PCR Notes

The Polymerase Chain Reaction in Molecular Biology

The Polymerase Chain Reaction: An Overview

  • The Polymerase Chain Reaction (PCR) is a technique to make many copies of a specific DNA region in vitro.
  • In vitro means in a controlled environment outside a living organism (e.g., in a test tube), while in vivo refers to processes happening within a living organism. There is also in silico which refers to computational analysis.
  • The DNA region can be any sequence of interest to the experimenter, such as:
    • A gene whose function a researcher wants to understand.
    • A genetic marker used by forensic scientists to match crime scene DNA with suspects.
  • The enzyme DNA polymerase is responsible for the synthesis of new DNA copies.
    • DNA polymerase "reads" the template DNA and adds the appropriate complementary base pair from 5’ to 3’, similar to DNA replication.
  • This process is repeated with many "cycles," resulting in many copies of the original template DNA.
  • The main goal of PCR is to create enough copies of a target DNA sequence for experimental uses, such as:
    • Cloning a fragment of foreign DNA into a plasmid to create a recombinant DNA molecule.
    • Identifying different microorganisms in a patient sample by sequencing.
    • Disease diagnostics and detection of mutations in specific genes (RFLPs).
    • Generating forensic profiles and allele marker analysis (fingerprinting; STRs).

History of PCR

  • 1976: Isolation of Taq DNA polymerase from Thermus aquaticus (T. aquaticus -> Taq).
    • T. aquaticus is a thermophilic bacterium with high thermostability.
    • It was found and isolated within hot springs.
    • Taq polymerase has an optimum temperature for activity between 75 – 80 °C and remains stable up to 95 °C.
    • Human DNA polymerase has an optimum temperature of 37 °C.
    • Taq polymerase can replicate a 1000 bp strand of DNA in approximately 30 seconds.
  • 1983: Kary Mullis created the technique of PCR.
    • He used Taq polymerase to demonstrate that forward and reverse primers can be used to produce many copies of a fragment of DNA from a specific gene region.
  • 1985: First publication using the PCR technique.
  • 1989: Taq polymerase was labeled molecule of the year.
  • 1993: Kary Mullis won the Nobel Prize.
  • 1988: Patent for Taq polymerase filed. First PCR thermocycler introduced.
  • 1953: Discovery of the DNA double helix structure.
  • 1967: Thomas Brock reports on the isolation of the extremophilic bacterium Thermophilis aquaticus.
  • 1971: Kleppe and co-workers first describe a method using an enzymatic assay to replicate a short DNA template with primers in vitro.
  • 1977: Frederik Sanger and colleagues introduce the "dideoxy" chain-termination method for sequencing DNA (also known as 'Sanger sequencing'). It utilizes DNA polymerase, nucleotide precursors, and one oligonucleotide primer.
  • 1985: Kary Mullis discovers that using two oligonucleotides instead of one -on opposite strands- enables DNA to be synthesized from a single, specific location in the genome.
  • 1991: Patent for Taq DNA polymerase is filed by Mullis et al. The first automated PCR cycler is introduced to the market by Perkin Elmer and Cetus (joint venture).
  • 1995: The first real-time PCR instrument is described.
  • 1995: The first complete genome of a free-living organism is sequenced by Venter and colleagues (Haemophilus influenzae).
  • 1994: Hot start PCR by wax technology described.
  • 1996: Antibody-based hot start technology.
  • 1996: Genome of the first eukaryotic organism, Saccharomyces cerevisiae, is sequenced. Two commercial real-time PCR instruments are launched to market.
  • 2003: Phusion High-Fidelity DNA Polymerase, the first PCR enzyme based on fusion protein technology, is launched by Finnzymes Oy.
  • 2005: Lynx Therapeutics publishes and markets "MPSS" - a parallelized, adapter/ligation-mediated, bead-based sequencing technology, launching "next-generation" sequencing.
  • 2009: The first complete human genome is sequenced by Levy et. al.
  • 2010: Gibson et al. create the first bacterial cell controlled by a chemically synthesized genome (using Phusion High Fidelity DNA Polymerase).
  • 2010: The MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) are published by Bustin et. al.

Components of PCR

  • The components of PCR are:
    • DNA polymerase
    • DNA template
    • Primers
    • Free nucleotides (dNTPs)
    • Free ions (K, Mg2+)
    • Sterile Water
DNA template
  • There are two types of template:
    • Genomic DNA (gDNA)
    • Complementary DNA (cDNA)
  • gDNA
    • Very stable dsDNA
    • Used for identification of an organism.
    • Used to identify mutations.
    • Used to identify genetic markers.
  • cDNA
    • Derived from RNA, which is unstable ssDNA.
    • RNA is converted to cDNA, which is very stable dsDNA.
    • Used to determine whether a gene is being transcribed.
    • Used to examine the response of genes to treatments (e.g., drug therapy).
Primers
  • Designed to amplify your target sequence.
    • ONLY your target sequence
  • Typically ~ 18 - 30 bp in length
  • G/C content should be around 35 – 55 % (GC clamp)
  • Annealing temperature must be within 1 °C of each other.
  • Primers instruct DNA polymerase where to bind and begin synthesis.
  • Upstream region:
    • Contains regulatory sequences to signal protein binding for transcription initiation.
  • Coding region (ORF):
    • Gene sequence which is translated into amino acids to make protein.
    • Has a start codon (ATG,…) and stop codon (TAA,…)
  • Downstream region:
    • Contains regulatory sequences to signal the end of transcription.
    • Terminator
  • To amplify the entire coding region of a gene (ORF):
    • Primers are designed appropriately at the start and end of the ORF.
Primer Design Top Tips
  • Where to design primers?
    • Thankfully, there are many software programs available to design primers!
    • Two of the most popular are Primer3 and Primer-BLAST.
  • GC content of 40 – 60 % (GC clamp)
    • GC has stronger H-bonding (3 vs. 2 of AT).
    • Improves primer binding and stability.
  • Length around 18 – 30 bp
    • Shorter primers bind more efficiently.
    • Longer primers are more target-specific.
  • TmT_m of primers between 50 – 65 °C
    • Temperature at which primer duplexes with ssDNA (annealing).
    • TmT_m of Forward and Reverse primers should be within 1 °C of each other.
  • Avoid runs of 4 or more of a single base or dinucleotide repeats.
    • e.g., ACCCC or ATATATAT
  • Avoid Forward & Reverse primer homology.
    • These result in undesirable interactions / secondary products.
    • Hairpins form when a primer is self-complementary.
    • Dimers form when both primers share homology.
Free nucleotides (dNTPs)
  • Free nucleotides (pictured as dNTPs) are the building blocks of nucleic acids.
  • As DNA polymerase reads the template, it recruits complementary dNTPs to synthesize the new strand:
    • dATP
    • dCTP
    • dGTP
    • dTTP
  • dNTP: deoxyribonucleotide triphosphate
Free ions (Mg2+, K)
  • Mg2+Mg^{2+} and K ions are cofactors, facilitating PCR reaction via:
  • Mg2+Mg^{2+}
    • Catalyzes phosphodiester bond of 3’ OH of primer and newly added dNTP.
    • Stabilizes complex formed between polymerase, primer, and template.
  • K
    • Stabilizes primer binding to template.

Take-Home Message: PCR Overview

  • What is PCR?
    • Amplify dsDNA; ssDNA must be converted to dsDNA.
    • The goal is to produce multiple copies of DNA for analysis.
  • History of PCR?
    • Technique created in 1983 by K. Mullis.
    • Taq polymerase enzyme extracted from thermophilic bacterium.
    • Taq is highly stable at much higher temperatures than human polymerase.
  • Components of PCR reaction
    • DNA polymerase – enzyme responsible for synthesizing new strands.
    • DNA template – starting material to be copied.
    • Primers – instruct polymerase where to bind and begin synthesis.
    • Free nucleotides – dNTPs added by polymerase to amplify and extend template DNA.
    • Free ions – stabilizes the PCR reaction.

The Polymerase Chain Reaction: An Insight

How PCR Works
  • There are three key steps in PCR:
    1. Denaturation (95 °C):
      • Breaks H-bonds holding DNA strands together into two single strands (ssDNA).
    2. Annealing (50 - 65 °C):
      • Cools reaction, allowing primers to bind (anneal) to the complementary sequence.
    3. Extension (68 - 72 °C):
      • Taq polymerase extends the primers, synthesizing new strands of DNA.
  • The steps of denaturation, annealing, and extension are repeated 25 – 35 times.
  • This process can take 2 – 4 hours, depending on the size of the template DNA being copied.
  • The reaction is exponential! With each cycle:
    • 1 copy becomes 2 copies, which becomes 4 copies, which becomes 8 copies… and so on.
  • A typical PCR program looks something like the following:
    1. Initial denaturation: 10 min @ 95 °C
    2. Denaturation: 30 secs @ 95 °C
    3. Annealing: 30 seconds @ 50-65 °C
    4. Extension: 30 seconds @ 68-72 °C
    5. Final extension: 5 min @ 68-72 °C
    6. End: Cool and hold @ 4 °C
PCR Thermocyclers
  • PCR machines, also known as thermocyclers, come in all different shapes and sizes.
Visualising PCR
  • The results of your PCR are visualized using gel electrophoresis.
  • This technique uses an agarose gel as a matrix.
  • DNA fragments are pulled through the matrix using an electric current.
  • DNA fragments migrate through the gel according to their size (bp).
    • Large fragments migrate slower than small fragments.
  • DNA has a negative charge.
    • The application of current allows DNA to migrate from the negative pole to the positive pole.
  • A control DNA ladder is loaded alongside your PCR reaction, allowing the size (bp) of your fragment to be determined.
  • Nucleic acids are naked to the visible eye.
    • However, they strongly absorb in UV (260 nm).
  • An intercalating dye is added to the gel, which binds with DNA and produces a fluorescent signal under UV light.
    • Gel Red
    • SYBR Safe
Optimising PCR
  • Errors happen, and experiments may not be successful the first time around. Your PCR may fail.
  • Steps you can take to optimize your reaction:
    • Adjust primer annealing temperature.
    • Adjust annealing time.
    • Adjust extension time.
    • Adjust Mg2+Mg^{2+} and/or K concentration.
    • Adjust the amount of template.
    • Adjust the amount of polymerase.
Annealing Temperature
  • Ensure you have the best annealing temperature for your primers.
    • Run a gradient PCR.
      • 45 – 55 °C
      • 55 – 65 °C
    • Lower the temperature, the less specific primer binding is.
    • Higher the temperature, the more specific.
Mg2+Mg^{2+} Concentration
  • Remember, Mg2+Mg^{2+}:
    • Catalyzes phosphodiester bond of 3’ OH of primer and newly added dNTP.
    • Stabilizes the complex formed between polymerase, primer, and template.
  • Adjusting Mg2+Mg^{2+} may help to produce single bands.

Take-Home Message: PCR Insight

  • How PCR works
    • An exponential reaction results in many copies of your template DNA.
    • Denaturation – separating dsDNA into ssDNA.
    • Annealing – primers hybridize to template DNA.
    • Extension – DNA polymerase adds dNTPs to extend the DNA template.
    • Cycles 25 – 35 times.
  • PCR Thermocyclers
    • Equipment responsible for carrying out PCR.
    • Runs your specific PCR cycling program.
  • Visualise PCR
    • Gel electrophoresis uses DNA dyes to visualise DNA under UV exposure.
    • Matrix separates DNA based on size; small fragments move faster than large fragments.
    • DNA is negatively charged and migrates from the negative pole to the positive pole.
  • Optimising PCR
    • If your PCR has failed or produces multiple bands, optimize the following:
      • Adjust primer annealing temperature, annealing time, extension time, ions concentration, template concentration, polymerase concentration.

Variations of PCR Technique & Scenarios

  • There are many variations of the technique; common examples include:
    • Conventional PCR
      • Amplification of gDNA.
    • RT-PCR
      • Converting RNA into cDNA.
    • qRT-PCR
      • Amplification and quantification of cDNA (RNA derived).
    • Microarrays
      • High throughput analysis of DNA.
Conventional PCR
  • This is the amplification of gDNA or a target sequence/gene of interest.
  • Also known as “endpoint detection.”
  • A single product is produced after many cycles.
  • It is non-quantifiable (e.g., cannot determine gene expression).
How?
  • DNA profiling via analysis of STRs from a DNA sample at a crime scene (blood, semen, saliva, etc.).
  • Short Tandem Repeats (STR) are repeat sequences of 2 – 6 bp present in non-coding DNA regions.
  • Approximately 5 – 20% of STRs at a given locus may be shared within a population.
  • By analysing multiple loci, you generate a unique identifier (DNA barcode).
  • For example, analysis of STRs across 10 loci gives a 1 in 1 billion chance of error!
  • Collect DNA sample from crime scene.
  • Perform PCR to amplify specific sequences of each locus.
  • Analyse PCR on gel and match DNA profiles produced to crime scene specimen.
RT PCR
  • Reverse transcriptase PCR (RT-PCR).
  • The conversion of RNA to complementary DNA (cDNA) using Reverse Transcriptase.
  • RNA is single-stranded and cannot be amplified directly by PCR, so it is converted to cDNA.
  • cDNA is then used in PCR.
  • RT-PCR is semi-quantitative.
  • Still looking at the “end point” but can be used to determine whether the mRNA transcript is present.
  • Effect on “gene X” transcript under UV exposure.
  • Suggests that the longer the UV exposure, the more transcript of “gene X” is present.
qRT PCR
  • Quantitative Real-Time PCR (qRT-PCR / qPCR).
  • Allows direct quantification of gene expression by quantifying a fluorescent signal produced during the exponential cycles of PCR.
  • SYBR green is a dye that fluoresces when bound to dsDNA.
  • The number of cycles required to detect a signal directly correlates to the amount of transcript:
    • Low cycle # = high transcript amount
    • High cycle # = low transcript amount
  • How?
    • Testing gene expression response to novel therapeutics.
    • Retrieve samples (e.g., treated and non-treated cancer cells).
    • Extract RNA, convert to cDNA, and run qRT-PCR.
    • Analyse data and determine which concentration of novel therapeutic elicits the greatest response on gene expression.
Microarrays
  • A high-throughput technique that allows the detection of thousands of genes or gene products simultaneously (approximately 30,000 spots).
  • Uses “DNA chips” comprised of a small glass plate in a plastic case (similar to a microchip in computers) loaded with DNA probes.
  • Can be used to investigate mutations in genes of interest or determine whether genes are switched on / off.
  • The hybridization technique is similar to northern blotting, but on a much larger scale!
  • Very expensive.
  • How?
    • Detection of a gene that when expressed is hypothesised to increase tumour growth.
    • Retrieve samples (e.g., normal and cancer cells).
    • Extract RNA, convert to cDNA, and label with a fluorescent probe.
      • Green for normal cell.
      • Red for cancer cell.
    • Combine samples and transfer to DNA chip containing synthetic DNA (original gene transcript of interest).
    • Allow hybridization to occur and analyze.

Take-Home Message: Variations of PCR Technique & Scenarios

  • Conventional PCR
    • Amplify target gDNA/cDNA sequence of interest.
    • Non-quantifiable - endpoint detection only.
    • e.g., Disease diagnostics and DNA profiling.
  • RT-PCR
    • Conversion of RNA to cDNA.
    • Semi-quantitative – end point detection.
    • e.g., Can see whether there are more/fewer copies of a gene transcript based on band intensity.
  • qRT-PCR
    • Uses cDNA to measure gene expression.
    • Useful fluorescent probes.
    • Quantitative – exponential detection.
    • e.g., Determine gene expression response to novel drugs and determine virus infection.
  • Microarrays
    • High throughput gene expression analysis.
    • Hybridization technique similar to northern blotting, but much larger scale.
    • Also uses fluorescent probes.
    • e.g., disease investigations such as cancer genetics.