PCR Analyzer: Introduction, Principle, Test Requirements, Handling Procedure, Uses, and Keynotes

PCR Analyzer: Introduction, Principle, Test Requirements, Handling Procedure, Uses, and Keynotes

Introduction

A PCR analyzer, also known as a thermal cycler or PCR machine, is a laboratory instrument that automates the polymerase chain reaction (PCR) process. The PCR technique is used to amplify and detect small fragments of DNA or RNA, making it an essential tool in various fields of research and diagnostics. A PCR analyzer allows researchers to amplify and detect nucleic acid sequences with a high degree of accuracy and sensitivity.

Principle 

PCR (polymerase chain reaction) analyzer is a device that performs PCR, a technique used to amplify and detect DNA sequences. The principle of a PCR analyzer is based on the three-step PCR process that includes denaturation, annealing, and extension. During denaturation, the double-stranded DNA template is heated to break it down into single strands. In the annealing step, the temperature is lowered to allow the primers to bind to the complementary sequences on the single-stranded DNA. Finally, during the extension step, the temperature is raised to allow the Taq polymerase enzyme to extend the primers and synthesize the new DNA strands. The amplified DNA is then detected and analyzed using various methods such as gel electrophoresis or fluorescent detection.

Types/Models

There are several manufacturers that produce PCR analyzers with different features and capabilities. Some popular types/models of PCR analyzers according to manufacturers are:

  1. Applied Biosystems 7500 Real-Time PCR System
  2. Bio-Rad CFX96 Real-Time PCR Detection System
  3. Qiagen Rotor-Gene Q Real-Time PCR System
  4. Thermo Fisher Scientific QuantStudio 6 Flex Real-Time PCR System
  5. Roche LightCycler 96 Real-Time PCR System
  6. Eppendorf Mastercycler RealPlex2 Real-Time PCR System
  7. Analytik Jena qTOWER³ Real-Time PCR System
  8. Agilent Technologies AriaMx Real-Time PCR System
  9. Promega Maxwell RSC48 Nucleic Acid Purification System
  10. Techne Prime Pro 48 Real-Time PCR System

Test Requirements

The test requirements for PCR analyzer are:

  1. PCR reagents: PCR analyzer requires PCR reagents such as Taq polymerase, dNTPs, primers, and buffer for performing the PCR reaction.
  2. Nucleic acid sample: The nucleic acid sample can be DNA or RNA and should be isolated from the target organism or material.
  3. Thermal cycler: A thermal cycler is required for performing the PCR reaction. The thermal cycler should have the capability to heat and cool the samples at specific temperatures for the PCR reaction.
  4. PCR tubes or plates: The PCR tubes or plates are required to hold the reaction mixture during the PCR reaction.
  5. PCR analyzer software: The PCR analyzer requires software for data analysis and interpretation. The software should have the capability to analyze and interpret the data generated from the PCR reaction.
  6. Positive and negative controls: Positive and negative controls are required for validating the PCR reaction and for ensuring the accuracy of the results.

Handling Procedure

The handling procedure of PCR analyzer may vary depending on the specific model and manufacturer. However, in general, the following steps may be involved:

  1. Preparation of the PCR reaction mix: The PCR reaction mix contains the template DNA, primers, Taq polymerase, nucleotides, and buffer. The mix is prepared in a sterile environment using appropriate laboratory equipment.
  2. Loading of the PCR reaction mix: The PCR reaction mix is loaded into the PCR tubes or plates, which are then placed into the PCR analyzer.
  3. Setting the PCR program: The PCR program is set according to the specific requirements of the experiment, such as the annealing temperature, extension time, and number of cycles.
  4. Starting the PCR reaction: The PCR analyzer is started to initiate the amplification of the DNA.
  5. Monitoring the PCR reaction: The PCR analyzer monitors the amplification of the DNA in real-time and displays the results.
  6. Data analysis: The data obtained from the PCR analyzer is analyzed using appropriate software.
  7. Interpretation of the results: The results obtained from the PCR analyzer are interpreted based on the specific experiment and application.
  8. Maintenance and cleaning: The PCR analyzer needs to be properly maintained and cleaned to ensure accurate and reliable results.

Applications

PCR analyzers are used in various applications such as:

  1. Molecular biology research: PCR analyzers are used to amplify DNA for downstream applications in molecular biology research.
  2. Medical diagnosis: PCR analyzers are used in medical diagnosis to detect genetic diseases, identify infectious agents, and monitor the efficacy of treatment.
  3. Forensic analysis: PCR analyzers are used in forensic analysis to analyze DNA samples from crime scenes, identify suspects, and provide evidence in court.
  4. Food safety testing: PCR analyzers are used in food safety testing to detect the presence of pathogens and other contaminants in food samples.
  5. Environmental testing: PCR analyzers are used in environmental testing to detect and quantify microorganisms in water, soil, and air samples.
  6. Agriculture: PCR analyzers are used in agriculture to detect and identify plant pathogens and genetically modify crops.
  7. Veterinary medicine: PCR analyzers are used in veterinary medicine to diagnose infectious diseases in animals.

Keynotes

Some possible keynotes on PCR analyzer are:

  • PCR analyzer is an essential tool for performing polymerase chain reaction (PCR) experiments, which are widely used in molecular biology and diagnosticss.
  • It works by thermocycling the reaction mixture through a series of temperature changes to denature the DNA template, anneal the primers, and extend the new DNA strand.
  • PCR machine typically consists of a thermal cycler with precise temperature control, fluorescence detection system, and software for data analysis and interpretation.
  • This instrument can be used for a wide range of applications, such as gene expression analysis, mutation detection, infectious disease diagnosis, genetic testing, and forensic analysis.
  • It  requires careful handling to prevent contamination and ensure reliable results, such as using appropriate protective equipment, working in a clean and sterile environment, and following good laboratory practices.
  • This analyzer can produce false positive or negative results if there is contamination, insufficient template or primers, non-specific amplification, or other technical issues, so it is important to validate and optimize the assay before using it for clinical or research purposes.
  •  Analyzer has revolutionized the field of molecular biology and enabled many groundbreaking discoveries, such as the identification of the human immunodeficiency virus (HIV) and the development of the first PCR-based diagnostic test for COVID-19.

Further Reading

  1. Bustin SA. (2017). The PCR revolution: Basic technologies and applications. Cambridge University Press.
  2. Chen H, Cheng JX, Li Y, et al. (2020). Recent advances in PCR technologies and their applications in forensic DNA analysis. Forensic Science Review, 32(1), 39-56.
  3. Rotondaro C, Cappabianca L, Colantoni A, et al. (2019). Real-time PCR: technical aspects, validation, and applications. European Review for Medical and Pharmacological Sciences, 23(2 Suppl), 14-21.
  4. Tariq MA, Everest-Dass A, Guragain M, et al. (2019). A brief review on the application of PCR as a molecular diagnostic tool for aquatic animal diseases. Aquaculture Research, 50(3), 645-657.
  5. Yang X, He X, Yi C, et al. (2019). The application of digital PCR in detection of infectious pathogens. Frontiers in microbiology, 10, 1132.
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