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Introduction to Polymerase Chain Reaction

Introduction to Polymerase Chain Reaction. Introduction to PCR. What is PCR? Reaction components How does PCR work? PCR vs. Cell Replication PCR applications in the real world Alu PV92 What is Alu? Movement of retrotransposons. What is the Polymerase Chain Reaction?.

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Introduction to Polymerase Chain Reaction

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  1. Introduction to Polymerase Chain Reaction

  2. Introduction to PCR • What is PCR? • Reaction components • How does PCR work? • PCR vs. Cell Replication • PCR applications in the real world Alu PV92 • What is Alu? • Movement of retrotransposons

  3. What is the Polymerase Chain Reaction? PCR is a scientific method of rapid amplification of a specific segment of DNA. The products of PCR are amplicons or “PCR products” Image: http://www.copernicusproject.ucr.edu/

  4. What do we need to copy a piece of DNA? Think about cellular DNA replication www.daviddarling.info/images/DNA_replication.gif

  5. Template: DNA Single-stranded DNA How do we make DNA single-stranded? • Acidity • Basicity • Enzymes (helicase) • Heat *

  6. Building Blocks: dNTPs Deoxynucleotide-triphosphates (dNTPs) are needed to create new DNA strands A “dNTP mix” contains equal amounts of : dATP dTTP dGTP dCTP Adenosine triphosphate Image: http://commons.wikimedia.org/

  7. Enzyme: DNA Polymerase Original reaction used bacterial DNA polymerase, which was denatured at high temperatures Identified a polymerase from a thermophilic organism that could withstand 95oC Thermus aquaticus, the enzyme is Taq polymerase Cute, but not this guy Image: www.tribuneindia.com/2004/20041207/world.htm

  8. Enzyme: DNA Polymerase, continued Processes in the 5’ to 3’ direction Requires a cofactor (Mg++) No proof-reading ability Adds nucleotides at a rate of about 2000 bp/ minute Hot-start Taq Molecular Model of Taq polymerase Image: Protein Data Bank DNA polymerases need a double-stranded piece of DNA…

  9. … … ….. ….. Double strand: Primers Primers are usually used in pairs (forward and reverse) and are typically 15-30 base pairs long Primers for PCR reactions are specific and complementary to the desired piece of DNA 3’ 5’ 5’ 3’

  10. Enzyme cofactor: Mg++ Mg++ or Mn++(depending on the enzyme) is required for DNA polymerase activity If not included in the PCR buffer mix, MgCl2 is added separately MgCl2 concentration must be optimized for successful DNA amplification

  11. Happy Medium: Buffer Buffer maintains an optimal pH environment Salts provide optimal ionic strength for enzyme activity

  12. Overview of PCR reaction components Template dNTPs Taq polymerase MgCl2 Primers Buffer Image: www.bridgebio.com/Plastic-TubePcr.php All components are needed in optimal concentrations

  13. How does PCR Work?

  14. Cycling temperature Functions Denature: 94-96oC Separates double helix into two strands Anneal: 37-65oC Primers bind to their target site on single stranded DNA Extension: 72oC DNA polymerase adds dNTPs according to the base pairing rules (also known as polymerization) The three temperatures are repeated 25-40 times

  15. Thermal cyclers through the ages Three hot water baths 95oC 55oC 72oC Image: http://swatlab.nmsu.edu/Equipment/bath-sml.html GeneAmp 2400 GeneAmp 2700 Stratagene Robocycler Image: www.labtrader.com Image: www.escience.ws/b572/L3/L3.htm Image: www.usedlabequip.com

  16. Steps of PCR

  17. Steps of PCR, continued

  18. Steps of PCR, continued

  19. After 30 cycles, DNA is amplified over a billion fold. Exponential Amplification Cycle 3 Cycle 2 Cycle 1

  20. Exponential Amplification* Assuming 100% efficiency each cycle Cycle 1 2 3 4 5 6 7 8 9 10 . . 20 . . 30 Amount of product (2n) where n = cycle # 2 4 8 16 32 64 128 256 512 1,024 . . 1,048,576 . . 1,073,741,824

  21. Animation of the polymerase chain reaction at the DNA Learning Center DNALC PCR animation http://www.dnalc.org/ddnalc/resources/pcr.html Sumanas, Inc. PCR animation http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

  22. The Plateau Effect Exponential growth of amplicons does not continue indefinitely. Why not? • AmpliTaq loses it’s activity after repeated denaturation temperatures • More target molecules than Taq molecules • Product strands compete with primers for annealing • Melting temperature of the reaction increases

  23. PCR vs. Cellular DNA Replication

  24. PCR in the Real World Screening Identification Basis for molecular techniques and advances

  25. PCR in the Real World: Screening • Detection of heritable diseases • Detection of viral diseases • Screening for bone marrow and organ compatibility • Detection of genetically engineered elements in crop plants

  26. PCR in the Real World: Identification • Genetic fingerprinting • Paternity testing • Identification of human remains • Analysis of ancient humans and other organisms • Study evolutionary relationships Image: http://news.nationalgeographic.com/news/ 2006/01/0131_060131_earliest_africans.html

  27. PCR in the Real World: Molecular Biology • Cloning genes for medicine (eg., insulin) • Site-directed mutagenesis • Genetic engineering of plants, animals, and microorganisms • Gene expression studies (RT-PCR and quantitative PCR) Image: http://www.ivy7.com/greenweb/ Issues/GMO.html

  28. PCR in the Real World: Genome Sequencing How do you amplify DNA if you don’t know the sequence? Image: http://www.gene-abc.ch/welt/tech/meth/gt036/index.html

  29. DNA Extraction and PCR Reaction Set-up Genomic DNA extraction • Saline mouthwash to obtain cheek cells • Centrifuge saline wash to pellet cheek cells • Add suspension to heated Chelex solution • 100 ºC ruptures cell membranes and denatures proteins Image: Frank Stephenson

  30. DNA Extraction and PCR Reaction Set-up PCR reaction • 20 µl of Master Mix Contains buffer, dNTPs, MgCl2, AmpliTaq Gold • 20 µl of Primer Mix Contains the forward and reverse primers, diluted to the proper concentration • 10 µl of your own genomic DNA Images: Frank Stephenson

  31. DNA Extraction and PCR Reaction Set-up PCR reaction, controls • Positive control: purchased human DNA This control allows us to distinguish between a failed PCR reaction and a failed DNA extraction • Negative control: water in place of DNA This reaction lets us know if we have any contaminated reagents

  32. DNA Extraction and PCR Reaction Set-up • Thermal Cycler Parameters for Alu PCR • 95oC - 10 minutes • 95oC - 30 seconds • 65oC - 30 seconds • 72oC - 2:00 minutes • 72oC - 10 minutes (final extension) Repeat 30 times

  33. What are we amplifying?

  34. Amplified Region 5’ Alu 3’ Exon 8 Exon 9 Exon 10 Alu PV92 Found on Chromosome 16 Located In a non-coding region of DNA A member of Alu-repeat family Not associated with any disease or disorder

  35. Alu repeats • 300 bp repeats (SINEs- Short Interspersed Elements) • Named after the Alu-1 restriction site within the element (AGCT) • Alu PV92 is stable and dimorphic • Over 1,000,000 Alu repeats scattered throughout the human genome; 10% of genome

  36. Movement of Retrotransposons Images: http://fig.cox.miami.edu/~cmallery/150/gene/c7.19.16b.transposon.jpg

  37. Evolutionary Significance of AluPV92 Highly conserved Inserted within the last 1,000,000 years Dimorphic (+/+, +/-, -/- ) Used in population genetics, paternity analysis, and forensics

  38. PCR of Alu PV92 Forward primer PCR Target Chromosome 16 Reverse primer 415 bp (-) Forward primer PCR Target Chromosome 16 Alu (300 bp) Reverse primer ?? (+)

  39. Analyzing Alu PV92 Results • Run class gels to determine individual genotype • Calculate the allele frequency for our class • Calculate the genotype frequency • Determine if our class population is in Hardy-Weinberg equilibrium • Write a formal lab report

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