1 Welcome! Our goals for today Review DNA structure and DNA amplification conceptsSolve a crime mystery using PCR!
2 PCR is at the heart of DNA analysis Molecular diagnosticsTextConsumer genomicsTextTextPersonalized medicinePCRFood and agricultureTextTextHuman evolutionTextForensics
3 Polymerase Chain Reaction (PCR) Genetic testingPathogen detectionDrug developmentCrop modificationForensic analysisSequencingEtc.Complex DNA sampleRegion of interestAmplified DNA (Billions of copies)ApplicationsA 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 complementaritySource: US National Library of Medicine, NIH, Thinkquest
5 How PCR works: 3 steps to copy DNA 1Denaturation50-60°C2Primer 1Primer 2Annealing72°C3Taq DNA polymerasedNTPsExtension
6 How PCR works: repeat the cycle denatured DNADNA + primersDNA + copySingle molecule94° C~1B copies50-60° C72° CDenaturationAnnealingExtensionRepeat x ~25-30 cycles
8 How miniPCRTM enables DNA amplification 1. Heated lidPrevents condensation12. Heating blockSeparates DNA strands , preparing them for copy233. Cooling fansCools DNA, priming it for copy44. MicroprocessorStores and controls temperature cycles
9 Today, we will use DNA analysis to solve a Crime Mystery PCR (DNA amplification)GelelectrophoresisDNA visualization
10 Missy Baker Gone Missing! Boston metroFreeMissy Baker, pastry shop owner goes missing, police at a lossThe 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 clonedTransgenic kiwi p.7solves world hunger
11 How to find Missy Baker: mutation in her CFTR gene CFTR1 primerCFTR2 primerHealthy gene(most people)CFTR GeneHealthy person: the amplified DNA fragment always has the same size, corresponding to the distance between the PCR primersCFTR1 primerCFTR2 primerDeletion mutation(very rare)mutated CFTR GeneCFTR GeneShort PCR fragmentDeletionPerson 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 amplifiedPair of DNA primersDNA polymerasedNTPsBuffer to maintain pH and provide Mg2+Thermal cyclerTaqCFTR1 primerCFTR2 primerATGC
14 Setting up your PCR reactions Label 2 PCR tubes per group “DETECTIVES”Groups1 through 4Tube “A”Tube “B”Hair DNA sample from suspect A’s carHair DNA sample from suspect B’s car“REFERENCE LAB”Groups5 through 8Tube “H”Tube “D”Control DNA from healthy CFTR geneControl DNA from CFTR deletion mutant
15 Let’s pipette PCR Mix 15 µL per tube Same in all tubes H20 Buffer DNA polymerasedNTPsPrimers µL per tubeSame primer in all tubesDNA µL per tubeTube A: Suspect A DNA sampleTube B: Suspect B DNA sampleTube H: Control DNA (healthy)Tube D: Control DNA (deletion)________________30 µL per tubeAB
16 Programming PCR parameters Initial denaturation: 94°C 30 secondsDenaturation: 94°C 5 secondsAnnealing 57°C 5 secondsExtension 72°C 8 secondsx30 cycles (25 cycles may also be enough if in a hurry)Final extension 72°C 30 seconds
18 Monitoring DNA amplification What is happening to DNA molecules at each PCR step?DenaturationAnnealingExtensionWhy 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 amplificationHow 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 oligonucleotideB. Typically bases in lengthC. Double stranded DNAD. Unique homology to the DNA templateE. Sequence with ~50% G:C content
20 Next step: visualize the DNA fragments amplified by PCR 1. Pour an agarose gel2. Load the PCR products3. Electrophoresise-- Pole+ Pole4. 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?