Release v2.4: January 2015 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Crime Lab Science for everyone, everywhere

1 © Copyright by Amplyus LLC, all rights reserved Welcome! Our goals for today 1.Review DNA structure and DNA amplification concepts 2.Solve a crime mystery using PCR!

2 © Copyright by Amplyus LLC, all rights reserved PCR is at the heart of DNA analysis PCR Text Forensics Food and agriculture Consumer genomics Molecular diagnostics Personalized medicine Human evolution Text

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

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

5 © Copyright by Amplyus LLC, all rights reserved How PCR works: 3 steps to copy DNA Denaturation 1 94°C Annealing °C Primer 2 Primer 1 Extension 3 72°C Taq DNA polymerase dNTPs

6 © Copyright by Amplyus LLC, all rights reserved How PCR works: repeat the cycle DenaturationAnnealingExtension DNA + primersdenatured DNADNA + copy 94° C 50-60° C 72° C Repeat x ~25-30 cycles Single molecule~1B copies

7 © Copyright by Amplyus LLC, all rights reserved PCR makes DNA visible (and useful)

8 © Copyright by Amplyus LLC, all rights reserved PCR experimentation chain Set-upVisualizationPCRElectrophoresis

9 © Copyright by Amplyus LLC, all rights reserved How miniPCR TM enables DNA amplification Heated lid Prevents condensation 2 2. Heating block Separates DNA strands, preparing them for copy 3. Cooling fans Cools DNA, priming it for copy 4. Microprocessor Stores and controls temperature cycles

10 © Copyright by Amplyus LLC, all rights reserved Today, we will use DNA analysis to solve a Crime Mystery PCR (DNA amplification) Gel electrophoresis DNA visualization

11 © Copyright by Amplyus LLC, all rights reserved Use of DNA in personal identification

12 © Copyright by Amplyus LLC, all rights reserved Missy Baker Gone Missing! Free Boston metro 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 in custody, 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. Wooly mammoth p.5 finally cloned Transgenic kiwi p.7 solves world hunger 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.

13 © Copyright by Amplyus LLC, all rights reserved CFTR protein: Cystic Fibrosis Transmembrane Receptor Gene

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

15 © Copyright by Amplyus LLC, all rights reserved How to interpret PCR results (example) Size in base pairs

16 © Copyright by Amplyus LLC, all rights reserved 1.Template DNA to be amplified 2.Pair of DNA primers 3.DNA polymerase 4.dNTPs 5.Buffer to maintain pH and provide Mg 2+ 6.Thermal cycler What goes in a PCR reaction CFTR1 primer CFTR2 primer Taq A A A A A A A T T T T G T T C C C C C C C G G G G G G G

17 © Copyright by Amplyus LLC, all rights reserved Prepare 4 PCR tubes (200µL tubes) DNA sample from hair found with Suspect A 15 µL 10 µL 5 µL A DNA sample from hair found with Suspect B B Control DNA from healthy CFTR gene H Control DNA from CFTR deletion mutant D PCR Master mix (NEB One Taq: PCR Buffer + Mg 2+ + Taq + dNTPs) Primer mix (Forward and Reverse primers) Also add your initials to side of tube

18 © Copyright by Amplyus LLC, all rights reserved Alternative approach: 2 PCR Tubes per group Label 2 PCR tubes per group A.Hair DNA sample from suspect A’s car Tube “A”Tube “B” “DETECTIVES” Groups 1 through 4 B.Hair DNA sample from suspect B’s car “REFERENCE LAB” Groups 5 through 8 Tube “H”Tube “D” D.Control DNA from CFTR deletion mutant H.Control DNA from healthy CFTR gene

19 © Copyright by Amplyus LLC, all rights reserved Programming PCR parameters Initial denaturation:94°C30 seconds Denaturation:94°C5 seconds Annealing57°C5 seconds Extension72°C10 seconds x30 cycles Final extension72°C30 seconds

20 © Copyright by Amplyus LLC, all rights reserved Programming

21 © Copyright by Amplyus LLC, all rights reserved 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

22 © Copyright by Amplyus LLC, all rights reserved 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

23 © Copyright by Amplyus LLC, all rights reserved Next step: visualize the DNA fragments amplified by PCR 1. Pour an agarose gel2. Load the PCR products 4. Visualization in a transilluminator3. Electrophoresis e- - Pole + Pole

24 © Copyright by Amplyus LLC, all rights reserved Questions to probe deeper – I (During gel run) What is the subcellular location of the CFTR gene product? How many different types of CFTR mutations can cause cystic fibrosis? How common do you think CFTR mutations are? 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?

25 © Copyright by Amplyus LLC, all rights reserved Medically relevant CFTR mutations

26 © Copyright by Amplyus LLC, all rights reserved CFTR mutations affect water balance in the lung airway lining

27 © Copyright by Amplyus LLC, all rights reserved 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.

28 © Copyright by Amplyus LLC, all rights reserved 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?

29 © Copyright by Amplyus LLC, all rights reserved Thank you We hope you enjoyed this lab!

30 © Copyright by Amplyus LLC, all rights reserved Additional resources Interpreting CFTR mutations The Innocence Project History of the use of DNA in crime solving crime-solving-judicial-and-legislative-history Deutsche Welle: 30 years of DNA Fingerprinting been-30-years-of-eureka/a

31 © Copyright by Amplyus LLC, all rights reserved Appendix: PCR cycles amplify DNA exponentially