PCR and Somatic Cell Nuclear Transfer (SCNT)

• PCR is a technique amplifying small DNA amounts into usable quantities for various applications like crime scene investigation

PCR Ingredients

• DNA template needing copying
• Primers
• DNA bases
• Taq polymerase to add bases
• Buffer

PCR Steps (Thermal Cycle)

• Repeated 20-40 times, doubling DNA each cycle
• Denaturing: Heating double-stranded DNA to 94-95°C separates it into single strands in 15-30 seconds
• Annealing: Lowering temperature to 50-65°C allows DNA primers to connect in 10-30 seconds, acting as the starting point for DNA synthesis
• Extension: Raising temperature to 72°C allows Taq polymerase adds bases
• Taq polymerase builds a complementary strand in 5' to 3' direction
• Takes about 1 minute for 1000 bases

Somatic Cell Nuclear Transfer (SCNT)

• SCNT involves transferring a somatic cell's nucleus into an enucleated egg cell's cytoplasm for organism cloning
• The cell is then placed in a surrogate
• Ethical Concerns: The cloning of animals raises ethical concerns of pain, suffering, and distress. Human cloning raises issues of safety, impact on family dynamics, and commodification

SCNT Steps

• Obtain a somatic cell and oocyte (egg cell) from the organism to be cloned
• Nucleus and genetic material are removed from oocyte with a micropipette
• Somatic cell nucleus is transferred to the oocyte
• The cell is stimulated with chemical or electrical pulse while put into surrogate for embryonic processes
• can be cultured in a lab setting

Gel Electrophoresis

• Technique separating molecules like DNA, RNA, or proteins by size and charge using an electric current through a gel
• Smaller molecules travel faster
• Used in crime scene identification

Gel Electrophoresis Steps

• Prepare DNA samples and buffer solution in box
• Load DNA sample with micropipette
• Run electrical current through buffer solution
• Observe bands and draw conclusions

Gel Electrophoresis Process

• Restriction enzymes break DNA into different-sized fragments, separated by electrical current
• Longer fragments stay closer to the start, smaller fragments travel towards the end

Restriction Enzymes

• Restriction enzymes, or "molecular scissors," are proteins from bacteria cutting DNA at specific nucleotide sequences
• Creates fragments with known sequences for genetic engineering

How Restriction Enzymes Work

• Cuts DNA between specific sequences, creating different sizes of DNA fragments for gel electrophoresis
• Ends can be sticky (staggered cut) or blunt (straight cut)

Bacterial Transformation

• Bacterial transformation involves a bacterial cell taking up foreign DNA and incorporating it into its genome
• May give new traits like antibiotic resistance
• Part of DNA cloning and creating recombinant DNA
• Used to make human proteins like insulin

Recombinant DNA

• Created by combining genetic material from multiple sources, enabling new gene combinations

Chi-Square Test

• Statistical test comparing observed results with expected results
• Determines if differences are due to chance or variable relationships

Creating Null Hypothesis for Chi-Square Test

• Null hypothesis states no relationship between variables
• Research hypothesis suggests a relationship between the variables

Chi-Square Steps

• Find expected value of offspring
• Plug values into formula: (observed - expected)² / expected
• Add the values
• Compare the p-value (result) to the critical value
• Reject the null hypothesis if value is lower, and if value is higher, then fail to reject

Gene Therapy

• All chi-square tests related to Gene Therapy have general null and research hypotheses that state no relationship, or relationship between the two variables being tested

Types of Gene Therapy

• In vivo: Corrected genes are delivered directly via IV or local delivery inside the body. ex: eye
• Ex vivo: Patient's cells are genetically modified outside the body in a lab and transplanted back in via vector to replace malfunctioning ones.

Selective Breeding

• Selective breeding (artificial selection) involves intentionally breeding plants/animals for desired traits
• Examples: increased crop yield or improved animal products

Ethical Concerns with Breeding

• Genetic engineering and selective breeding may violate animal rights by using them for human benefit

GMOs (Genetically Modified Organisms)

• GMOs are plants, animals, or microbes modified using genetic engineering
• Examples include crops like corn and fish like AquAdvantage salmon

Ethical Concerns about GMOs

• Health risks, environmental damage, impacts on farms, corporate dominance, and unnaturalness

Pros of GMO's

• Will help farmers make more of a profit
• crops will be bug / insect resistant

Cons of GMO's

• Allergic reactions, toxins, and increased herbicide use
• potential for antibiotic resistance, herbicide-resistant weeds and biodiversity loss

NGS (Next-Generation Sequencing)

• NGS (massively parallel/deep sequencing) is a DNA sequencing technology revolutionizing genomic research
• Entire human genome can be sequenced in a day

NGS Usage

• For understanding disease, improving medicine, and advancing research on cancer and rare conditions

Human Genome Project

• NGS stemmed from the earlier Human Genome Project
• Project took 32 years

Sanger Sequencing

• Fast, cost-effective sequencing method for small targeted genome regions
• Used to validate results from massively parallel sequencing and certain single-gene assays

How Sanger Sequencing Works

• Automated process: a computer reads each band that is fluorescing through an emitted light
• Steps: denaturing dsDNA, multiplying segment copies, attaching primer, adding polymerase solutions, amplifying and denaturing chains, electrophoreses solution

CRISPR

• Editing technology modifying DNA by cutting, inserting DNA sequences, adapted from bacterial defense system

CRISPR/Cas9

• Cuts DNA at specific location, deleting/inserting sequences, changing single DNA base, pieces of chromosomes, or gene expression
• Modifies point DNA sequences
• After observing bacteria, Scientists used bacteria's attacking mechanisms of deleting through Cas9 enzyme

Synthetic Biology

• Designs/builds new biological systems/redesigns existing ones, using DNA-encoded components for purposes like medicine/biofuels

Synthetic Biology Solutions

• Offers solutions for agriculture, medicine, and industry
• E.g., addressing food security, healthcare challenges, and sustainable production through gene editing and metabolic engineering

Transgenic Organisms

• Organism/cell with genome altered by introducing foreign DNA from another species artificially for research

Example of Transgenic Organisms

• Golden rice (genetically modified) engineered to reduce vitamin deficiency deaths in poor countries
• Created by mixing with corn and dirt bacteria that survive because of the vitamins they obtain, helping prevent disease.

DNA Fingerprinting

• Laboratory technique analyzing individual variations in DNA to create genetic profile
• Can then be used for indentification and forensics

Uses of DNA Fingerprinting

• Criminal investigations, forensics, and paternity testing