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Welcome! Our goals for today

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0 DNA amplification and analysis: miniPCRTM Crime Lab
Science for everyone, everywhere DNA amplification and analysis: miniPCRTM Crime Lab Release date: 16 October 2014 © Copyright by Amplyus LLC, all rights reserved

1 Welcome! Our goals for today
Review DNA structure and DNA amplification concepts Solve a crime mystery using PCR!

2 PCR is at the heart of DNA analysis
Molecular diagnostics Text Consumer genomics Text Text Personalized medicine PCR Food and agriculture Text Text Human evolution Text Forensics

3 Polymerase Chain Reaction (PCR)
Genetic testing Pathogen detection Drug development Crop modification Forensic analysis Sequencing Etc. Complex DNA sample Region of interest Amplified DNA (Billions of copies) Applications A process that identifies and copies (amplifies) a specific piece of DNA in a biological sample

4 PCR relies on DNA’s unique structure
DNA: a double helix... ...held together by base complementarity Source: US National Library of Medicine, NIH, Thinkquest

5 How PCR works: 3 steps to copy DNA
1 Denaturation 50-60°C 2 Primer 1 Primer 2 Annealing 72°C 3 Taq DNA polymerase dNTPs Extension

6 How PCR works: repeat the cycle
denatured DNA DNA + primers DNA + copy Single molecule 94° C ~1B copies 50-60° C 72° C Denaturation Annealing Extension Repeat x ~25-30 cycles

7 PCR makes DNA visible (and useful)

8 How miniPCRTM enables DNA amplification
1. Heated lid Prevents condensation 1 2. Heating block Separates DNA strands , preparing them for copy 2 3 3. Cooling fans Cools DNA, priming it for copy 4 4. Microprocessor Stores and controls temperature cycles

9 Today, we will use DNA analysis to solve a Crime Mystery
PCR (DNA amplification) Gel electrophoresis DNA visualization

10 Missy Baker Gone Missing!
Boston metro Free Missy Baker, pastry shop owner goes missing, police at a loss The mysterious disappearance of the baker raises serious concerns within the population. An enigma that befuddles police investigators. With two abduction suspects nabbed, local students volunteer to try to find the missing baker. Boston, October 20th Husband Ned reported Missy "Sugar-Cup" Baker missing, fretful after not finding her at the shop following his daily nap. The couple resides in the apartment above the pastry shop at 2 Middleborough Rd. a popular fixture in this usually quiet neighborhood. Anxiety takes hold in the community. Quickly following the report of the missing baker (wheat-blond and thin as a stick) investigators identified two suspects, but both have remained silent after harsh interrogation. Following extensive police searches, investigators found blond hair strands in each of their cars. A godsend for science students, who volunteer with high-tech DNA analysis equipment to identify the kidnapper. Wooly mammoth p.5 finally cloned Transgenic kiwi p.7 solves world hunger

11 How to find Missy Baker: mutation in her CFTR gene
CFTR1 primer CFTR2 primer Healthy gene (most people) CFTR Gene Healthy person: the amplified DNA fragment always has the same size, corresponding to the distance between the PCR primers CFTR1 primer CFTR2 primer Deletion mutation (very rare) mutated CFTR Gene CFTR Gene Short PCR fragment Deletion Person carrying a CFTR deletion: the amplified DNA fragment from the mutated gene is smaller than in most (healthy) individuals

12 How to interpret PCR results (example)
Size in base pairs

13 What goes in a PCR reaction
Template DNA to be amplified Pair of DNA primers DNA polymerase dNTPs Buffer to maintain pH and provide Mg2+ Thermal cycler Taq CFTR1 primer CFTR2 primer A T G C

14 Setting up your PCR reactions Label 2 PCR tubes per group
“DETECTIVES” Groups 1 through 4 Tube “A” Tube “B” Hair DNA sample from suspect A’s car Hair DNA sample from suspect B’s car “REFERENCE LAB” Groups 5 through 8 Tube “H” Tube “D” Control DNA from healthy CFTR gene Control DNA from CFTR deletion mutant

15 Let’s pipette PCR Mix 15 µL per tube Same in all tubes H20 Buffer
DNA polymerase dNTPs Primers µL per tube Same primer in all tubes DNA µL per tube Tube A: Suspect A DNA sample Tube B: Suspect B DNA sample Tube H: Control DNA (healthy) Tube D: Control DNA (deletion) ________________ 30 µL per tube A B

16 Programming PCR parameters
Initial denaturation: 94°C 30 seconds Denaturation: 94°C 5 seconds Annealing 57°C 5 seconds Extension 72°C 8 seconds x30 cycles (25 cycles may also be enough if in a hurry) Final extension 72°C 30 seconds

17 Programming 1 57.0 30 5 5 8 30 2 3

18 Monitoring DNA amplification
What is happening to DNA molecules at each PCR step? Denaturation Annealing Extension Why do we need to add an enzyme (Taq polymerase)? What temperature is optimal for most enzymes? What makes Taq unique? How many more molecules of DNA will we have with each PCR cycle? And at the end of the entire PCR reaction? We call this exponential amplification How will we know which suspect might be implicated? Which caveats should the investigators consider?

19 Quiz: Which of these are NOT characteristics of PCR primers?
A. Short synthetic oligonucleotide B. Typically bases in length C. Double stranded DNA D. Unique homology to the DNA template E. Sequence with ~50% G:C content

20 Next step: visualize the DNA fragments amplified by PCR
1. Pour an agarose gel 2. Load the PCR products 3. Electrophoresis e- - Pole + Pole 4. Visualization in a transilluminator

21 What are the effects of Cystic Fibrosis (CF) genetic mutations?
Cystic Fibrosis is due to one or more mutations in the CFTR gene, which encodes a channel protein involved in the passage of chloride ions through the cell membrane. The defective gene interferes with the body’s ability to transfer water and salt to and from cells. This causes secretions, which are normally thin and watery in healthy people, to become very thick and sticky. The thick secretions clog up organs and prevent them from working properly. An increase in the viscosity of cell secretions often causes respiratory diseases, which are the main cause of death from CF.

22 Questions to probe deeper – I (During PCR or gel run)
What is the subcellular location of the CFTR gene product? How common do you think CFTR mutations are? How many different types of CFTR mutations can cause cystic fibrosis? Are these mutations typically dominant? Recessive? What would we see if Missy Baker were heterozygous for the CFTR deletion? What can be done to treat people with CFTR gene mutations?

23 Questions to probe deeper – II (After gel run)
How do you think the investigation turned out? Is the evidence consistent with suspect A being guilty? Why? Is the evidence consistent with suspect B being innocent? Why? What’s the importance of running controls from the Reference Lab? What caveats should be applied when analyzing forensic DNA evidence? What was the most unexpected thing you learned today?

24 Thank you We hope you enjoyed this lab!

25 Additional resources University of Utah PCR Virtual Lab
The Innocence Project History of the use of DNA in crime solving

26 Appendix: Copy cycles amplify DNA exponentially

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