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Slide 01: Gene Sleuthing (Web)
Caption: Each living being contains the "blueprint" that determines what it looks like, what it is able to do, and how it responds to environmental challenges. This information is stored in the sequence of the four nucleotides in DNA. Just like language which stores information in the sequence of 26 letters.
The nucleotide sequences for a variety of organisms have been determined. Now, scientists identify the information in these sequences, foremost the location and the function of all the genes. Given the large number of nucleotide pairs in each genome (3,200,000,000 for human), computers are used to manage and analyze genome sequences and a new discipline, bioinformatics, aids the identification in biological molecules such as DNA, RNA, and proteins.
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Slide 02: Nucleus and DNA
Caption: Today, DNA receives a lot of attention by scientists. DNA testing is used to identify people for a number of reasons. What is DNA? Is DNA located anywhere else in the cell?
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Slide 03: Base pairing (James Watson)
Caption: James Watson used cardboard cutouts representing the shapes of the DNA bases to figure out how nucleotide bases pair. He realized that the adenine-thymine and cytosine-guanine pairs fit adhered best to the structure of the DNA molecule predicted from X-ray cristallography data.
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Slide 04: The double helix (James Watson)
Caption: "We knew if we just, even if we go up to the ceiling, we're building a tiny fraction of a molecule. Hundreds of millions of these base pairs in one molecule, all fitting into this wonderful symmetry, which we saw the morning of February 28, 1953."
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Slide 05: DNA packaging
Caption: Each chromosome consists of one continuous thread-like molecule of DNA coiled tightly around proteins, and contains a portion of the 6,400,000,000 basepairs (DNA building blocks) that make up your DNA.
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Slide 06: From ignorance to knowledge (Francis Collins)
Caption: Francis Collins, director of the Human Genome Project, tells of his excitement about the project.
Item: Video interview
Slide 07: DNA
Caption: DNA molecules contain the information for the shape and behavior of individual organisms and pass this information from generation to subsequent generation.
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Slide 08: Sequencing DNA
Caption: Techniques to read the sequence of DNA, letter by letter, have been available since the 1970s. However, the massive task of sequencing the three billion basepairs of the human genome required machines that could read and interpret the data.
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Slide 09: The revolution in DNA sequencing
Caption: Outputs from automated (top) and manual (bottom) DNA sequencing.
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Slide 10: Sequencing the entire human genome
Caption: The public genome project aimed to sequence the entire genome in an ordered, methodical manner.
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Slide 11: DNA sequencing game
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Reconstruct a piece of DNA using the fragments above. We've given you the first piece. You do the rest...
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Slide 12: Completion of a draft of the human genome (Bill Clinton)
Caption: A milestone for science and medicine was the completion of a draft sequence of the human genome in 2000. Further work was required to eliminate errors, resolve the sequences in centromeres and telomers, and of those regions consisting of highly repetitive DNA.
Shown with then-president Bill Clinton are the leaders of the two major genome sequencing efforts Dr. Francis Collins (Human Genome Sequencing Project) and Dr. Craig Venter (Celera, Inc.).
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Slide 13: What are genes?
Caption: Currently, a generally agreed upon definition for what constitutes a gene does not exist - it is not clear how one definition could cover all aspects of genes and gene expression in all types of organisms. The two gene concepts mostly used are as follows:
Stretches of DNA which encode the information for the building of amino acid sequences and, ultimately, proteins.
DNA sequences that are transcribed into RNA sequences (mRNA, tRNA, rRNA, RNAi, etc.)
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Slide 14: Transcription and translation
Caption: The Central Dogma of molecular biology:
DNA is transcribed into RNA which is translated into protein.
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Slide 15: More about genes in DNA Interactive
Caption: Learn more about genes working through the four chapters
meaning
DNA analysis
gene features
gene finding
in Genome Mining at the website http://www.dnai.org/c/index.html.
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Slide 16: Why do we care about genes?
Caption: Because they are a the base of all life. If we understand genes we will be able to better understand, prevent and treat diseases. We will be able to understand ourselves. We will be able to adjust our lives to our genetic make-up and choose a life style that makes the most out of our predispositions and to more successfully avoid relevant risk factors.
Understanding genes may help us to tackle environemtal problems and to find new ways of producing things such as plastic bags through bacterial polyhydroxy butyrate synthesis.
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Slide 17: Example Insulin
Caption: The A and B chains of insulin. Insulin can be isolated from the pancreas of pigs and cows for human use. Throughout most of the 20th century, millions of diabetics worldwide depended on insulin extracted from pigs or cows. Unfortunately, only so much insulin can be extracted from these animals, and some diabetics developed allergic reactions to cow and/or pig insulin. The availability of human insulin in unlimited quantities would certainly have a marketable value. Insulin itself is a small protein, and was the first to have its amino acid sequence and structure determined. Insulin is made up of two polypeptide chains. The A chain has 21 amino acids and the B chain has 30 amino acids. With so much known about it, many researchers thought that insulin should be easy to make in a laboratory.
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Slide 18: Synthesizing human insulin using recombinant DNA
Caption: In order to synthesize human insulin using recombinant DNA technology, the human DNA sequence for insulin was needed. The amino acid sequence of human insulin was known. The Genentech group deduced the human DNA sequence of insulin based on its amino acid sequence. They then used the DNA nucleotides and synthesized the human DNA sequence. This sequence was then inserted into a plasmid and transformed into bacteria to produce insulin. By synthesizing the DNA sequence, the Genentech group assembled a human DNA sequence of insulin without ever having to use \"real\" human DNA. They thus bypassed some of the restrictions on human recombinant DNA work resulting from the Asilomar conference.
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Slide 19: How insulin is made using bacteria
Caption: Synthetic human insulin was the first golden molecule of the biotech industry and the direct result of recombinant DNA technology. Currently, millions of diabetics worldwide use synthetic insulin to regulate their blood sugar levels. Synthetic insulin is made in both bacteria and yeast.
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Slide 20: Synthetic insulin
Caption: Making synthetic insulin is not as easy as it sounds. See what some of the challenges are.
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Slide 21: Where do genes live?
Caption: In genomes.
Human:
25 chromosomes: 1-22, X, Y, mt;
ca. 3,200,000,000 base pairs (bp);
ca. 30,000 genes;
size varies: 48 bp (RNA genes) to 2,800,000 bp (DMD);
ca. 25% of genome consist of genes;
ca. 1% of genome is coding for amino acid sequences.
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Slide 22: Paper stack (Human Genome)
Caption: As represented by this huge stack of paper, the human genome contains more than three billion nucleotides or DNA "letters."
1% of these letters encode amino acid sequences.
They are organized in about 30,000 genes, which make about 25% of the human genome.
Thus, even within genes the non-coding fraction of DNA amounts to 96%.
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Slide 23: To fnd genes in the human genome you need computers (Ewan Birney)
Caption: Interviewee: Ewan Birney
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Slide 24: Bioinformatics
Caption: Bioinformatics is the discipline of identifying the information in biological molecules (DNA, proteins), utilzing informatics tools and theory (computers, software). The analysis of biological molecules, in turn, informs the field of computer sciences.
Objectives
Understand disease, development, and evolution.
Find all genes and proteins.
Determine the function of genes and proteins.
Describe the interactions among genes and proteins.
Unveil descent and relationships.
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Slide 25: Strategies to find genes
Caption: Depends on evidence and tools available, and how you want to use these.
Sequences (DNA, RNA, amino acids),
databases,
bioinformatics tools.
Wet labs (reverse genetics, cloning) vs. sequence analysis.
Two principally different approaches (often used in combination):
predicting genes utilziing sequence characteristics,
predicting genes utilziing sequence similarity.
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Slide 26: Promoter region gene prediction tool
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Slide 27: Exercise: How is the DNA in genes different?
Caption: Objective: Find out whether there is anything special about DNA in genes.
Go to http://www.dnai.org/geneboy/.
Examine the composition of random nucleotide sequences, genic and intergenic DNA sequences. Instructions for this exercise are available at http://www.dnai.org/c/index.html in the chapters meaning and DNA analysis.
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Slide 28: Bioinformatics II
Caption: Almost all nucleotide databases
list only one nucleotide strand;
from left to right = from 5'-end to 3'-end;
use only A, C, G, T. Not U. Therefore, mRNA is presented as if it were cDNA.
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Slide 29: How is the DNA in genes different?
Caption: The DNA composition in genes is different from the composition of random nucleotide sequences and intergenic DNA.
In random nucleotide sequences all nucleotides are represented in roughly equal proportions.
The proportions of the four nucleotides in DNA varies. Genic regions contain elevated amounts of (C plus G) than (A plus T).
The proportions of the four nucleotides in the regions between genes, the intergenic regions varies, too. However, intergenic regions are in general higher in (A plus T) than (C plus G).
Scanning long DNA stretches for regions with a higher (C plus G)-content helps to locate gene-rich regions.
Scanning DNA for the occurence of CpG-islands (regions in which C is more often followed by G than expected by chance) is an even better indicator for genes.
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Slide 30: Model organisms
Caption: Each model organism has its own advantages and disadvantages. Choosing an appropriate model depends on the question being asked. Many laboratories find it useful to perform parallel experiments in two or more model systems to understand different aspects of a biochemical process.
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Slide 31: Gene finding by alignment
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Slide 32: Exercise: Resistant to medication (BLASTN, BLASTP)
Caption: Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis. In TB patients who do not respond to penicillin antibiotics any more M. tuberculosis has become resistant to this antibiotic type.
Imagine you are a microbiologist for a pharmaceutical company
Research objective: Identify the genetic basis for this resistance.
Grow bacteria in culture, isolate cDNA, sequence it, and determine what causes the bacterial penicillin resistance. (Instructions)
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Slide 33: The cracking of the code (Marshall Nirenberg)
Caption: Marshall Nirenberg, National Institute of Health, 1960. Having cracked the first codon, Marshall Nirenberg worked with a group of scientists (Maxine Singer, Marianne Grunberg-Manago and Phil Leder).
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Slide 34: What are characteristics of the DNA in single-ORF genes?
Caption: The composition of genic DNA is different from the composition of random nucleotide sequences and intergenic DNA.
The DNA in genes consisting of single ORFs begins with a start codon (ATG), followed by a nucleotide stretch whose number is a multiple of 3 (CDS), and ending in a stop codon (TAA, TAG, or TGA).
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Slide 35: Exercise: Identify "famous" bacterial genes
Caption: Identify a gene in bacterial DNA by finding ORFs, to determine the nature of the potential gene(s), and the nature of the organism. Identify a gene in bacterial DNA by finding ORFs, to determine the nature of the potential gene(s), and the nature of the organism. (Instructions)
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Slide 35: Developing programs that look at DNA sequence, Ewan Birney
Caption: Interviewee: Ewan Birney
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Slide 36: Exercise: Identify an organism through its genes (ORF-Finder, BLASTP)
Caption: Imagine that you work in a hospital and patients with a mysterious and ultimately lethal infection start coming in. You need to identify the infectious agent through a combined laboratory and bioinformatics approach. (Instructions)
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Slide 37: Exercise: What's that smell? (Keyword)
Caption: Use the names of genes, abilities, or diseases to find genes in the human genome. (Instructions)
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Slide 38: Tanscription/translation 1 on DNAi
Caption: Go to http://www.dnai.org/c/index.html. Click Genome Mining, then gene features. Work through the slides.
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Slide 39: Transcription: DNA codes for messenger RNA (mRNA)
Caption: What you are about to see is DNA's most extraordinary secret, how a simple code is turned into flesh and blood. It begins with a bundle of factors (transcription factors)assembling at the start of a gene. A gene is simply a length of DNA instructions stretching away to the left. The assembled factors trigger the first phase of the process, reading off the information that will be needed to make the protein. Everything is ready to roll: three, two, one, GO! The blue molecule (RNA polymerase) racing along the DNA is reading the gene. It's unzipping the double helix, and copying one of the two strands. The yellow chain (messanger RNA or mRNA) snaking out of the top is a copy of the genetic message and it's made of a close chemical cousin of DNA called RNA. The building blocks to make the RNA enter through an intake hole. They are matched to the DNA - letter by letter - to copy the As, Cs, Ts and Gs of the gene. The only difference is that in the RNA copy, the letter T is replaced with a closely related building block known as \"U\". You are watching this process - called transcription - in real time. It's happening right now in almost every cell in your body.
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Slide 40: RNA polymerase game
Caption: Be the RNA polymerase, and make your own RNA strand in this interactive game.
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Slide 41: Translation: RNA to protein
Caption: When the RNA copy is complete, it snakes out into the outer part of the cell. Then in a dazzling display of choreography, all the components of a molecular machine lock together around the RNA to form a miniature factory called a ribosome. It translates the genetic information in the RNA into a string of amino acids that will become a protein. Special transfer molecules, the green triangles, bring each amino acid to the ribosome. The amino acids are the small red tips attached to the transfer molecules. There are different transfer molecules for each of the twenty amino acids. Each transfer molecule carries a three letter code that is matched with the RNA in the machine. Now we come to the heart of the process. Inside the ribosome, the RNA is pulled through like a tape. The code for each amino acid is read off, three letters at a time, and matched to three corresponding letters on the transfer molecules. When the right transfer molecule plugs in, the amino acid it carries is added to the growing protein chain. Again, you are watching this in real time. And after a few seconds the assembled protein starts to emerge from the ribosome. Ribosomes can make any kind of protein. It just depends what genetic message you feed in on the RNA. In this case, the end product is hemoglobin. The cells in our bone marrow churn out a hundred trillion molecules of it per second! And as a result, our muscles, brain and all the vital organs in our body receive the oxygen they need.
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Slide 42: Ribosome game
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Be the ribosome and make part of a protein in this interactive game.
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Slide 43: Finding genes (Ewan Birney)
Caption: Interviewee: Ewan Birney
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Slide 44: Exercise: Finding genes in human DNA (Thalassemia)
Caption: Identify a gene in a human DNA sequence by first determining the gene structure and then the nature of the gene. (Instructions)
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Slide 45: What are characteristics of the DNA in spliced genes?
Caption:The composition of genic DNA is different from the composition of random nucleotide sequences and intergenic DNA.
The DNA in genes consisting of single ORFs begins with a start codon (ATG), followed by a nucleotide stretch whose number is a multiple of 3 (CDS), and ending in a stop codon (TAA, TAG, or TGA).
The DNA in spliced genes has a number of characteristic features, such as:
- The transcribed and, thus, the coding sequence is preceeded by a promoter which bears consensus sequences (example: TATA-box, TATAAA);
- the coding sequence begins with a start codon (ATG);
- introns begin with the nucleotide pair GT and end with the nucleotide pair AG;
- the coding sequence ends with a stop codon (TAA, TAG, TGA);
- the coding sequence is followed by a polyA-signal (A(A/T)TAAA);
- the final transcript (mRNA) contains a coding sequence which resembles an ORF in that it begins with a start codon which is followed by a nucleotide stretch consisting of multiples of three nucleotides, followed by a stop codon.
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Slide 46: Exercise: Without cover ... (Map Viewer)
Caption: You work with a patient who has had very patchy hair since birth. Hairloss can have a variety of causes and you will use bioinformatics tools to find out whether the herediatry hairloss of the patient can be related to anything in the his and the human genome sequence. (Instructions)
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Slide 47: Process of finding a gene (Mark Skolnick)
Caption: Mark Skolnick recounts the complicated process of verifying that the gene they had found was indeed BRCA1.
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Slide 48: Gene number and complexity (Eric Lander)
Caption: Eric Lander, Director of the Whitehead Institute at MIT, one of the more important and prominent contributors to the Human Genome Project.
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Slide 49: Reading our own code (John Sulston)
Caption: Nobel Laureate John Sulston reflects on the Human Genome Project from an evolutionary perspective.
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