Rohan_version_MKBS_314-_study_unit_2.2

Recombinant DNA Technology

  • Explores the intersection of DNA technology and industrial microbiology.

Polymerase Chain Reaction (PCR) Based Techniques

Learning Outcomes

  • Discuss PCR as a tool connecting DNA applications with replication.

  • Analyze quality and quantity of PCR products.

  • Explore principles of real-time PCR (QPCR).

  • Understand reverse transcription and gene expression basics.

Quality and Quantity of PCR Products

Reference Documents

  • Quality Assurance/Quality Control Guidance: EPA (Environmental Protection Agency).

  • BABEC PCR optimization.

Important Considerations for Setting Up a PCR

  1. Nucleic Acid Isolation and Storage

    • Not part of PCR but essential for ensuring PCR's effectiveness.

    • Aims to recover target nucleic acid efficiently with integrity, purity, and minimal hazardous chemicals.

  2. Procedure Parameters

    • Requires careful selection based on target nucleic acids for amplification (thermocycling conditions, reaction volumes, template concentration, and PCR reagent concentrations).

  3. Quality and Quantity of Reagents

    • Critical for obtaining reliable PCR results.

  4. Prevention of Contamination

    • Essential to avoid false results.

Nucleic Acid Isolation Goals

  • High target nucleic acid recovery.

  • Preserve integrity and minimize fragmentation.

  • Ensure purity free from PCR inhibitors.

  • Low or no dangerous chemicals.

  • Repeatable process.

Procedure Parameters

Thermocycling Conditions

  • Determine optimal denaturation, annealing, and elongation temperatures.

  • Use a single PCR thermocycling condition for convenience across primer sets.

  • Evaluate PCR cycle number for false negatives and positives.

Reaction Volumes

  • Typically 10 to 100 μL, depending on thermal cycler design.

  • Higher volumes may increase positive results but can also introduce inhibition.

Primer and Template Concentrations

  • Optimized to maximize amplification efficiency, especially in multiplex PCR.

  • High concentrations may inhibit the reaction.

PCR Reagents and Master Mix Preparation

  • Optimize key reagents: reverse transcriptase, DNA polymerase, magnesium, dNTP concentrations.

  • Consider commercial kits that match laboratory standards.

  • Add enhancers (like DMSO, BSA) to the master mix.

Amplicon Detection and Confirmation

Techniques for Detection

  • Electrophoresis: Common method for product detection.

  • Southern Blot: For hybridization confirmation.

  • Restriction Mapping: To ensure accurate enzyme digestion.

  • Real-time PCR: Quantitative detection during PCR.

  • Melting Curve Analysis: Determines amplicon melting temperature.

  • Sequencing: Most reliable method for confirmation.

Advantages and Disadvantages of Techniques

  • Electrophoresis: Easy and fast; confirms product size only.

  • Southern Blot: Detailed but time-consuming; requires preliminary steps.

  • qPCR: Quick with lower contamination risk; relies on probes.

  • Sequencing: Most accurate but costly and may require multiple confirmations.

Gel Electrophoresis Overview

Principles of Electrophoresis

  • Separation of DNA based on size and shape.

  • Visualize DNA using dyes like ethidium bromide or running dyes.

  • Generally not sufficient alone to confirm PCR products due to size overlap of amplicons.

Medium for Electrophoresis

  • Agarose Gel: Standard for DNA separation.

  • Polyacrylamide Gel: Higher resolution but more complex to prepare.

Quality Control in PCR

Importance of Quality Control

  • Routine evaluations with positive and negative controls.

Positive Controls

  • Verify recovery and amplification capability of the target nucleic acids.

  • Should be significantly concentrated compared to detection limits.

Negative Controls

  • Ensure no contamination was introduced during processing.

Corrective Actions

  • Document and repeat failed PCR runs.

  • Identify contamination sources and implement solutions (equipment blanks and wipe tests).

Additional Control Techniques

  • Equipment Blanks: Test equipment contamination.

  • Wipe Tests: Check for nucleic acid residue on surfaces.

  • Room QC: Regular checks for background contamination.