1. DNA Sequence Data Applications Disease Tracking, Treatment and Prevention

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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’ http://www.hhmi.org/biointeractive/vlabs/bacterial _id/index.html HHMI, 2008

21. 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

22. What technology did your sequencing use? A – Polymerization B – Polymeric Imaging C – Polymeric Systems D – Polymerase Chain Reaction

23. Sorry That was the wrong answer

24. Correct ! Congratulations

25. 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

26. Sorry That was the wrong answer

27. Correct ! Congratulations

28. 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

29. Sorry That was the wrong answer

30. Correct ! Congratulations

31. What is the name of the pathogen you identified? A – Escherichia coli B – Salmonella typhimurium C – Bartonella henselae D – Brucella canis

32. Sorry That was the wrong answer

33. Correct ! Congratulations

34. 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

35. Sorry That was the wrong answer

36. Correct ! Congratulations

37. 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

38. Sorry That was the wrong answer

39. Correct ! Congratulations

40. 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

41. Sorry That was the wrong answer

42. Correct ! Congratulations

43. 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. http://www.hhmi.org/biointeractive/vlabs/bacterial_id/index.html Russell W. How to use PowerPoint: multiple choice quizzes. http://presentationsoft.about.com/. Accessed 2009 May 10. http://presentationsoft.about.com/ 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. http://www.medicalnewstoday.com/articles/55446.php O’Donnell KL, Ward. DNA Profiling: Guarding against a plant disease epidemic. August 2000: 4-7.