MolbioLAB

• Polymerase Chain Reaction (PCR)


  A "polymerase-catalyzed" chain reaction to amplify sequence-specific DNA fragments in vitro

  
• Conventional Polymerase Chain Reaction

  A type of PCR

  
• Conventional PCR


  1. Introduction

  2. Components of PCR

  3. How does Conventional PCR work?

  4. Analysis of the PCR product

  5. Applications of PCR

  6. Modifications of PCR, including Reverse Transcriptase PCR
• PCR


  Allows exponential amplification of a DNA sequence
• Each PCR cycle theoretically doubles the amount of DNA
• DNA Amplification using PCR


  1. Separation of DNA strands

  2. Synthesis of a complementary strand

  
• Components of PCR


  • DNA Source
  • Primers
  • Nucleotide bases
  • DNA polymerase
  • Reaction buffer
  • Magnesium
• DNA Source


  • Fresh or preserved specimen (plant, animal, human tissues, blood, urine, stool, etc.)
  • DNA extraction and purification
• Primers


  • Define the DNA sequence to be amplified
  • Bind or anneal to the DNA template
  • Act as starting points for the DNA polymerase

  
• Good Primer


  • Melting temperature: 52-65⁰C
  • Absence of dimerization capability
  • Absence of significant hairpin formation
  • Lack of secondary priming sites
  • Low specific binding at the 3' end
• Melting Temperature (Tm)


  • Temperature at which ½ the DNA strands are single-stranded and ½ are double-stranded
  • Higher G+C content 🡪 higher Tm

  
• Annealing Temperature (Tanneal)


  • Temperature at which primers anneal to the template DNA
  • Optimal Tanneal is within 5⁰ of the Tm of the PCR primer
• The allowed maximum difference between annealing temperatures of both primers is 3⁰C
• Primer Pair Matching


  • PCR condition should be suitable for both primers
• Base Composition and Length


  • Average G+C content should be around 50-60%
  • Primer length has effects on uniqueness and melting/annealing temperature
• Nucleotide bases


  • Deoxynucleotide phosphates (dNTPs)
  • A pool of adenine, thymine, cytosine, guanine
  • The building blocks for the newly synthesized DNA strands

  
• DNA polymerase


  • An enzyme that synthesizes new DNA strands
  • Thermostable
  • Taq DNA polymerase is the most commonly used

  
• Reaction buffer


  A typical 10X reaction buffer contains: 500mM KCl, 100mM Tris-HCl, 1% Triton X-100
• Magnesium


  • The most important factor to optimize when performing the PCR
  • The optimal Mg++ concentration will depend upon the primers, template, DNA polymerase, and dNTP concentration
• Other necessary PCR components


  • Exogenous positive control
  • Endogenous positive control
  • Negative control
  • No template control
  • Internal control
  • No RT control
• How does Conventional PCR work?


  1. DNA Denaturation

  2. Primer Annealing

  3. Primer Extension
• These PCR steps are repeated for 30 or 40 cycles in a thermocycler/PCR machine
• Typical thermal cycler program

  1. Initial DNA denaturation at 95⁰C for 2 minutes

  2. 20-35 PCR cycles: Denaturation at 95 ⁰C for 30 seconds to 1 minute, Annealing at 42-65 ⁰C for 1 minute, Extension at 68-74 ⁰C for 1-2 minutes

  3. Final extension at 68-74 ⁰C for 5-10 minutes

  4. Soak at 4 ⁰C
• Exponential PCR Amplification
• Analysis of the PCR Product


  • Staining of the amplified DNA product using a chemical dye
  • Labelling the PCR primers or nucleotides with fluorescent dyes
• Applications of PCR


  • Diagnosis of diseases
  • Paternity testing
  • Identifying badly decomposed bodies or when only body fragments are found
  • Pathogen identification and diagnosis
  • DNA or RNA labeling
  • DNA or RNA cloning
  • Disease identification
  • Drug discovery
  • DNA and RNA detection
  • DNA and RNA quantification
  • Genotyping and DNA-based identification: Forensic Science
• PCR Modifications


  • Multiplex PCR
  • Reverse transcriptase PCR
  • Nested PCR
  • Real-time or quantitative PCR
• Reverse transcriptase PCR was introduced in 1977 and was developed due to the discovery of reverse transcriptase during the study of viral replication of a genetic material
• Labelling the PCR primers or nucleotides with fluorescent dyes (fluorophores)


  Prior to PCR amplification
• Gel Electrophoresis


  The most widely used method of analysis of the PCR product
• Applications of PCR


  • Diagnosis of diseases
  • Paternity testing
  • Identifying badly decomposed bodies or when only body fragments are found
  • Pathogen identification and diagnosis
  • DNA or RNA labeling
  • DNA or RNA cloning
  • Disease identification
  • Drug discovery
  • DNA and RNA detection
  • DNA and RNA quantification
  • Genotyping and DNA-based identification: Forensic Science
• PCR Modifications


  • Multiplex PCR – amplify several genes at the same run
  • Reverse transcriptase PCR – particularly useful for RNA viruses
  • Nested PCR – uses 2 primers, more specific amplification
  • Real-time or quantitative PCR
• Reverse transcriptase PCR (RT-PCR) was introduced


  1977
• Reverse transcriptase


  Discovery during the study of viral replication of a genetic material led to the development of RT-PCR
• RT-PCR


  Used to detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA
• Conventional PCR


  1. Amplification

  2. Conversion of mRNA to cDNA
• Real-time PCR (qPCR)


  • Tells us "how much" - Allows to follow the amplicon accumulation kinetics and assess when the exponential phase occurs for each sample
  • Tells us "what" - End-point analysis of PCR products (by visualization dye-stained gels, primers labelled with fluorescent dyes, etc.)
• In qPCR, the same procedure with conventional PCR occurs but with 2 major differences:
• Difference 1


  The amplified DNA is fluorescently labelled
• Difference 2


  The amount of fluorescence released during amplification is directly proportional to the amount of amplified DNA