Human Molecular Genetics - PCR Overview

Topic = Polymerase Chain Reaction

SCH2226 Human Molecular Genetics Lecture Notes

Polymerase Chain Reaction (PCR)

  • Overview

    • Definition: PCR is a technique that amplifies a specific sequence of DNA from a small initial sample to produce a larger amount for analysis.

    • In vitro Technique: The process occurs outside of living organisms in a controlled environment.

  • Historical Context

    • Developed by Kary Banks Mullis in 1985

    • Awarded the Nobel Prize in Chemistry in 1993.

    • Mullis conceived the idea while driving to California, and it was introduced to the scientific community in 1985.

    • Mullis received $10,000 for his invention and his company sold the patent for $300 million.

  • Purpose of PCR

    • To amplify numerous identical double-stranded DNA molecules (fragments) using enzymatic methods through cycling conditions.

  • Chemical Components of PCR

    • Magnesium Chloride: 0.5-2.5 mM

    • DNA Polymerase: 1-2.5 units

    • Buffer: pH 8.3 - 8.8

    • dNTPs (deoxynucleotide triphosphates): 20-200 µM

    • Primers: 0.1-0.5 µM

    • Template DNA: <1 mg

  • PCR Equipment and Storage

    • PCR Tubes: 0.2 ml, 0.5 ml

    • Mastermix: 1.5 ml tubes

    • Storage Temp: -20 °C

    • Pipette Tips and Autopipettes: Used in various volume ranges.

Steps of PCR

  • 1) Denaturation

    • Process: Reaction mixture is heated to 90-98 °C.

    • Breaks hydrogen bonds, causing double-stranded DNA to separate into single strands.

    • Duration: 5-15 minutes.

  • 2) Annealing

    • Process: Temperature is cooled to 50-70 °C.

    • Primers base-pair with complementary sequences, re-forming hydrogen bonds.

    • Note: Annealing refers to the renaturing of DNA.

  • 3) Extension

    • Process: Temperature is shifted to 72 °C, optimal for DNA Polymerase function.

    • DNA Polymerase extends the primer by adding dNTPs complementary to the template DNA strand.

    • This phase can be cycled up to 40 times in a thermocycler.

  • Example of PCR Program

    • Initial Denaturation: 95 °C for 5 minutes

    • Repeat the following 30 cycles:

    • Denaturation: 95 °C for 30 seconds

    • Annealing: 55 °C for 30 seconds

    • Extension: 72 °C for 30 seconds

    • Final Extension: 72 °C for 5 minutes

    • Holding: 4 °C for storage.

Troubleshooting PCR

  • If no product (of correct size) produced:

    • Check DNA quality.

    • Reduce annealing temperature.

    • Change magnesium concentration.

    • Add dimethylsulphoxide (DMSO) to assay (around 10%).

    • Use different Taq polymerase.

    • Create new primers.

  • If extra bands present:

    • Increase the annealing temperature.

    • Reduce magnesium concentration.

    • Reduce the number of cycles.

    • Try a different Taq polymerase.

Optimizing PCR Conditions

  • 1) Primers: Correctly designed pairs are crucial. Avoid primer dimerization and hairpin formation.

  • 2) DNA Polymerase: Taq polymerase is commonly used due to heat resistance, but it lacks proofreading capability. Other options include Tma DNA Polymerase and Pfu DNA Polymerase.

  • 3) Annealing Temperature: Critical for hybridization success. It should be set 1-2 °C below the melting temperature of the primer-template duplex.

  • 4) Melting Temperature: Can be experimentally determined or calculated using:
    Tm=(4(G+C))+(2(A+T))−5T_m = (4(G+C)) + (2(A+T)) - 5

  • 5) G/C Content: Ideally, the primer should have a balanced nucleotide composition with about 50% GC content.

Variations of PCR

  • 1) Inverse PCR: Amplifies unknown sequences adjacent to a known sequence, useful for genomic insert identification.

  • 2) Anchored PCR: Involves adding a small sequence to the target DNA to facilitate amplification using a primer.

  • 3) Reverse Transcriptase PCR (RT-PCR): Used to amplify RNA, helping study gene expression.

    • Process:

    • OligodT binds to polyA of RNA.

    • Reverse transcriptase synthesizes cDNA.

    • cDNA undergoes PCR to generate an amplicon.

  • 4) Quantitative Real-Time PCR (qPCR): Quantifies and detects DNA. Signal proportional to PCR product amount. Monitors during reaction, not just at the end product.

  • 5) Allele-Specific PCR: Amplifies specific alleles to identify single nucleotide polymorphisms (SNP).

Limitations and Advantages of PCR

  • Advantages:

    • Used in clinical diagnostics, DNA sequencing, forensic medicine, gene manipulation, genomics comparisons, and other studies requiring minimal amounts of DNA.

  • Limitations:

    • Limited sequence information, constraints on amplicon size, error rates during amplification, sensitivity to inhibitors, potential contamination, and artefacts may arise.

  • Additional PCR Types Mentioned:

    • Overlap Extension PCR, Helicase-dependent amplification, Ligation-mediated PCR, Methylation-specific PCR, among others.

Additional Resources

  • Future Lecture Topics: Discussion on The Human Genome Project and Genetic Linkage.