Molecular Biology Techniques Quiz

Biomedical Techniques CLSB-222

• Course instructor: Dr. Ahmad Alamri, M.H.S, Ph.D.
• Instructor contact: [email protected]

Electrophoretic Techniques

• Electrophoresis is the migration of charged molecules in an electric field.
• It's commonly used in clinical labs for isolating and quantifying serum proteins, isoenzymes, hemoglobin, and lipoproteins.
• Factors affecting electrophoresis include the net electric charge on molecules (pH and ionic strength of buffer), size and shape of molecules, electric field strength, and the nature of the supporting media, and temperature of operation.

A) Paper Electrophoresis

• A form of electrophoresis performed on filter paper.
• Useful for separating small charged molecules like amino acids and small proteins.
• Samples are spotted, and a high voltage is applied, separating molecules based on charge.
• Separated components are detected by staining methods based on their chemical identity.

B) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

• A widely used technique in biochemistry, forensics, genetics, and molecular biology for separating proteins.
• Primarily separates proteins based on their molecular weights.
• SDS (Sodium Dodecyl Sulfate) denatures proteins, binds to hydrophobic regions, disrupting non-covalent bonds, and giving proteins a negative net charge.
• Proteins then migrate through the gel based on their size, with smaller proteins moving faster.
• The rate of migration during SDS-PAGE is determined by the molecular weight.

Polyacrylamide Gel

• Formed by co-polymerization of acrylamide and N,N'-methylene-bis-acrylamide.
• Polymerization requires ammonium persulfate and tetramethylene ethylene diamine (TEMED) as catalysts.

Staining the Gel

• Staining the gel involves using a buffer containing glacial acetic acid, methanol, and Coomassie brilliant blue 250-R.
• Destaining the gel uses a buffer containing glacial acetic acid and methanol to remove excess stain.
• Ethidium bromide stains DNA, allowing visualization under UV light. Caution: Ethidium bromide is a powerful mutagen and is moderately toxic.

C) Agarose Gel Electrophoresis

• Used to separate DNA fragments based on their size using a gel made of agarose.
• Separates DNA by their rate of movement through the gel in an electric field.
• Commonly used to determine the presence and size of PCR products.

Sample Preparation

• Mix DNA samples with 6X sample loading buffer containing bromophenol blue (for color) and glycerol (for weight).
• This improves visibility during loading and increases sample density for better placement in the gel wells.

Loading the Gel

• Carefully place the pipette tip over a well and gently expel the sample.
• The sample should sink into the well, avoiding puncturing the gel with the tip.

Running the Gel

• Place the cover on the electrophoresis chamber, connecting the electrical leads to the power supply.
• Ensure the leads are correctly attached (DNA migrates towards the anode, red).

DNA Ladder Standard

• A DNA ladder containing DNA fragments of known sizes is included on the gel.
• This allows for easier determination of unknown DNA sizes.
• Bromophenol blue runs at approximately the same rate as a 300 bp DNA molecule.

Southern Blotting

• Method for detecting specific DNA sequences in DNA samples.
  • Digests genomic DNA with restriction enzymes.
  • Separates DNA fragments by agarose gel electrophoresis.
  • Denatures DNA fragments.
  • Transfers DNA to a solid support (nylon or nitrocellulose).
  • Hybridizes immobilized DNA to a labeled probe.
  • Detects complementary probes (e.g., by autoradiography).
  • Estimates the size and number of bands after digestion.

F) Northern Blotting

• Method routinely used for detecting specific RNA sequences in RNA samples.
• Isolates intact mRNA.
• Separates RNA by size (using denaturing agarose gel).
• Transfers RNA to a solid support.
• Fixes RNA to the solid matrix.
• Hybridizes with complementary probes.
• Removes unbound probe molecules.
• Detects, captures, and analyzes images of specifically bound probe molecules.

F) Western Blotting

• "Immunoblotting" is a technique for determining the molecular weight and quantity of proteins in a complex mixture; it is also used in medical diagnostics.
• Separates proteins by SDS-PAGE (polyacrylamide gel electrophoresis).
• Transfers proteins to a membrane (e.g., nitrocellulose).
• Uses primary and secondary antibodies to detect target proteins.
• Detects target proteins using colorimetric or chemiluminescent probes.

Test your knowledge on key molecular biology techniques, including SDS-PAGE, Southern blotting, and Northern blotting. Answer questions regarding the principles and purposes of these techniques as well as the factors affecting protein and nucleic acid separation. This quiz covers essential concepts and methods used in the laboratory.