BIO 121: Lecture 2 (Biotechnology Part 2)

Initial Step in Creating Bacterial Cells
- Use plasmid DNA for pharmaceutical and research purposes.
- Plasmids contain non-coding DNA with a sequence for a human gene of interest.
Restriction Enzymes
- Severs DNA at specific sticky ends with nucleotide overlaps, which allows the attachment of the gene of interest.
- This enables the construction of recombinant DNA by using ligase (DNA lipase) to join DNA strands, creating a stable recombinant DNA that can be inserted into microbes for protein expression.

Pharmaceutical Production
- Primary products include insulin, cytokines, and adrenalines from genetically modified bacteria.
Biomanufacturing with Microbes
- Bacteria used to produce vast quantities of target proteins through plasmid engineering.
Advantages
- The process is robust and relatively simple, allowing for self-organization of DNA due to sticky ends.
- Bacteria are suitable hosts for plasmid uptake using methods like bacteriophages or electroporation.

Gel Electrophoresis
- Technique used for assessing the success of DNA insertion based on band size corresponding to proteins of interest.
- DNA fragments move through a gel matrix; size of DNA is inferred from their movement.
- Examples of fluorescent markers include GFP (Green Fluorescent Protein) used alongside the human gene to visualize successful incorporation into bacteria.

Basic Concept
- PCR mimics natural DNA replication using temperature and specialized enzymes.
- Consists of three main steps: denaturation, annealing, and extension.
Key Mechanisms
- Denaturation: Heating DNA to 95°C breaks hydrogen bonds separating strands.
- Annealing: Primers attach to the single-stranded DNA as temperature lowers.
- Extension: DNA polymerase synthesizes new strands, creating double-stranded DNA from single strands.
- Taq polymerase is used due to its heat stability, allowing it to function at elevated temperatures during PCR processes.
Amplification
- Multiple cycles result in exponential amplification of the targeted DNA segment, approximated by the formula $(2^n)$ , where n is the number of cycles.
Applications of PCR
- Used for diagnostics, gene cloning, and in research such as the observation of genetic expressions in response to various conditions.

Reverse Transcription
- Converts mRNA into cDNA for stability and use in PCR.
- mRNA is tagged with a poly A tail; oligo dT primers are commonly used to exploit this feature during synthesis.
Quantitative PCR (qPCR)
- Allows measurement of gene expression levels using fluorescence to quantify amplified DNA at each cycle.
- Helps in assessing the levels of expression against control genes for relative expression analysis.

Overview
- A modern technique used to analyze all expressed RNAs simultaneously, providing comprehensive data about gene activity in a sample.
Process
- RNA where mRNA is reverse-transcribed to cDNA, then sequenced using next-gen technologies allowing for massively parallel analysis.
- Eliminates the need for specific primers unlike PCR, as sequencing relies on inherent properties of mRNA.
Advantages over Traditional Methods
- High-throughput capacity allows simultaneous analysis of thousands of genes and does not require knowing gene sequences beforehand.
- Capable of detecting low abundance transcripts that would otherwise not be observable with PCR alone.

Human Genome Project
- Historical context for advances in sequencing technology; originally took 13 years but rapid advancements in sequencing have decreased this time significantly.
- Illustrates the power and necessity of sequencing in understanding genetic information at a broad scale.

Mechanism
- Enables precise editing of the genome by knocking out genes or inserting functional ones.
- Enhances control over genetic modification compared to previous methods.
Clinical Applications
- Involved in trials for various diseases and has been applied to model diseases in lab animals.
- Promising results in targeted therapies including regenerative medicine and genetic disorders.

Concerns with CRISPR use
- Numerous failed trials highlight the unpredictability of gene editing success.
- Ethical considerations about potential unforeseen consequences in ecology or individual health raise important questions on its application.

Integration of Technologies
- Emphasis on combining various techniques from plasmid engineering to CRISPR for advancing biotechnological applications.
- Education on these processes remains vital for future scientific endeavors and therapeutic developments.

Notes on the methodology, results, and real-world implications will be provided in future classes.