Molecular Biology Techniques Quiz

Gel Electrophoresis

• Gel electrophoresis is a method used to separate and analyze macromolecules (nucleic acids and proteins) and their fragments based on molecular weight.
• It is used to analyze virus components, as the molecular weights of viral capsid proteins and nucleic acids are distinctive features of viruses.

Gel Types

• Agarose: A polysaccharide extracted from seaweed, used for large molecule separation, such as nucleic acids. It is non-toxic.
• Polyacrylamide: A cross-linked polymer of acrylamide, used for smaller molecules. It is potent neuro-toxic. Nucleic acid and protein separation can be performed.

Gel Casting

• Agarose gels are cast horizontally.
• Polyacrylamide gels are cast vertically.

Toxicity

• Agarose gels are non-toxic.
• Polyacrylamide gels are potent neuro-toxic.

Separation

• Agarose gels separate large molecules like nucleic acids.
• Polyacrylamide gels separate smaller molecules like nucleic acids and proteins.

Staining

• Staining methods, like ethidium bromide or RedSafe, are used to visualize DNA under UV light. These bind to DNA and cause fluorescence.
• Staining can be done before or after running the gel. Post-run staining is common for protein separation.

Determination of Molecular Mass of Coat Proteins by Polyacrylamide Gel Electrophoresis (PAGE)

• Steps: Denaturation involves purifying the virus and adding denaturation buffer to denature the viral capsid into polypeptide subunits.
• Purpose:
  • Denature the viral capsid into polypeptide subunits.

Electrophoresis (additional notes from page 2)

• Buffers: Denaturation buffer may contain SDS (Sodium dodecyl sulfate) a detergent that gives proteins a net negative charge, leading to protein unfolding; and 2-ME (2-mercaptoethanol) a reducing agent to break disulfide bonds.
• Stacking Gel: The concentration of acrylamide is lower (approximately 4%) in the stacking gel. This gel concentrates proteins while they migrate.
• Resolving Gel: The lower resolving gel contains a higher concentration of acrylamide (approximately 12%), allowing separation of protein fragments based on size.

General Staining Steps (page 3)

• Remove electrophoresis buffer (water wash).
• Fix (Use acid or alcohol to prevent protein diffusion).
• Wash.
• Stain (Use a dye or chemical to bind to proteins).
• Destain (remove excess dye). Examples include Coomassie dye stain (turns proteins blue) and silver stain (high sensitivity).

Microarray (page 4)

• Microarrays are collections of thousands to millions of microscopic spots containing known nucleic acid fragments (probes or reporters) attached to a solid surface, called a 'chip'.
• They are used for detecting the presence of viral samples, gene expression, and comparative gene hybridization (CGH).
• Hybridization is the process of forming double-stranded DNA molecules through base pairing between complementary sequences.
• Antibody microarrays use protein-specific capture antibodies on a solid surface to detect interaction with target antigens.

Blotting (page 5)

• Blotting is a technique to transfer macromolecules (DNA, RNA, or proteins) from a gel to a solid support (e.g., nitrocellulose, PVDF) for further analysis.

• Types of blotting include Southern (DNA), Northern (RNA), and Western (protein) blotting.

• Sample Preparation: involves extracting and preparing proteins to visualize them further via techniques.

• Gel Electrophoresis: PAGE or agarose gels are used based on the separation of the macromolecules.

• Blotting (page 6): Various methods exist for transfer.

  • Wet Transfer: uses a large amount of buffer; vertical transfer; and is slow. High efficiency (80-100%).
  • Semi-Dry Transfer: uses a small amount of buffer; horizontal placement; and is faster. Slightly less efficient (60-80%)

• Immobilization: Fixing the fragments onto the membrane. can be done through UV radiation or heat.

Hybridization (page 7)

• Western Blotting: A primary antibody is incubated with the membrane overnight at 4°C. Then washing occurs to remove unbound antibody. Secondary antibody (labeled with a detectable signal, e.g., radioactively, fluorescent dye, or enzyme) is added, and further washing is performed. HRP (horseradish peroxidase) is often used as a signal.
• Southern/Northern Blotting: Labelling DNA/RNA with a homologous sequence and detection using specific substrates are common.
• Methods like colorimetric (using TMB, developing a purple-black color), or chemiluminescent substrates are used. These are different for specific use-cases.

Dot Blot (page 7)

• A technique to detect and identify proteins, DNA, or RNA directly on a membrane without prior separation via electrophoresis.

PCR (Polymerase Chain Reaction (pages 8-9))

• A method used to amplify a specific DNA sequence. It involves cycles of heating (denaturation) and cooling (annealing and extension).
• Components: Template DNA, DNA polymerase (e.g., Taq polymerase), primers, nucleotides (dNTPs), buffer, and metal ions (e.g., Mg2+).
• Steps: (initial denaturation, denaturization, annealing, extension/elongation)
• Cycles/steps: are repeated to exponentially amplify the target DNA.

RT-PCR (Reverse Transcription PCR) (page 10)

• Combines reverse transcription of RNA into cDNA followed by PCR amplification. This allows for studying gene expression levels.
• Multiplex PCR: simultaneously detects multiple targets (genes) in a single reaction using different primer pairs for each target. The primer pairs need to be highly specific.

qPCR (Quantitative PCR) (page 11)

• A real-time PCR technique combines DNA amplification and quantification of DNA.
• Components: Real-time chemistries, and conventional PCR components.
• Threshold Line: The level to detect signal when fluorescence intensity is above the background.
• Ct value:
  • The cycle number where the fluorescence of the PCR product crosses the threshold line.

Specific Probes (page 12)

• TaqMan probes: consist of a fluorophore and quencher, which prevents fluorescence until the probe is cleaved. cleavage of the probe occurs after priming/elongation, which is measured.

Test your knowledge on key molecular biology techniques, including PCR, microarrays, and blotting methods. This quiz covers principles behind SYBR green, TaqMan probes, and the use of fluorescent dyes in protein detection. Ensure you understand hybridization and gene expression concepts as well.