1. Basic LaboratoryTechniques: PCR BIOL 2115Catherine Loc-Carrillo, Ph.D.

2. Class ObjectivesBy the end of this class you should: Understand what PCR is Understand how it can be used to identify bacteria Have hands-on experience on performing a routine PCR assay Be able to analyze the results obtainedYou will also be presented with some of itsapplications and participate in an fact or fiction quiz 3. Polymerase Chain Reaction (PCR)PCR allows us to produce many copies of a specificDNA sequence in a short period of timeThe protocol involves 4 components: an enzyme (polymerase) nucleotides (dNTPs) oligonucleotides (primers) Template DNA 4. A closer look at the major players:DNA polymerase catalyzes template-dependent synthesis ofDNAA pair of synthetic Forward Primer 5 -GACGGTGAGTAATGCCTA-3oligonucleotides to prime Reverse Primer 5-CACTGGTGTTCCTTCCTATA-3DNA synthesisDeoxynucleosidetriphosp 3 phosphateshates (dNTPs) equalamounts are needed sugarbases 5. Denaturing step:The mixture is heated to 94-96C to separate DNAinto single strands 6. Annealing step:The temperature is lowered to 50-65C to allow theleft and right primers to pair (by H bonds) to theircomplementary sequencesThe primer set is designed to bracket the DNA regionto be amplified 7. Extension step:The temperature is raised to 72C to allow the Taqpolymerase to attach at each priming site andsynthesize a new DNA strand 8. Proceeding cycles: 9. Turning Theory into Practice 10. How can PCR be used toidentify bacteria? Bacteria have conserved genes that determine their genus and species The sequence of the specific gene must be knownPrimers are designed based on thesequence A PCR assay can be used to identify the presence of the specific genes sought 11. Your Mission:You are provided with 4 unidentified bacteria: Pseudomonas aeruginosa X 2 Pseudomonas fluorescens Escherishia coliEach group will have 1 set of primers (A or B) One set will detect the Pseudomonas genus One set will detect Pseudomonas aeruginosaUsing your results as a class, you can determine thebacteria on your agar plates 12. Master Mix set-up Components: Per 20l rxn For 5 samples Molecular grade water 13.8 l 69.0 l NEB standard buffer 2.0 l10.0 l Primer, forward 0.8 l 4.0 l Primer, reverse 0.8 l 4.0 l dNTP mix0.4 l 2.0 l Taq polymerase0.2 l1.0 lVol. Req. = Req. Conc.X Total Vol.V1 = C2V2C1V1= C2V2Actual Conc.C1 13. How to perform aroutine colony PCR assay 14. Its PCR time! 15. How to analyze yourresults-956 bp fragmentsIdentify Ps. aeruginosa618 bp fragments IdentifyPseudomonasgenus 16. Good Bacteria, Bad BacteriaThe Pseudomas genus > 191 described species Ubiquitous in nature Naturally resistant to some antibioticsP. fluorencesand agriculture Some strains have biocontrol propertiesP. aeruginosa and cystic fibrosis Infection occurs ~85% of adolescent patients 17. Applications of PCRMedical: Used for genetic testing to detect presence of genetic disease i.e. sickle cell anemiaInfectious Disease: Used to screen donated blood for HIVForensic: Used to match an individual with their close relativesResearch: Gene expression using qPCR 18. The Taq polymerase enzyme was isolated fromYellow Stone National Park.An individual can be identified from a strand of hair.DNA fingerprinting can be performed in 5 minutes.There is a 70-90% similarity between mouse andhuman genes. 19. The Taq polymerase enzyme wasisolated from Yellow StoneNational ParkThe thermophile(Thermusaquaticus) was isolatedfrom hot springs The enzyme is able to withstandthe protein denaturingconditions required during PCR 20. An individual can be identifiedfrom a strand of hairThere are 2 different types of DNA in astrand of hairNuclear DNA can be obtained from thefollicleMitochondrial DNA can be obtained fromthe shaftContains less information than nDNA but ismore stableCan be even be extracted from mummiesInherited from maternal side, thereforemother and her off-springs will haveidentical mDNA profiles 21. DNA fingerprinting can beperformed in 5 minutesNot with our currenttechnology!Profile is compared to areference sample/databaseTypical labs can take up to 14days But some research labs have reported in doing it within a couple of hours (automated) Bin Ladens body could have been identified by the FBI with a few hours 22. There is a 90% similarity between mouse and human genesIn 2001 the human genome wassequencedIn 2002 the next mammal sequencedwas the mouseComparative analysis in 2009: Identified 15,187 human and mouse genes with orthologous relationships, representing 75% of mouse and 80% of human genesMouse models are used to understandhuman disease and development 23. To recap!What are the 4 main components for PCR?What are the 3 main processes during a PCR cycle?Why was the discovery of Taq polymerase soimportant?Which bacteria in your experiment was the negativecontrol?Which PCR product will have the biggest fragmentsize?