PCR

Polymerase Chain Reaction (PCR)

• Technique used to amplify DNA sequences.

History of PCR

• Inventor: Kary B. Mullis, 1985.

• Purpose: Allows scientists to create millions of copies of a limited DNA sample.

• Prior Method: DNA cloning, which was time-consuming and required living bacterial cells.

How PCR Works

• Key Element: Heat is essential for the PCR process.

• Processes:

  • Melting of DNA: Double-stranded DNA is heated to become single-stranded.

  • Annealing: Primers anneal to target sequences on single-stranded DNA.

  • Extension: Taq polymerase synthesizes new DNA strands.

• Chemical Reactions:

  • Thermal cycles: DNA subjected to heating and cooling repeatedly.

PCR Components

Reagents Needed:

• Template DNA: Source DNA to amplify.

• Primers: Short DNA sequences that initiate DNA synthesis.

• dNTPs: Deoxynucleoside triphosphates, the building blocks of DNA.

• Taq Polymerase: Enzyme that synthesizes new DNA strands.

• Buffer: Maintains pH and salt concentrations.

• MgCl2: Essential for polymerase activity.

• sH2O: Sterile water.

Steps in PCR

1. Denaturation

• Heating to approximately 95°C breaks hydrogen bonds, separating DNA into single strands.

2. Annealing

• Cooling to 30-65°C allows primers to bind to complementary DNA regions.

3. Extension

• Heating to 60-75°C enables Taq polymerase to add dNTPs, synthesizing new DNA strands.

• New copies of target DNA double with each cycle, generally repeated up to 35 times.

PCR Design and Optimization

• Target DNA Amplification: Ensure optimal conditions to produce desired DNA without non-specific products.

• Optimization Variables:

  • Primer Design: Specificity and Tm calculation are critical.

  • Template Concentration: Must be balanced to avoid excess or deficiency.

  • MgCl2 Concentration: Affects binding and enzyme activity.

  • Taq Polymerase Concentration: Avoid both high excess and deficiency.

  • Annealing Temperature: Should be below the lowest Tm of the primer pair for effective binding.

  • Cycle Number: Up to 35 cycles, monitoring yields and possible second rounds of PCR are needed.

Controls in PCR

• Reagent Control: Confirm no contamination occurred.

• Negative Control: Use template DNA without target sequence to ensure no non-specific amplification.

• Positive Control: Use template DNA with known target sequence for confirmation of reaction success.

Troubleshooting PCR Issues

• Possible problems include no bands, faint bands, fuzzy bands, and unexpected large bands.

• Common causes range from errors in DNA volume, master mix preparation, degraded reagents, or incorrect cycling conditions.

Applications of PCR

• Medical Diagnostics: For genetic disorders, cancer diagnosis, and prenatal testing.

• Forensics: DNA amplification for human identification and crime scene analysis.

• Research: Amplification of ancient DNA, sequencing, and HLA typing for immune response studies.

Modifications of PCR

• Includes techniques like Hot-start PCR, Nested PCR, Multiplex PCR, Reverse Transcriptase PCR (RT-PCR), Quantitative real-time PCR (qPCR), and Digital PCR.

Real-Time PCR (qPCR)

• Allows for real-time monitoring during amplification using fluorescent signals, which can define the concentration of initial template DNA based on cycle threshold (CT) values.

Conclusion

• PCR is a powerful tool in molecular biology with wide-ranging applications in healthcare and research, reliant on precise techniques and optimization to yield accurate and efficient DNA amplification.