PCR

Nucleic Acids Used in Diagnostics

Polymerase Chain Reaction (PCR)

• Definition: The polymerase chain reaction is an extremely versatile technique for copying DNA.
• Functionality: PCR allows a single DNA sequence to be copied millions of times or altered in predetermined ways.
• Variations:
    - Reverse Transcription PCR (RT-PCR): Used for the amplification of RNA.
    - Real-Time PCR (QPCR): Allows for quantitative measurement of DNA or RNA molecules.

PCR Analysis

• Process Overview:
    - Follows the principle of DNA replication.
    - Involves exponential amplification of target DNA through cycles of temperature changes.
• Cycles of Amplification:
    - Each cycle doubles the amount of target DNA.
    - Example of amplification:
      - 1st Cycle: 2 copies
      - 2nd Cycle: 4 copies
      - 3rd Cycle: 8 copies
      - 4th Cycle: 16 copies
      - Continues,
      - 35th Cycle: $2^{35} = 34,359,738,368$ or approximately 34 billion copies. (Adapted from Andy Vierstraete, 1999)

Primers in PCR

• Definition: A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis.
• Characteristics of Primers:
    - Usually short, chemically synthesized oligonucleotides, approximately 20 bases in length.
    - Minimum primer length used in most applications is 18 nucleotides.
    - Replication starts at the 3'-end of the primer, copying the opposite strand.
    - In natural DNA replication, the primer is typically a short strand of RNA.

Applications of PCR

• Gene Expression Studies: Commonly used for studying patterns of gene expression.
• DNA Sequencing: Assists in the sequencing of DNA.
• DNA Cloning: Numerous applications related to traditional DNA cloning methodologies.
• Phylogenetic Analysis: Enables the analysis of DNA from ancient sources.
• Genetic Mapping: Useful for studying genetic mapping patterns.
• Parental Testing: Involves matching an individual with their close relatives.

Gel Electrophoresis

• Basic Principle: DNA, RNA, and proteins can be separated using an electric field.
• Types of Gel Electrophoresis:
    - Agarose Gel Electrophoresis: For separation of DNA and RNA based on size.
    - SDS-PAGE Gel: For separation of proteins based on size.
    - 2D Gel Electrophoresis: Separates proteins based on size and electric charge.

Macromolecule Blotting & Probing Techniques

• Components:
    - Genomic DNA
    - Restriction Enzyme (Endonuclease)
    - Agarose Gel
    - Blotting Buffer
    - Nitrocellulose or Nylon Filter
• Process Overview:
    - DNA is separated via agarose gel electrophoresis.
    - Transferred to a membrane through capillary action.
    - Membrane is probed with labeled sequences (e.g., radioactive or fluorescent).
    - Visualization through X-ray film or other methods to reveal bands representing DNA present.
    - Capable of detecting high and low molecular weight DNA.

Southern Blotting

• Definition: A method for probing the presence of a specific DNA sequence within a sampling of DNA.
• Process:
    - DNA samples are separated through gel electrophoresis.
    - Transferred to a membrane by capillary blotting.
    - Membrane is exposed to a labeled DNA probe that complements the target sequence.
• Relevance:
    - Less commonly used due to advances in techniques like PCR but still utilized for applications such as:
      - Measuring transgene copy numbers in transgenic mice.
      - Engineering gene knockout embryonic stem cell lines.

Northern Blotting

• Definition: A technique used to study the expression patterns of a specific type of RNA molecule.
• Process:
    - RNA is separated based on size and transferred to a membrane.
    - Probed with labeled complementary sequences.
    - Results are visualized depending on the label used, typically resulting in bands corresponding to RNA sizes detected.
• Significance:
    - Intensity of bands indicates the amount of target RNA in analyzed samples.
    - A fundamental technique to determine gene expression timing and conditions in living tissues.

Western Blotting

• Definition: A method for detecting specific proteins in a sample.
• Process:
    - Proteins are separated by size using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
    - Proteins are transferred to a support membrane (nitrocellulose, nylon, etc.).
    - The membrane is probed with antibodies that bind specifically to the target protein.
    - Visualization techniques include colored products, chemiluminescence, or autoradiography.
• Key Feature: Antibodies can be labeled with enzymes to facilitate detection through chemiluminescence, allowing for both detection and quantitative analysis of proteins.