L8 Gene therapy and DNA techniques

Polymerase Chain Reaction (PCR)​

• How it works: Amplifies DNA by repeatedly denaturing, annealing primers, and extending DNA using replication machinery.

• Best for: Detecting or amplifying specific DNA sequences when only a small sample is available.

Real-Time PCR (qPCR)​

• How it works: Monitors DNA amplification in real-time using fluorescent probes.

• Best for: Quantifying DNA or RNA levels in a sample.

Southern Blotting​

• How it works: DNA is electrophoresed, transferred to a membrane, and probed for specific sequences.

• Best for: Identifying specific DNA fragments in a mixture.

Northern Blotting​

• How it works: Similar to Southern blotting but used for RNA detection.

• Best for: Studying gene expression by detecting RNA transcripts.

Western Blotting​

• How it works: Proteins are separated by electrophoresis, transferred to a membrane, and detected using antibodies.

• Best for: Identifying specific proteins and analyzing gene expression at the protein level.

DNA Sequencing (Dideoxy Method)​

• How it works: Uses chain-terminating dideoxynucleotides to randomly terminate DNA synthesis, and fragments are analyzed by size.

• Best for: Determining the exact nucleotide sequence of DNA.

Genomic Libraries​

• How it works: A genome is fragmented, and the pieces are cloned into vectors.

• Best for: Identifying and isolating specific genes from an organism's genome.

Protein Tagging for Purification​

• How it works: Tags (e.g., GST tags) are fused to proteins to simplify their purification.

• Best for: Isolating and studying specific proteins.

Transfection Methods​

• Techniques include electroporation, liposome-mediated delivery, and biolistics.

• Best for: Introducing foreign DNA into eukaryotic cells for therapeutic or research purposes.

Gene Therapy (In Vivo and Ex Vivo)​

• How it works: In vivo introduces genes directly into a patient’s cells, while ex vivo modifies cells outside the body and reintroduces them.

• Best for: Treating genetic disorders or diseases.

Replication:

• Process and regulation

• Process: DNA replication involves unwinding the double helix and synthesizing complementary strands using DNA polymerases.

• Regulation: Ensures fidelity through proofreading and repair mechanisms.

Transcription:

• Process and regulation

• Process: DNA is transcribed into mRNA by RNA polymerase.

• Regulation: Includes promoter activation, enhancers, and repressors. Eukaryotic cells also modify mRNA post-transcriptionally (e.g., splicing, capping).

Translation:

• Process and regulation

• Process: mRNA is translated into proteins by ribosomes using tRNA to decode codons.

• Regulation: Controlled by mRNA stability, translation initiation factors, and post-translational modifications.

Gene Expression and Modification​

• Prokaryotes cannot process eukaryotic introns, necessitating cDNA for proper expression.

• Protein tagging (e.g., GST) and blotting methods help study gene and protein expression.