Learning Objectives Draw or model the first three cycles of PCR, including the correct directionality (5’- and 3’-ends) of the primers and single-stranded PCR products. Diagram how single-stranded products from the first cycle of PCR are used as templates for subsequent PCR cycles. Demonstrate which parts of the primers will anneal to the original DNA template and subsequent PCR products. Model and demonstrate when the primer restriction enzyme sites are incorporated into double-stranded PCR products. Calculate the number of desired-length PCR products and long PCR products for each amplification cycle. Demonstrate how the incorporation of primer restriction enzyme sites into PCR products is a useful tool for subsequent cloning of the product into a vector.

PCR = in vitro synthesis of DNA by thermostable DNA polymerase Uses? Amplification: exponential or linear? http://schoolworkhelper.net/pcr-uses-steps-purpose/ Amplify ~1 million-fold after 20 cycles

One cycle: 3 steps (times may vary) Denaturation: 95oC, 30 sec Annealing: variable temp: 45-65oC, 30 sec Extension: 68-72oC, time: 60 sec http://biosistemika.com/workshops/qpcr-basics/

Compare PCR to DNA replication in a cell DNA Replication in the cell PCR in a test tube Enzyme used? DNA polymerase Heat resistant DNA polymerase Template needed? What is composition of template? Primer needed? What is composition of primer? Synthesis direction? Nucleotides needed? Which ones? What separates template strands? Is entire template replicated?

In-class Activity Scenario: Each group has been assigned a gene to amplify from the chromosome of Whodatamonas saintsatium, a newly discovered (obviously fictitious) bacterium that helps grown men win football games on Sunday. We have isolated “chromosomal DNA” for you because we are such wonderful instructors.

Objective: Demonstrate the 3 steps of a PCR reaction for 3 cycles PCR components (in “toolkit” envelope) Double-stranded chromosomal DNA = twisted yarn Black stripes show fragment to be amplified Primers = 3-inch colored Bendaroos (flexible, waxed sticks) Forward and reverse primers – different colors Restriction enzyme site to be added: yellow piece at end of colored Bendaroo Newly synthesized DNA = 3 sheets of bendaroos – different colors for different cycles Polymerase = your hands (What components are we missing?)

Homework Cycle Number of new single-stranded products generated during each cycle Total number of single-stranded products (cumulative cycles) Total number of single-stranded products of correct length that include the restriction enzyme site Total number of single-stranded products that are too long Percentage of correct length products/total products 1 2 0% 3 4 5 6

Homework Answer Key Cycle Number of new single-stranded products generated during each cycle Total number of single-stranded products (cumulative cycles) Total number of single-stranded products of correct length that include the restriction enzyme site Total number of single-stranded products that are too long Percentage of correct length products/total products 1 2 0% 4 6 33% 3 8 14 57% 16 30 22 73% 5 32 62 52 10 84% 64 126 114 12 90%