MCB Lecture [In-person]-20250305_090111-Meeting Recording

Overview of Chapter Six

• Focus on studying the slides in conjunction with the textbook.

• Quiz on Friday and test on the entire chapter on Monday.

• Quiz serves as practice for the test format, similar multiple choice questions expected.

PCR and Its Applications

• PCR (Polymerase Chain Reaction) is central to the chapter.

  • Adaptability: Different approaches developed using PCR beyond just DNA amplification.

  • Restriction enzymes: Added to DNA primers to enhance cloning.

  • Quantitative PCR (qPCR): Allows quantification of DNA templates based on the initial amount of the template.

Amplification Insights

• Less abundant templates require more cycles to detect amplicons.

• High initial template abundance yields amplicons sooner.

• qPCR useful for detecting bacterial or viral infections.

Transcript Analysis with PCR

• qPCR excels in analyzing gene expression levels.

• Reverse Transcriptase: Converts RNA into double-stranded cDNA for PCR.

• Basic process includes:

  • Isolating RNA (mRNA).

  • Using a poly A-tail primer for cDNA synthesis.

• Two-step vs one-step RT-PCR reactions.

Advantages of qPCR

• Monitors amplicon generation in real-time using fluorophores.

• Can analyze gene expression from a pool of RNAs extracted from tissues.

• Helps determine transcriptional activity and responses to stimuli.

Microarray Analysis (ChIP Technology)

• Used to identify active genes by analyzing mRNA abundance in samples.

• Hybridization: Specific oligonucleotides allow identification of RNA sequences in experimental conditions.

• Example: Tumor tissue analysis can highlight active genes involved in tumor progression.

Experimental Setup

• Compare responses of two cell cultures to serum for growth factor analysis.

  • RNA extraction followed by fluorescently labeled cDNA synthesis to assess gene expression.

• Data output includes colored dots indicating gene expression levels.

Cloning and Gene Expression

• Use of bacterial cells to produce proteins (e.g., GFP and therapeutic proteins like G-CSF).

• Expression Vectors: Enable the production of proteins of interest in bacteria.

• Some proteins require mammalian cells for correct post-translational modifications.

Transfection Techniques

• Transient Transfection: Plasmid remains outside the genome, high initial efficiency but decreases with cell division.

• Stable Cell Lines: Required integration of the plasmid into the genome for consistent expression, often requiring antibiotic resistance markers.

Viral Vector Technologies

• Use of viral systems to achieve high-efficiency stable transfections.

• Retroviruses: Infect cells and integrate their RNA into the host genome, utilizing homologous recombination.

• Example: VSVG protein enables infection across a variety of mammalian cells.

Gene Function Analysis and Reverse Genetics

• Reverse genetics involves disrupting known genes to study resulting phenotypes.

• Homologous Recombination: Allows for replacement of genes with antibiotic resistance markers to select for successful disruptions.

• Example: Analysis of yeast genes to uncover functions and essentiality.

Pooled Assays

o- Implementing pooled assays allows for stress-testing numerous gene disruptions simultaneously.

CRISPR Technology

• CRISPR acts as a bacterial immune system adapted for gene editing in eukaryotes.

• Uses guide RNA to target specific genomic sequences leading to double-stranded breaks.

• Repairs occur via non-homologous end joining (NHEJ) or homologous recombination (HR) options, potentially allowing corrections or gene disruptions.

Summary

• Understanding PCR, qPCR applications, transcript analysis, cloning, and CRISPR systems are critical for mastery of chapter six’s concepts.