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Hotspots of Genetic Diversity
• DNA profiling is based on the concept that every genome is unique
• DNA consists of paired nucleotides, which members of the same species share approximately 99% commonality
• Almost all genetic diversity exists in small polymorphic regions
DNA Polymorphisms
• Single-nucleotide polymorphisms (SNP – pronounced snips)
• Short tandem repeats (STR)
• Variable number of tandem repeats (VNTR)
• DNA profiling relies on these regions known as molecular markers
DNA Profiling in Fighting Disease
• Identifying markers like SNPs help medical researchers to distinguish different types of disease causing agents
• Cataloging the appropriate genetic markers is called scoring a gene
• Until recently it took 3 months to score a gene
• Now it it possible to score thousands in a single day
New DNA Profiling Applications
• Protecting against threats to food supply
• Connecting strains of pathogens to the disease they cause
• Determining the appropriate course of treatment for different genetic variations of cancer
DNA Sequencing
• DNA fingerprinting is helpful when comparing variable sections of similar genomes within the same species
• When a scientist is identifying an unknown pathogen, a different technique is used
• Technicians use sections of genome that are distinct for different species of bacteria
Fusariam Infection: A Case Study in Using DNA to Guard Against a Plant
Epidemic
How new technology is being used to blockade an ancient fungal disease that threatens the world’s food supply
Guarding Against a Plant Epidemic
• The Fusarium toxin causes a disease in barley and wheat called ‘head blight’ (or scab) and ‘stalk rot’ in corn
• Human exposure to these toxins in infected grain results in a variety of symptoms including acute dermatitis, diarrhea, and hemorrhaging
A Fungal Toxin in Action
• A healthy wheat head (left) stands in contrast to one inoculated with Fusarium graminearum showing symptoms of head blight disease (right)
• This illustrated damage
causes $3 billion in the U.S. alone
O’Donnell, 2000
One Disease – Eight Causes
• In the past, all scab epidemics worldwide were thought to be caused by a single pathogen
• The scab species could not be distinguished from the toxins they produced
• DNA profiling revealed that at least eight genetically distinct scab pathogens exist
O’Donnell, 2000
How Profiling Helps• Based on DNA profiling, scientists created a
DNA-based diagnostic procedure• It precisely distinguishes between the eight
species allowing technicians to determine the pathogen's origin
• By determining which plants from various parts of the world harbor the pathogens, officials are able to prevent them from spreading epidemics to the U.S.
O’Donnell, 2000
Lung Cancer : A Case Study in DNA Analysis to Treat Genetic Diseases
• Approximately 20% of lung cancer are classified as small-cell carcinomas
• The rest are classified as non-small-cell carcinoma (NSCLC), for which therapies often lead to unpredictable results
• Until DNA profiling, there was no way to predict NSCLC’s response to therapy
Medical News Today, 2009
Non-Small-Cell Carcinoma
• Using DNA microarray analysis researchers discovered that NSCLCs have genetic patterns that predetermine their behavior
• Some are genetically disposed to spread aggressively, and to metastasize early
• Others are likely to be easily treated
Medical News Today, 2009
Tumor Subtypes Detected
• Bronchoid– Associated with the likelihood of improved
survival in early-stage disease– Tumors that are least likely to respond to
chemotherapy because many genes associated with resistance to chemotherapy agents
• Squamoid– Associated with better survival in advanced
disease
Medical News Today, 2009
Escherichia coli O157:H7 A Case Study in Identifying the
Evolution of a Pathogen
• E. coli is a waterborne and foodborne bacteria whose virulence seems to have been increasing
• Recent outbreaks have been marked by greater hemolytic uremic syndrome (HUS)
• DNA profiling was used to prove newly evolved strains are increasingly deadly
Manning, 2008
E. coli O157:H7 virulence• Novel avenues of infection with new
animals and produce have resulted in ‘relentless evolution’
• O157 strains have increased geographically and in diversity– Viruses are responsible for insertions into the
bacterial DNA adding toxins that cause HUS– The 2006 spinach outbreak is an example – it
demonstrated that the pathogen could subsist on produce as well as meat
Manning, 2008
E. coli Evolution
DNA sequences have been used to trace:
– the decline in one strain of hemolytic E. Coli (Clade 2)
– while another becomes more prevalent (Clade 8)
– DNA collected during outbreak proved new clade increased 500%
Changing patterns revealed through DNA profiling
Manning, 2008
E. coli Evolution (continued)
• Distribution of Shiga toxin variants among O157 strains by clade
• As clades vary by toxin, profiling the DNA of the pathogen is critical to save lives with prompt identification of the appropriate toxins
Manning, 2008
Training Exercise
• Pathologists use techniques like DNA fingerprinting and sequence analysis to identify pathogens or their specific strain
• In this lab, you will act as a pathologist at a well-equipped research hospital. Your task is to identify a bacterial sample received from a clinician at the site of a mysterious outbreak through sequence analysis
Virtual Lab: Identify a Pathogen
• You will follow a multi-step process to identify potential bacterial pathogens by collecting and searching for its DNA sequence
• Pay close attention – the public is anxious about reports of an unidentified outbreak and the news media is going to be asking questions when you finish
• To begin visit: http://www.hhmi.org/biointeractive/vlabs/bacterial_id/index.html and click on ‘enter the lab’
HHMI, 2008
News Conference• After completing your research, the news media
expects answers to questions based on your findings.
• For every question, click on the correct answer. If you make a mistake you will be returned to the question to correct your statement.
• Good luck.
Software courtesy Russell, W
What technology did your sequencing use?
• A – Polymerization
• B – Polymeric Imaging
• C – Polymeric Systems
• D – Polymerase Chain Reaction
What do proteolytic enzymes accomplish when preparing samples?
• A – Energizes the cell wall to activate DNA
• B – Digests the cell wall to make access to DNA possible
• C – Strengthens cell wall to protect DNA
• D – Denatures the cell wall to inactivate DNA
Why is the 16S rRNA Gene Chosen to Identify Unknown Bacteria ?
• A – Its DNA sequences are completely distinct for every species, making identification simple
• B – The gene is so common that it is economical to process
• C – Its gene sequences are all shared (conserved) by most bacteria so it is a good candidate for a universal primer needed to copy the DNA
• D – Its sequences are mostly conserved making it easy to bind with primers while distinct regions help with identification
What is the name of the pathogen you identified?
• A – Escherichia coli
• B – Salmonella typhimurium
• C – Bartonella henselae
• D – Brucella canis
If sandflies are common at the site of the outbreak, what disease will the pathogen likely cause?
• A – Lyme Disease
• B – Yellow Fever
• C – Oroya Fever
• D – Angiomatosis
How do BLAST searches work?
• A – A sample is compared to the GenBank public DNA sequence database available through
the National Library of Medicine • B – BLAST assigns a numerical value to the
degree of similarity between two DNA sequences
• C – BLAST returns a numerical score based on a set formula (algorithm). The higher the score, the better is the match
• D – All of the above
What is the name of the process by which PCR quickly multiplies the number of DNA copies?
• A – Amplification• B – Compensation• C – Stratification• D – Expansion
Works Cited
• Howard Hughes Medical Institute. 2008 Virtual Bacterial ID Lab. http://www.hhmi.org/biointeractive/vlabs/bacterial_id/index.html. Accessed 2009 May 10.
• Russell W. How to use PowerPoint: multiple choice quizzes. http://presentationsoft.about.com/. Accessed 2009 May 10.
• Manning SD et al. 2008 Variation in virulence among clades of Escherichia coli O157:H7 associated with disease outbreaks. PNAS. 105(12): 4868-4873.
• Medical News Today. DNA Profiling Study Identifies Three New Lung Tumor Subtypes. http://www.medicalnewstoday.com/articles/55446.php. Accessed 2009 May 4.
• O’Donnell KL, Ward. DNA Profiling: Guarding against a plant disease epidemic. August 2000: 4-7.