Workshop 3 Methods in Molecular Biology - DNA extraction, PCR analysis
DNA Structure and Terminology
Terminology: Understanding of terms such as 3' end, 5' end, bases, sugar, phosphodiester bond, and hydrogen bond.
3' end: Refers to the end of a DNA strand with a free hydroxyl group.
5' end: Refers to the end of a DNA strand with a free phosphate group.
Bases include adenine (A), thymine (T), guanine (G), and cytosine (C).
Phosphodiester bond connects the sugar of one nucleotide to the phosphate of the next.
Hydrogen bonds hold base pairs together.
Learning Outcomes
General Outcomes: Key areas of focus in molecular biology methods.
DNA Isolation: Understanding genomic isolation techniques (Workshop 3).
Restriction Enzymes: Their role in cutting DNA (Workshop 3).
DNA Analysis Techniques: Various methods including agarose gel electrophoresis, nucleic acid hybridization, Southern and Northern blotting, and DNA fingerprinting (RFLP).
Core Topics in Molecular Biology
Introduction to Molecular Biology
The significance of DNA in generating diversity among living organisms.
Location of DNA: Prokaryotic (in cytoplasm) vs Eukaryotic (in nucleus).
The central dogma of molecular biology: DNA → RNA → Protein.
DNA Synthesis: Involves processes such as replication, transcription, and translation.
DNA Isolation Techniques
Organic Extraction: A method to extract DNA using various chemical agents such as ethanol, SDS, EDTA, and proteinase K.
Polymerase Chain Reaction (PCR)
Overview: A revolutionary technique allowing for the amplification of DNA sequences.
Process Steps:
Denaturation: DNA strands are separated by heating. Heats double-stranded DNA to 94-98°C, breaking hydrogen bonds and separating DNA strands into two single strands.
Annealing: Primers bind to the target sequences. Lowers temperature to 50-65°C, binding primers to specific sequences on DNA templates.
Extension: DNA polymerase synthesizes new DNA strands. Increases temperature to 72°C, allowing Taq polymerase to synthesize new DNA strands.
• Repeat Cycles: Repeated steps for 25 to 35 cycles, doubling the number of DNA copies.
• Analysis: Amplitude confirmed using gel electrophoresis or sequenced.
Applications of PCR:
Medical diagnostics: Detection of pathogens like HIV and Mycobacterium tuberculosis.
Forensics: DNA fingerprinting for criminal investigations.
Molecular evolution studies.
Specific Applications of PCR
Quantitative PCR (qPCR): Used to quantify the amount of PCR product after each cycle using fluorescence to measure the amplification.
Example: Detection of RNA levels through reverse transcription.
Genetic Diseases and Diagnosis
Phenylketonuria (PKU): A genetic disorder resulting from PAH gene mutations that cause phenylalanine build-up.
Overview of mutation types include missense, deletion, splicing, nonsense, and silent mutations.
Special note on the dangers of aspartame for individuals with PKU.
Splice-site mutations causing functional impairments of the PAH enzyme.
Molecular Diagnosis Procedures
Procedure for Identifying Genetic Disorders:
Techniques used include DNA extraction, PCR, and gel electrophoresis to analyze the presence of mutations.
In DNA extraction, the following components are crucial:
SDS (Sodium Dodecyl Sulfate): SDS is a detergent that helps to lyse cells, breaking down cell membranes and denaturing proteins. This allows for the release of DNA and other cellular contents.
EDTA (Ethylenediaminetetraacetic acid): EDTA chelates divalent metal ions (like Mg²⁺) that are required as cofactors for nucleases (enzymes that degrade nucleic acids). By binding these metal ions, EDTA helps to stabilize the DNA and prevent its degradation during the extraction process.
Proteinase K (Prot-K): This enzyme digests proteins, further breaking down cellular debris and any proteins bound to DNA, thus purifying the DNA for downstream applications.
Phenol-Chloroform (Phe-chl): This organic solvent mixture is used in a separation process to remove proteins and lipids from the aqueous phase containing DNA. This step helps to enhance the purity of the extracted DNA.
Ethanol (EtOH): Ethanol is used to precipitate DNA from the solution. When mixed with the aqueous phase after separation, it causes the DNA to aggregate, allowing it to be collected by centrifugation.
TE Buffer/H₂O: TE buffer (Tris-EDTA) is used to resuspend the DNA after precipitation, providing a stable pH environment and preventing degradation. Alternatively, water can be used, but TE is preferred for long-term storage.
DNA Analysis: Agarose Gel Electrophoresis
Overview
Agarose gel electrophoresis is a widely employed technique for separating DNA fragments based on size. It uses a gel matrix that allows for the movement of DNA through its porous structure when subjected to an electric field.
Principle
DNA fragments are negatively charged due to their phosphate backbone. When an electric current is applied, the DNA moves towards the positive electrode. Smaller fragments migrate faster and further than larger ones, resulting in size-based separation.
Materials Needed
Agarose powder
Buffer solution (e.g., TAE or TBE buffer)
DNA samples
Loading dye (to visualize the DNA and track migration)
DNA ladder (a set of known DNA fragment sizes) for comparison
Gel electrophoresis apparatus (tank, power supply)
UV transilluminator or gel documentation system for visualization of DNA
Procedure
Prepare the Agarose Gel
Calculate the concentration of agarose needed (typically 0.7% to 2%).
Mix agarose powder with buffer solution and heat until it dissolves completely.
Allow the solution to cool slightly before pouring into a gel casting tray.
Insert a comb into the gel to create wells for the samples and let it solidify at room temperature.
Prepare DNA Samples
Mix the DNA sample with loading dye (which typically contains a tracking dye to visualize migration).
The loading dye also increases the density of the sample, allowing it to sink into the wells.
Load the Gel
Carefully remove the comb from the solidified gel and place the gel into the electrophoresis chamber filled with buffer solution.
Use a pipette to load the DNA samples into the wells created earlier, alongside a DNA ladder (marker) in one of the wells for size reference.
Run the Electrophoresis
Connect the electrophoresis chamber to a power supply.
Set the voltage (typically between 80-150 volts) and run the gel for a specified time, usually until the tracking dye has migrated an adequate distance through the gel (usually 30 minutes to 2 hours).
Visualize the DNA
Once the run is complete, turn off the power and carefully remove the gel from the tank.
Stain the gel with a DNA-binding dye (e.g., ethidium bromide or SYBR Safe) if not already included in the gel preparation.
Place the gel on a UV transilluminator to visualize the bands of DNA. Record or capture images for analysis.
Applications
Genetic fingerprinting: Used in forensics to compare DNA from crime scenes.
Molecular cloning: Verifies the presence and size of inserted DNA fragments after ligation.
Check PCR results: Confirms successful amplification by comparing product sizes.
Mutational analysis: Helps identify mutations by analyzing changes in the banding pattern.
Advantages
Simple and cost-effective.
High resolution for size separation.
Versatile for different types of DNA analysis.
Limitations
Limited to separating macromolecules like DNA, RNA, and proteins.
Requires careful handling of samples and reagents.
Staining methods using ethidium bromide pose safety concerns due to its mutagenicity.
Conclusion
Agarose gel electrophoresis is a fundamental tool in molecular biology, providing a reliable method for analyzing and characterizing DNA fragments. Its efficiency and versatility make it essential for many applications in research, diagnostics, and forensic science.