DNA Sequence Data Applications

Disease Tracking, Treatment and Prevention

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

Sorry

That was the wrong answer

Correct !

Congratulations

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

Sorry

That was the wrong answer

Correct !

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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

Sorry

That was the wrong answer

Correct !

Congratulations

What is the name of the pathogen you identified?

• A – Escherichia coli

• B – Salmonella typhimurium

• C – Bartonella henselae

• D – Brucella canis

Sorry

That was the wrong answer

Correct !

Congratulations

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

Sorry

That was the wrong answer

Correct !

Congratulations

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

Sorry

That was the wrong answer

Correct !

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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

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That was the wrong answer

Correct !

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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.