Lab Genetics - PCR

Traditional PCR

  • Definition: Traditional PCR (Polymerase Chain Reaction) is a method used to amplify specific DNA sequences.

  • Main Purposes: It is employed in various fields including diagnostics, forensics, and biological research.

Types of PCR

Different types of PCR serve different functions in molecular biology. The following types are notable:

  1. AFLP PCR: Amplified Fragment Length Polymorphism PCR

  2. Allele-specific PCR: Amplifies specific alleles.

  3. Alu PCR: Utilizes Alu repeats for amplification.

  4. Assembly PCR: Assembles DNA fragments through PCR.

  5. Asymmetric PCR: Produces an excess of one strand of DNA.

  6. COLD PCR: Increases sensitivity for low-abundance sequences.

  7. Colony PCR: Amplifies DNA from bacterial colonies.

  8. Conventional PCR: Standard method for amplifying DNA sequences.

  9. Digital PCR (dPCR): Quantifies nucleic acids with high precision.

  10. Fast-cycling PCR: Allows rapid amplification with shorter cycle times.

  11. High-fidelity PCR: Minimizes errors during DNA synthesis.

  12. Hot-start PCR: Reduces non-specific amplification by inhibiting polymerase during preheating.

  13. In situ PCR: Amplifies DNA within the original tissue section.

  14. Intersequence-specific (ISSR) PCR: Utilizes microsatellite markers for genetic diversity studies.

  15. Inverse PCR: Amplifies DNA from unknown regions using known sequences.

  16. LATE PCR: Linear after exponential PCR for reducing background.

  17. Ligation-mediated PCR: Uses linkers for amplification of fragmented DNA.

  18. Long-range PCR: Amplifies longer DNA fragments, typically up to 30 kb.

  19. Methylation-specific PCR (MSP): Detects methylation status of DNA.

  20. Miniprimer PCR: Uses shorter primers to amplify small DNA segments.

  21. Multiplex-PCR: Simultaneously amplifies multiple targets in a single reaction.

  22. Nanoparticle-Assisted PCR (nanoPCR): Enhances sensitivity using nanoparticles.

  23. Nested PCR: Increases specificity by two rounds of amplification.

  24. Overlap extension PCR: Constructs DNA with overlapping ends.

  25. Real-Time PCR (qPCR): Quantitatively measures DNA as it is amplified.

  26. Repetitive sequence-based PCR: Targets repetitive DNA sequences for analysis.

  27. Reverse-Transcriptase (RT-PCR): Converts RNA into DNA for amplification.

  28. Reverse-Transcriptase Real-Time PCR (RT-qPCR): Combines RT with quantitative measurement.

  29. RNase H-dependent PCR (rhPCR): Uses RNase H to enhance sensitivity.

  30. Single cell PCR: Amplifies DNA from individual cells.

  31. Single Specific Primer-PCR (SSP-PCR): Uses a single specific primer for amplification.

  32. Solid phase PCR: Conducts PCR on a solid support for amplification.

  33. Suicide PCR: Prevents re-amplification of specific products.

  34. Thermal asymmetric interlaced PCR (TAIL-PCR): Used for amplifying specific DNA regions from heterogeneous samples.

  35. Touch down (TD) PCR: Gradually lowers annealing temperature to increase specificity.

  36. Variable Number of Tandem Repeats (VNTR) PCR: Targets regions of DNA with repeating sequences.

Traditional PCR vs. RAPD PCR

  • Traditional PCR: Generates copies of a known DNA sequence by amplifying a gene with prior knowledge of its sequence.

  • RAPD PCR (Random Amplified Polymorphic DNA PCR): Amplifies unknown DNA sequences without prior knowledge, resulting in multiple fragments of DNA across the genome.

Overview of PCR Process

Components Used in PCR:

  • Polymerase Enzyme: Catalyzes the synthesis of DNA or RNA polymers.

  • Chain Reaction: Involves generating multiple exact copies of the original target sequence.

PCR Cycle: 30-40 Cycles of 3 Steps

  1. Denaturation: Heat the reaction to 94 °C for 1 minute to separate the DNA strands.

  2. Annealing: Cool to 54 °C for 45 seconds allowing primers to bind to the template strands.

  3. Extension: Raise the temperature to 72 °C for 2 minutes for DNA polymerase to synthesize the new strands using dNTPs.

Components of a PCR Kit

  • The components can vary based on the PCR kit but typically include:

    • Magnesium

    • dNTPs (deoxynucleotide triphosphates)

    • Enzymes: such as Taq polymerase.

    • Primers: both forward and reverse.

    • Buffer

    • Controls: Internal control and probes.

Detailed PCR Process Cycle

  • Denaturation: Strands separate at 95 °C.

  • Annealing: Primers bind to the template at 35 °C.

  • Extension: New strands are synthesized at 72 °C.

Reverse-Transcriptase PCR vs. Traditional PCR

  • Traditional PCR: Utilizes two specific primers for amplification with known target sequences.

  • RAPD PCR: Uses one nonspecific primer to amplify unknown DNA sequences creating a unique banding pattern.

Comparisons and Applications

  • PCR Applications:

    • Useful for detecting specific genes, particularly in disease diagnostics.

    • Can analyze evolutionary relationships among samples.

  • Banding Patterns:

    • PCR: Displays specific segments of amplified DNA on a gel.

    • RAPD PCR: Shows multiple bands representing various segments of DNA

PCR Gel Electrophoresis

  • DNA Ladder: Size standard in gel electrophoresis to identify fragments.

  • Example sizes in base pairs (bp)

    • 5000 bp, 3000 bp, 2000 bp, etc.

Knowledge Check Questions

  • Differences between forward and reverse primers in PCR.

  • Distinction between traditional PCR and RAPD PCR.

  • Mechanisms of denaturing, annealing, and extension.

  • Components needed for PCR and their functions.

  • Differences in banding patterns and results from traditional PCR gel compared to RAPD PCR gel.