Bio Rad PCR Song Lyrics There was a time when to amplify DNA, You had to grow tons and tons of tiny cells. (Oooh) Then along came a guy named Dr. Kary Mullis, Said you can amplify in vitro just as well. Just mix your template with a buffer and some primers, Nucleotides and polymerases too. Denaturing, annealing, and extending, Well it s amazing what heating and cooling and heating will do. [Chorus] PCR when you need to detect mutation (detect mutation) PCR when you need to recombine (recombine) PCR when you need to find out who the daddy is (who s your daddy?) PCR when you need to solve a crime (solve a crime) [x2] STUDENT SHEET POLYMERASE CHAIN REACTION (PCR) 1. Before Briefly explain the steps involved in normal DNA replication in a cell. 2. What is the function of each of the following in a PCR reaction? HEAT: PRIMER: taq POLYMERASE:
3. Briefly describe what the thermocycler (PCR machine) does and what happens in each of the following three steps of Polymerase Chain Reaction: DENATURING: ANNEALING: ELONGATION/EXTENSION: 4. Suppose you start with one double-stranded molecule of DNA and you want to amplify this one DNA molecule using PCR. You add an excess of primers, each of which will anneal to the DNA molecule in only one place, copying the segment of DNA between them. Draw representations of the DNA and primers during the three steps of PCR during one cycle.
5. Illustrate the exponential growth of the DNA from question 4 through 3 cycles of PCR. 6. How many molecules of double-stranded DNA will you have after three cycles? After five cycles? After 30 cycles?
STUDENT WORKSHEET ANSWER SHEET 1. Briefly explain the steps involved in normal DNA replication in a cell. In a cell, the double-stranded DNA helix unwinds and unzips at the replication origin. DNA polymerase makes a new copy of the template strand by adding complimentary bases, producing a replication fork. ANSWER SHEET 2. What is the function of each of the following in a PCR reaction? Heat: turning up the temperature causes the double stranded DNA to separate or denature Primer: these are the hooks that provide the starting point for the polymerase. There are two primers, a forward and a reverse which flank the target sequence. Taq polymerase: heat-stable DNA polymerase 3. Briefly describe what the thermocycler (PCR machine) does and what happens in each of the following three steps of Polymerase Chain Reaction: Denaturing: 94 C, raising the temp separates the DNA strands from double- stranded to single-stranded Annealing: 50-65 C, dropping the temperature allows the primers to anneal to their complimentary sequences Extension: 72 C, bring the temperature back up allows the Taq polymerase to extend the template strands by adding complimentary base pairs to the singlestranded DNA molecules 4. Suppose you start with one double-stranded molecule of DNA and you want to amplify this one DNA molecule using PCR. You add an excess of primers, each of which will anneal to the DNA molecule in only one place, copying the segment of DNA between them. Draw representations of the DNA and primers during the three steps of PCR during one cycle. Student drawings will vary. 5. Illustrate the exponential growth of the DNA from question 4 through 3 cycles of PCR. Student drawings will vary. 6. How many molecules of double-stranded DNA will you have after three cycles? After five cycles? After 30 cycles? 3 cycles = 8 ds DNA (2 3 ); 5 cycles = 32 ds DNA (2 5 ); 30 cycles = 1,073,741,824 (2 30 )
Types of PCR: Traditional vs. Real Time PCR OVERHEAD Advantages of Real-Time PCR vs. Traditional PCR
Types of PCR: Traditional vs. Real Time PCR LESSON OVERHEAD PLANS Overview Quantitative? Real-time PCR Measures PCR amplification as it occurs. Yes, because data is collected during the exponential growth (log) phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid. Traditional PCR Measures the amount of accumulated PCR product at the end of the PCR cycles. No, though comparing the intensity of the amplified band on a gel to standards of a known concentration can give you 'semi-quantitative' results. Applications Quantitation of Gene Expression Microarray Verification Quality Control and Assay Validation Pathogen detection SNP Genotyping Copy Number Variation MicroRNA Analysis Viral Quantitation sirna/rnai experiments Summary Advantages of Real-Time PCR Increased dynamic range of detection No post-pcr processing Detection is capable down to a 2-fold change Collects data in the exponential growth phase of PCR An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated The cleaved probe provides a permanent record amplification of an amplicon Amplification of DNA for: Sequencing Genotyping Cloning Disadvantages of Traditional PCR Poor Precision Low sensitivity Short dynamic range < 2 logs Low resolution Non-Automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Post-PCR processing