Restriction Enzymes & PCR

• Restriction enzymes are proteins that cut DNA at specific nucleotide sequences called recognition sites.
• Restriction enzymes are naturally produced by bacteria.
• Each restriction enzyme recognizes a specific nucleotide sequence.
• Restriction enzymes cut locations determine if DNA fragments have sticky or blunt ends.
• Sticky ends result from staggered cuts; blunt ends result from straight cuts.
• Recognition sites are typically 4-6 base pairs long and are palindromic.
• DNA ligase catalyzes the joining of DNA fragments at the sugar-phosphate backbone.
• Blunt end fragments can join any other blunt end fragment.
• Sticky ends must be cut by the same restriction enzyme for complementary base pairing.
• PCR amplifies a DNA sample by producing many copies.
• PCR involves heating and cooling cycles with primers, free nucleotides, and heat-resistant DNA polymerase.
• Heating breaks hydrogen bonds between DNA strands.
• Cooling allows primers to attach to complementary regions, initiating polymerase binding.
• DNA polymerase adds free nucleotides to copy the DNA sample.
• Primers are synthetic, single-stranded DNA or RNA complementary to a specific nucleotide sequence.
• Gel electrophoresis separates DNA fragments by size.
• DNA fragments and dye are placed in wells of an agarose gel.
• An ion solution covers the gel, allowing DNA movement in an electric field.
• A negative electrode is placed at the wells and a positive electrode at the opposite end.
• Negatively charged DNA moves toward the positive electrode, with smaller fragments moving faster.
• Dye migration indicates when to stop the charge.
• A secondary dye binds to DNA and fluoresces under UV light for visualization.
• A DNA standard (ladder) contains DNA fragments of known length for comparison.
• Controlled parameters include voltage, agarose gel percentage, temperature, solution pH and concentration, gel length, and time.

DNA Profiling

• DNA profiling compares and analyzes DNA regions from different individuals, using gel electrophoresis.
• Short tandem repeats (STRs) and variable number tandem repeats (VNTRs) are used in profiling due to length and sequence variation among individuals.
• Human profiling uses 13 VNTR regions extracted from cells.
• PCR amplifies VNTRs, followed by cutting and gel electrophoresis.

Genetic Screening

• Genetic screening identifies if an individual carries a specific gene or disorder.
• Examples include testing fetuses or newborns for Down syndrome, hemophilia, and cystic fibrosis.

Implications of DNA Profiling and Genetic Screening

• Ethical considerations include informed consent, secure data storage, and analysis reliability.
• Social implications include potential misidentification, privacy concerns, inheritance knowledge, and discrimination risks.
• Economic implications involve treatment costs, impacting decisions about having children with potential disorders.
• Biological implications involve potential genome alterations to prevent inheritance of certain disorders.

Gene Cloning

• A plasmid is a circular DNA found in bacteria.
• A vector carries genetic material into another organism.
• Recombinant DNA combines host DNA and donor DNA (carried by a vector).
• The host of recombinant DNA is genetically modified.
• Gene cloning uses plasmids and DNA manipulation techniques.
• Cloning the human insulin gene into bacteria is used for type I diabetes treatment.
• The process involves:
  • Cutting a plasmid with a specific restriction enzyme.
  • Isolating and cutting out the insulin gene using the same enzyme if sticky ends are required.
  • Mixing the insulin gene and cut plasmid with DNA ligase to form a recombinant plasmid.
  • Mixing recombinant plasmids with host bacteria and calcium ions.
  • Bacteria that successfully absorbed the recombinant plasmid replicate.
  • Identifying transformed bacteria using antibiotic resistance genes on an agar plate.
  • Extracting and purifying insulin.

Implications of Gene Cloning

• Ethical concerns: animal welfare, modifying genes, injecting animal products into humans.
• Social impacts: greater access to therapeutic treatments, job creation in biotech.
• Economic impact: cheaper medicine.
• Biological impact: purer products, development of new treatments.
• Downside: potential for antibiotic-resistant bacteria, such as in insulin production.

Genetically Modified vs. Transgenically Modified Organisms

• GMOs have altered genomes or contain DNA from donors of the same species.
• TMOs contain DNA from donors of different species. Producing a GMO involves:
  • Identifying the desired gene.
  • Synthesizing or obtaining the gene from a donor (same species).
  • Amplifying the gene using PCR.
  • Inserting the gene into a vector using DNA ligase.
  • Inserting the vector into a recipient organism.
  • Identifying transformed cells (e.g., using antibiotic resistance).
  • Cloning and growing transformed cells into GMOs.

Increasing Crop Productivity

• Genetic modification can increase crop productivity.
• Modifying plants to repair heat damage increases biomass and survival in hotter climates.
• Transgenic modification increases crop productivity.
• Golden rice is a TMO that produces beta-carotene, reducing vitamin A deficiency.

Improving Disease Resistance

• Genetic modification produces crops resistant to diseases.
• Wheat and barley plants can be resistant to wheat scab by adding a gene from wild wheatgrass.

Implications of Genetically Modifying Plants

• Ethical: manipulation of genetic information, safety of consumption, cross-pollination, corporate control.
• Social: increased yield and nutrition, farming accessibility, consumer choice between GM and non-GM foods.
• Economic: increased income, reduced treatment costs and expensive seeds.
• Biological: less space for agriculture, reduced insecticide/pesticide use, reduced biodiversity, gene transfer to native crops.

CRISPR-Cas9

• Genome editing involves insertion, removal, or replacement of DNA within a living cell's genome.
• CRISPR is a DNA section with short nucleotide repeats involved in bacterial defense.
• CRISPR-Cas9 combines CRISPR with the Cas9 enzyme.
• Cas9 cuts specific DNA regions guided by guide RNA (gRNA).
• gRNA contains a 20-nucleotide sequence that binds to complementary DNA.
• Cas9 cuts both DNA strands simultaneously.
• Repair enzymes introduce deletions or additions that disrupt the gene.
• Short DNA regions with desired nucleotide sequences can be inserted to modify a gene.
• Bacteria use Cas9 to cut up viruses; CRISPR stores parts of the virus.
• The CRISPR-Cas9 system allows artificial gRNA creation to target any DNA sequence, disrupting or modifying genes.
• Development breakthroughs include:
  • Inactivating Cas9 active sites to transport additional enzymes for single nucleotide alterations.
  • Adding transcription activators to increase DNA transcription rates.
  • Adding fluorescent proteins to locate specific genes.
  • Treating diseases, editing cancer genes, and removing HIV genes.