploits both bioinformatics and laboratory- based experimental approaches in its search to define the function of DNA sequences.
- 31. •Several methods for identifying genes and assessing their functions are available including: •In situhybridization, •DNA footprinting, •Experimental mutagenesis, •Use of transgenic animals and knockouts.
- 32. Predicting Function from SequencePredicting Function from Sequence • The nucleotide sequence of a gene can be used to predict the amino acid sequence of the protein that it encodes. •The protein can then be synthesized or isolated and its properties studied to determine its function. •This biochemical approach to understanding gene function is both time consuming and expensive. •A major goal of functional genomics has been to develop computational methods that allow gene function to be identified from DNA sequence alone, •This will bypassing the laborious process of isolating and characterizing individual proteins.
- 33. Homology searchesHomology searches Relies on comparing DNA and protein sequences from the same and different organisms. Genes that are evolutionarily related are said to be homologous. Homologous genes found in different species that evolved from the same gene in a common ancestor are called orthologs For example, both mouse and human genomes contain a gene that encodes the alpha subunit of hemoglobin; The mouse and human alpha hemoglobin genes are said to be orthologs, because both genes evolved from an alpha-hemoglobin gene in a mammalian ancestor common to mice and humans.
- 34. Homologous genes in the same organism are called paralogs By duplication of a single gene in the evolutionary past Within the human genome is a gene that encodes the alpha subunit of hemoglobin and another homologous gene that encodes the beta subunit of hemoglobin. These two genes arose because an ancestral gene underwent duplication and the resulting two genes diverged through evolutionary time, giving rise to the alpha- and beta-subunit genes; these two genes are paralogs. Homologous genes (both orthologs and paralogs) often have the same or related functions; so, after a function has been assigned to a particular gene, it can provide a clue to the function of a homologous gene.
- 35. Database Search for OrthologousDatabase Search for Orthologous Databases containing genes and proteins found in a wide array of organisms are available for homology searches. Powerful computer programs have been developed for scanning these databases to look for particular sequences. A commonly used homology search program is BLAST (Basic Local Alignment Search Tool). Suppose a geneticist sequences a genome and locates a gene that encodes a protein of unknown function. A homology search conducted on databases containing the DNA or protein sequences of other organisms may identify one or more orthologous sequences.
- 36. Database Search for ParalogousDatabase Search for Paralogous computer programs can search a single genome for paralogs. Eukaryotic organisms often contain families of genes that have arisen by duplication of a single gene. If a paralog is found and its function has been previously assigned, this function can provide information about a possible function of the unknown gene.
- 37. Other sequence comparisonsOther sequence comparisons Complex proteins often have specific domains. Each domain has its characteristic amino acid arrangement For example, certain DNA-binding proteins attach to DNA in the same way; All these proteins have a common DNA-binding domain Many protein domains have been characterized, and their molecular functions have been determined. Newly identified gene can be scanned against a database of known domains. If it encodes one or more domains of known function, the function of the domain can provide important information about a possible function of the new gene.
- 38. Phylogenetic Profile :Phylogenetic Profile : Another computational method forAnother computational method for predicting protein functionpredicting protein function Phylogenetic profiling is a bioinformatics technique in which the joint presence or joint absence of two traits across large numbers of species is used to infer a meaningful biological connection, such as involvement of two different proteins in the same biological pathway It was first introduced by Pellegrini and co-workers in 1999 In this method, the presence- and-absence pattern of a particular protein is examined across a set of organisms whose genomes have been sequenced.
- 39. Phylogenetic ProfilePhylogenetic Profile If two proteins are either both present or both absent in all genomes surveyed, the two proteins may be functionally related. The genes (proteins) found and lost together should be involved in a common function ◦ 1) being involved in the same biological pathway which is therefore incomplete without all its members in a given genome, ◦ 2) being beneficial for the phenotype in a particular environment. Consider the following proteins in four bacterial species : E. coli: protein 1, protein 2, protein 3, protein 4, protein 5, protein 6 Species A: protein 1, protein 2, protein 3, protein 6 Species B: protein 1, protein 3, protein 4, protein 6 Species C: protein 2, protein 4, protein 5
- 40. The phylogenetic profile reveals that proteins 1, 3, and 6 are either all present or all absent in all species; so these proteins might be functionally related. proteins P2 and P7 have the same pattern of presence/absence in the analyzed genomes. We could therefore infer that they are involved in the same biological process.
- 41. Gene Neighbor AnalysisGene Neighbor Analysis Genes that encode functionally related proteins are often closely linked in bacteria. For example, if two genes are consistently linked in the genomes of several bacteria, they might be functionally related.
- 42. 42 mRNA Expression: Two dominant approaches RNA sequencing DNA Microarrays
- 43. Microarrays Monitors the level of each gene: Is it turned on or off in a particular biological condition? Is this on/off state different between two biological conditions? Microarray is a rectangular grid of spots printed on a glass microscope slide, where each spot contains DNA for a different gene
- 44. Gene Expression and MicroarraysGene Expression and Microarrays The development of microarrays has allowed the expression of thousand of genes to be monitored simultaneously. Microarrays rely on nucleic acid hybridization, in which a known DNA fragment is used as a probe to find complementary sequences The probe is usually fixed to some type of solid support, such as a nylon filter or a glass slide. A solution containing a mixture of DNA or RNA is applied to the solid support; any nucleic acid that is complementary to the probe will bind to it. Nucleic acids in the mixture are labeled with a radioactive or fluorescent tag so that molecules bound to the probe can be easily detected.
- 46. How can we examine changes in geneHow can we examine changes in gene expression?expression? Let we have two types of cells: Experimental cells mRNA is converted into cDNA and labeled with red fluorescent nucleotides Control cells cDNA, labeled with green fluorescent nucleotides. Labelled cDNA are mixed and Hybridized to DNA chip, which contain DNA Probes form different genes Hybridization of the red (experimental) and green (control) cDNAs is proportional to the relative amounts of mRNA in the samples.
- 48. Red indicates the over expression of a gene in the experimental cells (more red-labeled cDNA hybridizes), Green indicates the under expression of a gene in the experimental cells (more green-labeled cDNA hybridizes). Yellow indicates equal expression (equal hybridization of red- and greenlabeled cDNAs. No or black color indicates no expression. ◦ Microarrays allow the expression of thousands of genes to be monitored simultaneously, ◦ To study which genes are active in particular tissues. ◦ To investigate how gene expression changes in the course of biological processes such as development or disease progression.
- 49. Genome wide MutagenesisGenome wide Mutagenesis One of the best methods for determining the function of a gene is to examine the phenotypes of individual organisms that possess a mutation in the gene. To conduct a mutagenesis screen, random mutations are induced in a population of organisms, creating new phenotypes. Random inducement of mutations on a genome wide basis and mapping with molecular markers—are coupled and automated in a mutagenesis screen. Mutagenesis screens can be used to search for specific genes encoding a particular function or trait.