Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction overview

• Function
  • can amplify a selected region of DNA 
• Process
  • solution prepared containing
    • DNA primers specific for selected DNA region
    • DNA sample of interest
    • heat stable DNA polymerase
    • deoxyribonucleotides
  • denaturation of dsDNA by heating
  • annealing of DNA primer specific for region of interest and slowly cooling the solution
  • replication of DNA at the primer by heat stable DNA polymerase
  • repetition of the process several times 
  • gel electrophoresis used to separate the various components of the solution
• Clinical use
  • in all uses PCR functions to amplify the amount of DNA present in a sample
  • high specificity bacterial/viral infection testing
    • HIV
      • first step is ELISA (high sensitivity)
      • PCR is used to determine viral load
      • test examines the amount of viral DNA integrated into host cell DNA
      • advantages over ELISA
        • PCR becomes positive earlier in disease course
          • Positive ELISA result is dependent on antibody formation
        • PCR does not require that the patient have a competent immune system
          • ELISA requires the host to make antibodies
      • important specific cases when PCR should always be used
        • a newborn whose mother is HIV+
          • will have antibodies even if not infected, so ELISA does not work
        • when earliest possible detection is required
  • genetic identification
    • forensic/paternity testing
    • use of variable number tandem repeats (VNTRs) or short tandem repeats (STRs)
      • unique copies of non-coding regions of DNA between individuals
      • since they exist on both chromosomes, individuals have two copies at each locus
        • 1 paternal and 1 maternal
    • can only prove with certainty that the sample DOES NOT belong to the test subject
      • cannot prove with 100% certainty that DNA belongs to individual of interest because there is a small chance that someone shares the same VNTR or STR
  • direct mutation
• if DNA region is known, PCR can amplify that region for sequencing

RT-PCR overview

• Function
  • used to measure the amount of RNA present in a sample
• Process 
  • reverse transcriptase is added to solution containing RNA, dNTPs, primers for specific sequence of interest, and heat stable DNA polymerase
  • RNA is converted to DNA and DNA sequence of interest is amplified
  • When combined with quantitative (real-time PCR), the combined qRT-PCR (quantitative reverse transcription PCR) assesses the amount of RNA in the original sample by measuring the amount of amplified PCR product after a set number of PCR cycles, which is directly proportional to the concentration of RNA in the original sample
• Clinical use
  • HIV viral load
    • measures transcriptional activity of the virus by detecting the amount of RNA present
• gives a more detailed picture of the infection and treatment results

Introduction – Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify a specific segment of DNA from a complex mixture of DNA molecules. The technique was first developed in the 1980s and has since revolutionized many areas of biological research, including genomics, forensics, and medical diagnostics.

Polymerase Chain Reaction is based on the ability of a DNA polymerase enzyme to copy a DNA template strand by adding complementary nucleotides to the growing DNA strand. The reaction requires a short piece of DNA called a primer, which anneals to the template strand and provides a starting point for the DNA polymerase to extend the new DNA strand.

Components Of Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a complex process that requires several components to work effectively. The main components of a PCR reaction are:

1. Template DNA: The DNA molecule containing the region of interest that needs to be amplified.
2. Primers: Short DNA sequences, usually 18-22 nucleotides in length, that anneal to the template DNA at the start and end of the target region, providing a starting point for DNA synthesis by the DNA polymerase enzyme.
3. DNA polymerase enzyme: The enzyme that synthesizes the new DNA strand by adding nucleotides to the 3′ end of the primers. Taq polymerase is the most commonly used DNA polymerase for Polymerase Chain Reaction, as it is heat-stable and can withstand the high temperatures used in the reaction.
4. Deoxynucleoside triphosphates (dNTPs): The building blocks of DNA, which are added to the new DNA strand during synthesis.
5. Buffer solution: A solution that provides the optimal pH, salt concentration, and cofactors for the DNA polymerase enzyme to work effectively.
6. Magnesium chloride (MgCl2): A cofactor required by the DNA polymerase enzyme to function.
7. Thermal cycler: A machine that precisely controls the temperature and duration of each step in the PCR reaction, allowing for repeated cycles of denaturation, annealing, and extension.

Additional components may also be included in PCR reactions depending on the specific application, such as fluorescent dyes for real-time PCR detection, or specific enzymes or reagents for specialized Polymerase Chain Reaction techniques.

Types Of Polymerase Chain Reaction

There are several types of Polymerase Chain Reaction (PCR) techniques that have been developed to suit different applications. Some of the most common types of PCR are:

1. Standard PCR: Also known as conventional Polymerase Chain Reaction, this is the most basic form of PCR that amplifies a specific DNA sequence. It involves denaturation, annealing, and extension cycles, as described in the PCR introduction.
2. Real-time PCR: Also known as quantitative PCR (qPCR), this technique allows for the quantification of DNA during the PCR process. It involves the use of fluorescent dyes or probes that bind to the DNA during amplification and produce a signal that can be measured in real-time. Real-time PCR is often used for gene expression analysis, pathogen detection, and viral load quantification.
3. Reverse transcription PCR (RT-PCR): This technique allows for the amplification of RNA molecules, which are first converted into complementary DNA (cDNA) using a reverse transcriptase enzyme. RT-PCR is commonly used for gene expression analysis and viral RNA detection.
4. Nested PCR: This technique involves two rounds of PCR amplification, with the first round using outer primers to amplify a larger region of DNA, followed by a second round using inner primers that anneal to the product of the first round. Nested PCR is often used for detection of low-copy number DNA, such as in forensic analysis.
5. Multiplex PCR: This technique allows for the simultaneous amplification of multiple DNA targets in a single reaction, using multiple sets of primers and fluorescent dyes or probes. Multiplex PCR is often used for pathogen detection and genotyping.

Studies – Polymerase Chain Reaction

1. Purpose: Polymerase Chain Reaction is used to amplify a specific DNA sequence, which can then be analyzed in various ways, such as sequencing or gel electrophoresis. PCR is commonly used in applications such as genetic testing, gene expression analysis, and disease diagnosis.
2. Procedure: PCR involves a series of temperature-dependent steps that enable DNA amplification. The basic steps include denaturation (heating the DNA to separate the double-stranded template), annealing (cooling the DNA to allow primers to bind to the template), and extension (raising the temperature to enable DNA polymerase to extend the primers and synthesize new DNA strands).
3. Components: PCR requires a DNA template, primers (short DNA sequences that anneal to the template), DNA polymerase (the enzyme that synthesizes new DNA strands), nucleotides (the building blocks of DNA), and buffer (a solution that provides optimal reaction conditions).
4. Variations: PCR has evolved to include several variations, such as quantitative PCR (qPCR) which can measure the amount of DNA present, reverse transcription PCR (RT-PCR) which converts RNA to cDNA for analysis, and nested PCR which amplifies a specific target sequence multiple times for increased sensitivity.

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