Genomics.ppt

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Key Concepts

Once a genome has been completely sequenced, researchers use a variety of techniques to identify which sequences code for

products and which act as regulatory sites.

Bacterial and archaeal genomes are relatively small. Among

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Key Concepts

Eukaryotic genomes are large and complex. They include many sequences that have little to no effect on the fitness of the

organism, and many transcribed sequences whose function is not known.

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Introduction

• The complete DNA sequence of an organism is its genome. The human genome sequence was published in February 2001 as part of the Human Genome Project.

• Genomics is the scientific effort to sequence, interpret, and compare whole genomes.

• Genomics provides a list of the genes present in an organism.

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Whole-Genome Sequencing

• Improved automation has increased the speed and reduced the cost of DNA sequencing.

• The primary international repositories for DNA sequence data now contain over 194 billion nucleotides.

• With about 3 billion nucleotides, humans have the largest haploid genome sequenced to date.

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How Are Complete Genomes Sequenced?

• Most genome sequencing projects use a whole-genome shotgun sequencing approach.

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The Shotgun Sequencing Process

1. Sonication (use of high-frequency sound waves) breaks a genome into pieces approximately 160 kilobases long.

2. Each piece is inserted into a plasmid called a bacterial artificial chromosome (BAC). A BAC library is created by inserting

each BAC into a different Escherichia coli cell. Colonies of each cell are allowed to grow, creating multiple copies of each BAC library.

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The Shotgun Sequencing Process

4. Each 1-kb segment is cloned into a plasmid. These plasmids are then inserted into E. coli cells and replicated, producing shotgun clones.

5. The fragments from each clone are then sequenced and analyzed by computer programs.

6. The computer puts the sequences in order, thus reconstructing the BACs.

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The Shotgun Sequencing Process

• In essence, the shotgun strategy consists of breaking a genome into tiny fragments, sequencing the fragments, and then putting the

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The Role of Next-Generation Sequences Strategies

• Pyrosequencing is a cheaper and faster alternative to traditional sequencing.

• It takes place on a single DNA fragment rather than multiple copies of the same fragment.

• However, it only works with fragments that are too small to be pieced back together to reconstruct a complete genome accurately.

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How Are Complete Genomes Sequenced?

• Bioinformatics is the effort to manage, analyze, and interpret

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Which Genomes Are Being Sequenced, and Why?

• The first genome of an organism to be sequenced was that of the bacterium Haemophilus influenzae in 1995; it consists of about 1.8 million base pairs.

• The first eukaryotic genome to be sequenced was that of the yeast Saccharomyces cerevisiae in 1996.

• To date, complete genomes have been sequenced from over 800 species.

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Which Sequences Are Genes?

The most basic task in annotating or interpreting a genome is to identify which bases constitute genes.

• Identifying genes is relatively straightforward in bacteria and

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Identifying Genes in Bacterial and Archaeal Genomes

• Computer programs are used to scan a genome sequence in both directions in order to identify open reading frames (ORFs). ORFs are possible genes—long stretches of sequence that lack a stop

codon but are flanked by a start codon and a stop codon.

• The computer programs also look for sequences typical of promoters, operators, and other regulatory sites.

• Researchers can confirm that an ORF is actually a gene by

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Identifying Genes in Eukaryotic Genomes

• In eukaryotic organisms, genes contain introns, and most of the

genome does not code for a product—thus, it is not possible to scan for ORFs.

• The most effective strategy for identifying genes is to use reverse transcriptase to produce a cDNA version of each mRNA, and

sequence a portion of the resulting molecule to produce an

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Bacterial and Archaeal Genomes

• By sequencing the genomes of various strains of the same

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The Natural History of Prokaryotic Genomes

In bacteria, there is a general correlation between the size of the genome and the metabolic capabilities of the organism.

• The function of many bacterial genes is still unknown.

• There is tremendous genetic diversity among bacteria and

archaea. About 15 percent of the genes in a prokaryotic genome are unique to its own species.

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The Natural History of Prokaryotic Genomes

• Multiple chromosomes and plasmids are more common than expected.

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Lateral Gene Transfer

• The movement of DNA from one species to another species is called lateral gene transfer.

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Evidence for Lateral Gene Transfer

• Two general criteria support the hypothesis that sequences in bacterial or archaeal genomes originated in another species:

1. A gene is much more similar to genes in distantly related species than it is to those in closely related species.

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How Does Lateral Gene Transfer Occur?

• Lateral gene transfer often results because genes are carried on plasmids.

• Another way lateral gene transfer occurs is through transformation, taking up DNA fragments from the environment.

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Environmental Sequencing

• Environmental sequencing, or metagenomics, is the practice of cataloging all of the genes present in a community of bacteria and archaea. The subject of these studies is genes—not organisms.

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Eukaryotic Genomes

Many eukaryotic genomes are dominated by repeated DNA

sequences that occur between genes or inside introns and do not code for products used by the organism.

• Sequencing eukaryotic genomes presents unique challenges.

– Eukaryotic genomes are much larger than the genomes of bacteria and archaea.

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Parasitic and Repeated Sequences

• Protein-coding sequences constitute a very small percentage of the human genome, and repetitive sequences make up more than 50 percent. In contrast, over 90 percent of the prokaryotic genome consists of genes.

• Repeated sequences in the human genome are often the result of

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Characteristics of Transposable Elements

• Transposable elements are examples of selfish genes—parasitic DNA sequences that survive and reproduce but that do not increase the fitness of the host genome.

• Transposable elements are classified as parasitic because they decrease their host’s fitness:

– It takes time and resources to copy them along with the rest of the genome.

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How Do Transposable Elements Work?

• Long interspersed nuclear elements (LINEs) are one type of transposable element.

• An active LINE contains all the sequences required to make copies of itself and insert them into a new location in the genome.

• Analyses of the human genome have revealed that only a handful of LINEs appear to be complete and potentially active.

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Repeated Sequences

• Eukaryotic genomes have several thousand loci called short

tandem repeats (STRs). These are small sequences repeated down the length of a chromosome. There are two types of STRs.

1. Microsatellites, or simple sequence repeats, are repeating units of 1 to 5 bases.

2. Minisatellites, or variable number terminal repeats

(VNTRs), are repeating units of 6 to 500 bases.

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Repeated Sequences

• One hypothesis for why microsatellites and minisatellites have so many different alleles is that these highly repetitive stretches may misalign when chromosomes synapse during meiosis.

– _{This misalignment then causes}_{unequal crossover}_.

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Repeated Sequences and DNA Fingerprinting

• DNA fingerprinting refers to any technique for identifying individuals on the basis of unique features of their genomes.

• Because microsatellite and minisatellite loci vary so much among individuals, they are now the markers of choice for DNA

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DNA Fingerprinting Process

• A sample of DNA is acquired from the individual.

• PCR is performed using primers that flank a region containing an STR.

• The region is cloned.

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Gene Families

• In eukaryotes, the major source of new genes is duplication of existing genes.

• Within a species, genes that are extremely similar to each other in structure and function are considered to be part of the same gene family.

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How Do Gene Families Arise?

• When gene duplication occurs, an extra copy of a gene is added to the genome.

• The most common type of gene duplication results from unequal crossing over during meiosis.

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New Genes—New Functions?

• Gene duplication is important because the original gene is still functional and produces a normal product.

• The duplicated gene may:

1. Retain its original function and provide additional quantities of the same product.

2. Undergo mutation resulting in a beneficial altered protein, thus creating an important new gene.

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Insights from the Human Genome Project

• Scientists do not know the function of more than half of the genes found in the human genome.

• Two recent discoveries are changing biologists’ thinking about the human genome:

1. Genes for miRNAs are much more common than previously thought.

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Why Do Humans Have So Few Genes?

• A surprising observation about eukaryotic genomes is that

organisms with complex morphology and behavior do not appear to have large numbers of genes.

• Before the human genome was sequenced, scientists expected that humans would have at least 100,000 genes. However, the actual sequence revealed that we have only about 20,000 genes.

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Similarities between Human and Chimp Genomes

• At the level of base sequence, the human and chimpanzee genomes are 98.8 percent identical.

• This raises the question of how humans and chimps can be so similar genetically but so different in morphology and behavior.

• One hypothesis proposes that even though many structural genes

(those that code for products) in humans and chimps are identical,

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Functional Genomics and Proteomics

• Whole-genome data can be used to answer fundamental questions about how organisms work.

• Large-scale analyses of gene expression are called functional genomics.

• One of the basic tools of functional genomics is a DNA

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How Are DNA Microarrays Used?

• mRNAs produced in two contrasting types of cells are isolated, and then cDNAs produced from these mRNAs are used to probe the

microarray.

• Researchers can thus identify differences in which genes are expressed in the two cell types.

• A microarray allows researchers to study the expression of

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What Is Proteomics?

• A transcriptome is the complete set of genes that are transcribed in a particular cell.

– _A_proteome_{is the complete set of proteins that are produced.}

• Proteomics is the large-scale study of protein function.

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Applied Genomics: Understanding Cancer

Researchers are using tools created by advances in genomics to deepen our understanding of cancer.

– _{Microarrays allow researchers to compare gene expression in}

normal versus cancerous cells.

– The Human Genome Project has revealed common sets of genes that are mutated in cancerous cells.

– _{The complete genome sequences of cancerous and}