1. Standard (Conventional) PCR * Purpose: Basic DNA amplification. * Process: 1. Denaturation: DNA is heated to 94-98°C to separate the strands. 2. Annealing: Primers bind to the target sequence at 50-65°C. 3. Extension: DNA polymerase extends primers at 70-75°C to synthesize new DNA strands. * Applications: Cloning, gene expression analysis, genetic testing. 2. Real-Time PCR (qPCR) * Purpose: Quantifies DNA in real-time during the amplification process. * Process: Uses fluorescent dyes or probes to monitor the amplification in each cycle. * Applications: Gene expression analysis, quantifying pathogens, viral load detection. 3. Reverse Transcription PCR (RT-PCR) * Purpose: Converts RNA into complementary DNA (cDNA) for amplification. * Process: 1. Reverse Transcription: RNA is reverse transcribed into cDNA using reverse transcriptase. 2. PCR Amplification: The cDNA is then amplified using standard PCR. * Applications: Gene expression analysis, RNA virus detection (e.g., HIV, SARS-CoV-2), studying RNA biology. * Note: RT-PCR is critical for studying RNA as it allows researchers to study the transcriptome of a cell or organism. 4. Multiplex PCR * Purpose: Amplifies multiple DNA targets in a single PCR reaction. * Process: Uses more than one pair of primers to target different sequences simultaneously. * Applications: Disease diagnosis (e.g., detecting multiple pathogens), genetic testing, SNP analysis. * Challenges: Primer design and optimization to prevent primer interference. 5. Nested PCR * Purpose: Increases specificity and sensitivity by using two rounds of PCR. * Process: 1. First round: Amplifies a larger fragment. 2. Second round: Uses primers from within the first amplified fragment to increase specificity. * Applications: Low-abundance DNA detection, diagnostics, pathogen detection. 8. Digital PCR (dPCR) * Purpose: Provides absolute quantification of DNA or RNA. * Process: DNA or RNA is partitioned into many individual reactions; results are quantified based on the number of positive reactions. * Applications: Rare mutation detection, precise quantification of low-abundance targets. 10. LAMP (Loop-Mediated Isothermal Amplification) * Purpose: Isothermal amplification technique that does not require thermal cycling. * Process: Uses a set of primers and a strand-displacing DNA polymerase to amplify DNA at a constant temperature (60-65°C). * Applications: Rapid diagnostics, field testing, pathogen detection. 11. Degenerate PCR * Purpose: Amplifies a DNA sequence with degenerate primers, allowing detection of related sequences with unknown or varied nucleotide composition. * Process: Uses primers that contain ambiguous bases (e.g., R = A/G, Y = C/T) to target homologous sequences in related species or unknown genes. * Applications: Amplification of conserved genes across different species, detection of homologous gene families. * Note: Especially useful when the exact sequence of the target gene is unknown or highly variable. 12. Fast PCR * Purpose: Speeds up the PCR process by reducing cycle times. * Process: Optimizes the denaturation, annealing, and extension steps to reduce the overall PCR reaction time. * Applications: High-throughput screening, time-sensitive experiments, rapid diagnostics. * Note: Requires specially formulated polymerases and optimized protocols. 13. Random Amplification of Polymorphic DNA (RAPD) PCR * Purpose: Amplifies random DNA fragments from a genome using short, arbitrary primers, often used for genetic fingerprinting. * Process: A single arbitrary primer is used to amplify random genomic regions, generating a unique pattern of bands that can be analyzed. * Applications: Genetic diversity studies, DNA fingerprinting, phylogenetic studies, population genetics. * Note: RAPD PCR is used when a comprehensive genomic sequence is unavailable and can provide insight into genetic variation. huh