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

What is Polymerase Chain Reaction (PCR)?

PCR is a technique used to amplify a specific segment of DNA, generating thousands to millions of copies of a particular DNA sequence.

What is standard PCR?

Standard PCR is the basic form of PCR that amplifies DNA using a DNA polymerase enzyme and thermal cycling.

What is reverse transcription PCR (RT-PCR)?

RT-PCR is a technique used to convert RNA into DNA and then amplify the resulting DNA, often used for gene expression analysis.

What is multiplex PCR?

Multiplex PCR allows simultaneous amplification of multiple DNA targets in a single reaction by using multiple primer pairs.

What is nested PCR?

Nested PCR involves two sets of primers and two amplification steps to increase specificity and sensitivity of the target DNA.

What is digital PCR (dPCR)?

dPCR allows for the absolute quantification of DNA by partitioning the sample into many individual reactions.

What is degenerate PCR?

Degenerate PCR is a variation of PCR that uses primers with multiple, mixed sequences to amplify DNA that may have unknown or variable regions, allowing for the amplification of similar sequences from different organisms.

What is the significance of regular PCR in molecular biology?

Regular PCR is essential for cloning, gene expression analysis, and the detection of genetic variations, serving as a fundamental tool in research and diagnostics.

What is isothermal PCR?

Isothermal PCR is a nucleic acid amplification technique that amplifies DNA at a constant temperature, eliminating the need for thermal cycling.