Advanced Biomedical Techniques Module 9 - Droplet PCR

Importance of Early Biomarker Detection

  • Advantages of Early Detection:

    • Identifies molecular changes before they appear on scans.

    • Minimally invasive procedure (only requires a blood draw), avoiding surgical biopsy.

    • Real-time monitoring: Tests can be repeated frequently to track treatment response.

    • Can detect minimal residual disease by identifying microscopic changes that are undetectable by scans.

  • Comparison with Scans:

    • Scans show only anatomical changes (size/shape of tumours), potentially missing small or early lesions.

    • Liquid biopsy reveals molecular alterations, offering higher sensitivity and earlier insights.

    • Enables personalized treatment decisions rather than solely visual assessments.

What is Liquid Biopsy?

  • Definition: A non-invasive test that analyzes cancer biomarkers (cell-free DNA [cfDNA], circulating tumor DNA [ctDNA], RNA, exosomes, etc.) derived from body fluids (such as blood).

Overview of DNA Fragments in Bloodstream

Key Terminology

  • cfDNA (cell-free DNA): Any free DNA present in plasma.

  • ctDNA (circulating tumor DNA): DNA fragments released from tumor cells into the plasma.

  • Length: cfDNA/ctDNA is typically 160-200 bp (base pairs) long.

  • Half-Life: The half-life of ctDNA in the blood is very short, ranging from approximately 16 minutes to 2.5 hours.

  • References: Keller et al. & Maia et al.

ctDNA Levels

  • Proportion of ctDNA within cfDNA: Varies significantly, ranging from 0.003% to 95%.

  • Early-stage tumors typically have low ctDNA levels, constituting up to 1% of cfDNA.

  • In patients with advanced tumors, ctDNA can exceed 10% of cfDNA and potentially reach up to 40%.

  • Reference: Pavel et al., 2023

Intro to Droplet Digital PCR (ddPCR)

  • Concept: An innovative method that starts with a droplet and ends in molecular discovery.

  • Operational Details:

    • PCR reactions are partitioned into tens of thousands of sub-reactions.

    • Enables direct molecular counting.

    • Does not require a reference curve for quantitation, leading to high quantitative accuracy—highest among current techniques.

What is Digital PCR?

  • Definition: A direct molecular counting method combined with statistical distribution analysis.

  • Key Formula: A relationship used in the computation includes (N=−racln(N<em>neg)V)(N = - rac{ln(N<em>{neg})}{V}), where N is the number of molecules, $N{neg}$ is the number of negative droplets, and V is the volume of one droplet.

Preparation for ddPCR

  1. Reaction Components:

    • SuperMix

    • Assay

    • Sample

    • Water

  2. Primers/Probes:

    • Wild-type probe and primer sequence examples given.

ddPCR Process

Droplet Generation

  • Mix reaction components with oil to create droplets.

  • Utilize an automated droplet generator for partitioning.

Thermocycling

  • Standard Cycling Protocol:

    • 95°C for 10 minutes

    • 40 cycles of 94°C for 30 seconds and 55°C (or specific) for 1 minute

    • 98°C for 10 minutes

    • Hold at 4°C.

Droplet Reading

  • Post-PCR Fluorescence Detection:

    • Use the QX200 Droplet Reader to analyze fluorescent signals in the droplets post-PCR.

  • Result Interpretation: Count positive vs. negative droplets to estimate target concentration.

Data Analysis and Interpretation

  • Counting Approach: Examples of expected results from various samples according to the number of positive droplets counted.

  • Poisson Distribution Application: Relevant for determining target concentration based on the number of positive droplets counted. Correct partitioning is critical for accuracy.

Sample Partitioning Analysis

  • Examples of expected outcomes and distributions for low, medium, and high target DNA copies across droplets.

  • Summary Statistics: Probability fractions lead to concentration estimates illustrated across various experimental designs.

Applications of ddPCR

  • Examples: Absolute quantification of:

    • DNA or RNA (numerical results)

    • Liquid biopsies

    • Pathogen/Viral load quantification

    • GMO food stock testing

    • Residual DNA analysis

    • Environmental monitoring

    • Immunotherapy monitoring

  • Capable of supporting singleplex or multiplex analyses.

  • References: Calapre, L., et al., 2019. Clinical benefits of ctDNA analysis by mutation using ddPCR, Dr. Leslie Beasley, Prof. Elin Gray.

Useful Resources

  • Droplet Digital PCR Using the Bio-Rad QX200 and AutoDG ddPCR Systems

  • The Power of Partitioning: The World of Droplet Digital PCR Applications

  • Using Droplet Digital PCR for Cancer and Liquid Biopsy Studies

Note: The above notes are structured for detailed understanding and can serve as a comprehensive study guide on droplet digital PCR and its application in melanoma treatment and detection.