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.