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.
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