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Objective # 16 Module 4C – Genomics Explain the difference between the following types of genome maps, and explain how each type of map i constructed: is d a) genetic maps b)physical b) physical maps The complete set of genetic instructions for an organism is referred to as its genome.. In this module, we will ggenome examine genomics – the science of mapping and studying entire genomes. 1 2 Objective 16 Objective 16 Genome maps are used to determine the location of genes. Maps of genomes are constructed at different levels of resolution using different kinds of information. Depending on the type of map we use, a gene can be located on a particular chromosome, in a specific region of the chromosome, or at its precise location in the chromosome’s DNA sequence. The 3 2 main types of genome maps are genetic maps and physical maps. Genetic maps are linkage maps. They show the relative location of genes on a chromosome as determined by b recombination frequencies. Distances on genetic maps are measured in centimorgans (cM). One cM equals 0.01% recombination frequency. Objective 16 4 Objective 16 Physical maps show distances between DNA landmarks. The resolution of landmarks range from recognition sites for restriction enzymes to the ultimate level of detail: the actual DNA sequence. Distances between landmarks on a physical map are measured in basebase-pairs (1000 basebase-pairs equals 1 kilobase kb). The 5 first physical maps were created by cutting genomic DNA with different restriction enzymes. The fragments were then analyzed to determine their size and how they fit together into a continuous segment of the genome called a contig. contig. Such a map shows the physical distance between the different recognition sites of the restriction enzymes: 6 1 2. The fragments produced by enzyme A only, by enzyme B only, and by enzymes A and B simultaneously are run out sideby-side on a gel, which separates them according to size, smaller fragments running faster faster. Objective 16 DNA Molecular weight marker 14kb 10kb 6kb 2kb 3. The fragments are arranged so that the smaller ones produced by the simultaneous cut can be grouped to generate the larger ones produced by the individual enzymes 4. A physical map is constructed. The ultimate physical map of an organism’s DNA is the actual basebase-pair sequence of the entire genome. Although g automated sequencers q have been developed to determine the basebasepair sequence of DNA, they are only accurate on fragments of DNA up to about 500 basebase-pairs in length. enzyme B 1. Multiple copies of a segment of DNA are cut with restriction enzymes. 14kb 9kb 8kb 9kb 5kb 5kb 3kb 2kb 2kb 2kb 8kb 9kb A A 5kb 14kb B 2kb 3kb 5kb 9kb A B A A B A 2kb 5kb 10kb 19kb 7 8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Objective 16 Clone-by-clone method In order to sequence an entire genome, which may contain billions of base pairs, the DNA must be cut into small fragments that can be sequenced with automatic sequencers. There are 2 different ways to do this: clone clone--byby-clone sequencing shotgun sequencing 1. Cut DNA segment into large fragments, then determine the order of the large fragments by identifying regions that overlap. 2. Cut each large 2 fragment into smaller fragments, then sequence the small fragments with an automatic sequencer. 3. Determine the order of the small fragments by identifying sequences that overlap. 9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 10 Objective # 17 Shotgun method 1. Cut DNA of entire chromosome into small fragments. Discuss some of the main findings of the Human Genome Project. j 2. Sequence each small fragment with an automatic sequencer, then order the fragments based on overlapping nucleotide sequences. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 11 12 2 Objective 17 Objective 17 The project to sequence the entire human genome is called the human genome project. This project has already led to some important findings about genomes: The Th h human m genome m iis surprisingly rpri i l similar to other genomes. Many of the genes in humans are identical or similar to genes found in a wide variety of other organisms from bacteria to chimps. Scientists use computer analysis to help locate genes within the DNA sequence of the entire human genome. The branch of biotechnology gy that uses computer analysis to search for genes, to compare genomes, and to assemble entire genomes from smaller fragments is called bioinformatics. bioinformatics. 13 14 Objective 17 Objective 17 Although eukaryotic genomes are much larger and have many more genes than prokaryotes, the size and complexity of an organism is not directly related to the number b r off genes it has. h F Forr example, pl the human genome has around 25,000 genes, about the same number as mice and far fewer than rice. Based on current analysis, the human genome seems to have far fewer genes than expected. For many years geneticists had estimated the number of human genes to be around 100,000. However, current research indicates that the number is only around 25,000. 15 Objective 17 Estimated Number o of Genes The size and complexity of an organism is not directly related to the number of genes it has: prokaryotes eukaryotes 50,000 Genomes Rice (Oryza sativa) 40,000 Puffer fish (Fugu rubripes) Thale cress (Arabidopsis thaliana) 30,000 Fission yeast (Schizosaccharomyces pombe) 20,000 Bakers yeast (S. cerevisiae) Protozoan (Encephalitozoon cuniculi) 10,000 Human (Homo sapiens) Mouse (Mus musculus) Nematode (Caenorhabditis elegans) Mosquito (Anopheles sp.) Fruit fly (Drosophila melanogaster) Malaria microbe (Plasmodium falciparum) Slime mold (Dictyostelium discoideum) 0 0 1 10 100 1000 16 10,000 contain both coding and non-coding DNA. Surprisingly, only nonabout 1.5% of the human genome actually codes for proteins. Types of coding di DNA include: i l d Single copy genes – many genes exist as single copies on a particular chromosome Size of Genome (million base-pairs) Log Scale Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 17 18 3 Objective 17 Segmental duplications – whole blocks of genes that were copied from one chromosome to another Multigene g families – ggroups p of related but distinctly different genes that often occur close together. These related genes seem to have arisen by the duplication of a single ancestral gene: 19 20 Objective 17 Objective 17 Tandem clusters – these are identical copies of the same gene that occur near each other. They are transcribed simultaneously , increasing the amount of mRNA available for protein synthesis. Tandem clusters also include genes that do not encode proteins, such as clusters of rRNA genes. Most DNA does not code for proteins. Although its exact function is not entirely clear, scientists have recognized several types of nonnon-coding DNA in humans: Introns (24% of genome) are non non-coding regions that make up most of each human gene. 21 22 Objective 17 Objective 17 Segmental duplications (5% of genome) are long nucleotide sequences that have been duplicated and moved either within a chromosome, or to a nonhomologous chromosome chromosome. Pseudogenes (2% of genome) seem like normal protein protein--coding genes that may have lost their function due to mutations. 23 Structural DNA (20% of genome) remains tightly coiled throughout the cell cycle (constitutive heterochromatin) and tends to be localized near the centromeres and telomeres telomeres. Simple sequence repeats (3% of genome) are composed of a short sequence of nucleotides that is repeated thousands of times. 24 4 Objective 17 Transposable elements or transposons (45% of genome) are segments of DNA that move around from one location to another within the genome. In some cases cases, the transposon is duplicated and the copy moves to a new location. In other cases, the transposon is removed from its original location and is then inserted in a new location. 25 26 Objective 17 Although humans have only about 25,000 protein protein--encoding genes, these genes can code for at least 87,000 different proteins. How is this possible? Alternative splicing of exons can produce several different mature mRNAs from a single gene: 27 28 Alternative splicing of exons from the same gene produces different mature mRNAs and therefore different proteins 1 2 3 4 5 6 7 8 9 10 11 12 13 Primary y RNA transcript p 5´ cap mRNA splicing exons introns Processed RNA in brain 3 4 5 6 8 9 10 12 1 2 4 5 6 8 9 10 13 Mature mRNA in brain 5´ cap 29 3´ poly-A tail 3´ poly-A tail Processed RNA in muscle Mature mRNA in muscle 5´ cap 3´poly-A tail Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 24 5 Objective 17 the human genome is a major step towards understanding the genetic control of human traits. Among g other benefits, it has the potential to vastly improve the prediction, diagnosis, prevention, and treatment of disease in humans. The practical applications of this information will be enormous. Objective # 18 Sequencing Explain what DNA microarrays (biochips) are, and discuss their usefulness to scientists. scientists 31 32 Objective 18 Objective 18 Such a chip can be used to screen a genome for the presence of thousands of specific alleles at once. To screen a person person’ss genome, genome a cDNA sample is prepared, cut into fragments, tagged with a fluorescent dye, and then flooded over the chip. A DNA microarray, microarray, is a small square of glass that is covered with thousands of short pieces of singlesingle-stranded DNA arranged rr d iin a grid. rid The short chains of DNA nucleotides, which are attached to the glass surface, are called oligonucleotides. oligonucleotides. 33 34 35 36 Objective 18 By determining which oligonucleotides on the chip the cDNA binds to, scientists can determine which specific p alleles are being expressed in the cells that were used to prepare the cDNA: 6 37 7