Chapter 15: Studying and Manipulating Genes - Notes

15.1 Personal DNA Testing

Key Concepts:

  • Human DNA Similarity: Approximately 99% of human DNA is identical across all individuals.
  • SNP (Single-Nucleotide Polymorphism):
    • A SNP is a variation in a single base pair within DNA.
    • To be classified as an SNP, this variation must be present in at least 1% of the population.
    • Around 4.5 million known SNPs exist in human DNA.

Significance of SNPs:

  • SNPs influence observable traits such as hair and eye color.
  • They affect an individual's predisposition to various diseases, including diabetes and cancer.
  • SNPs also impact how individuals respond to drugs (pharmacogenomics).

Application:

  • SNP Chips (genotyping microarrays):
    • SNP chips contain probes designed to detect specific SNP sequences.
    • These chips are utilized in consumer DNA tests like 23andMe.
    • SNP chips help identify genetic predispositions and ancestry.
  • Personalized medicine:
    • Personalized medicine involves using a person’s SNP profile to determine the most effective treatment.
    • For example, certain cancer drugs show greater efficacy depending on specific SNPs present in a patient.

15.2 Cloning DNA

Cutting and Pasting DNA:

  • Restriction Enzymes:
    • Restriction enzymes are naturally produced by bacteria.
    • They cut DNA at specific sequences such as GAATTC.
    • These enzymes create "sticky ends," characterized by short, single-stranded overhangs.
  • DNA Ligase:
    • DNA ligase is an enzyme that joins DNA fragments by forming covalent bonds in the sugar-phosphate backbone.

Recombinant DNA:

  • Recombinant DNA is formed by combining DNA sequences from different organisms.
  • An example includes inserting the human insulin gene into bacterial DNA to enable insulin production.

DNA Cloning Process:

  1. Extract the gene of interest from an organism’s DNA.
  2. Cut the gene and plasmid vector using the same restriction enzyme.
  3. Mix and ligate: sticky ends pair up, and DNA ligase seals the fragments.
  4. Introduce the recombinant plasmid into a host cell, such as E. coli.
  5. Bacteria multiply, creating clones containing the recombinant gene.

Cloning Vectors:

  • The most common cloning vectors are plasmids, which are small, circular DNA molecules found in bacteria.
  • Plasmids often contain:
    • Antibiotic resistance genes, used for selection.
    • Multiple cloning sites, which provide many restriction sites.
    • Reporter genes, such as lacZ for blue-white screening.

cDNA Cloning:

  • cDNA stands for Complementary DNA.
  • cDNA is synthesized from mRNA and represents only expressed genes without introns.
  • Steps:
    • Reverse transcriptase is used to create DNA from mRNA.
    • DNA polymerase is used to produce double-stranded cDNA.
    • cDNA is then cloned into plasmid vectors.
  • Useful for:
    • Studying gene expression.
    • Producing proteins without introns, since bacteria cannot process introns.

15.3 Isolating Genes

DNA Libraries:

  • Genomic Library: Contains all DNA fragments from an organism’s genome.
  • cDNA Library: Contains all genes expressed in a specific tissue at a specific time.

Probes and Hybridization:

  • Probe: A labeled, single-stranded DNA fragment.
  • It binds to a complementary DNA sequence in the library.
  • This process uses nucleic acid hybridization, which is the base pairing between the probe and target DNA.
  • This helps locate the clone containing the gene of interest.

PCR (Polymerase Chain Reaction):

  • PCR is a rapid DNA amplification technique.
  • Steps:
    1. Denaturation (approximately 95°C95°C): DNA strands separate.
    2. Annealing (approximately 55°C55°C): Primers bind to the target DNA.
    3. Extension (approximately 72°C72°C): Taq polymerase extends the new DNA strand.
  • After approximately 30 cycles, billions of copies are produced.
  • Used in forensics, diagnostics, and genetic testing.

15.4 DNA Sequencing

Goal: Determine the exact sequence of bases in DNA.

  • Uses dideoxynucleotides (ddNTPs):
    1. ddNTPs lack a 3' OH group, which terminates DNA synthesis.
    2. ddNTPs are labeled with fluorescent dyes, each color representing a different base (A, T, C, G).
  • Process:
    1. DNA is replicated with a mix of regular nucleotides and ddNTPs.
    2. Random termination produces fragments of different lengths.
    3. Electrophoresis is used to separate fragments by size.
    4. A computer reads the color tags to determine the sequence.

Human Genome Project:

  • Completed in 2003.
  • Sequenced 3 billion base pairs.
  • Identified approximately 29,000 genes.
  • The function of many genes remains unknown.

15.5 Genomics

Genomics: Study of entire genomes.

  • Compares:
    • Human genes with those of other species.
    • Individual genetic variation (e.g., SNPs, STRs).
  • Applications:
    • Medical diagnostics and evolutionary biology.

DNA Chips (Microarrays):

  • Thousands of DNA spots on a glass plate.
  • Used to detect gene expression patterns.
  • Example: Comparing cancerous cells with normal cells.

DNA Profiling:

  1. SNPs:
    • Analyzed using SNP chips.
    • Hybridization reveals the presence of specific SNPs.
  2. STRs (Short Tandem Repeats):
    • Repeated sequences (e.g., GATA-GATA-GATA…).
    • Vary significantly between individuals, creating a DNA fingerprint.
    • Analyzed by PCR and electrophoresis.
    • Used in forensics, paternity testing, and population studies.

15.6 Genetic Engineering

  • Genetic engineering involves the deliberate modification of an organism’s genome.
  • Produces:
    • GMOs: Genetically modified organisms.
    • Transgenic organisms: Organisms containing genes from another species.
  • Applications:
    • Bacteria producing human insulin.
    • Fluorescent proteins for tracking gene expression.
    • Industrial enzymes (e.g., brewing, cheese-making).

15.7 Designer Plants

How to Add Genes:

  • Physical methods: gene gun, electric shock.
  • Biological method: Ti plasmid from Agrobacterium tumefaciens, which inserts genes into the plant genome.

Genetically Engineered Crops:

  • Traits:
    • Herbicide resistance.
    • Pest resistance (Bt toxin).
    • Drought resistance.
    • Nutrition enhancement (e.g., Vitamin A in Golden Rice).
  • Concerns:
    • Herbicide-resistant weeds.
    • Gene flow to wild relatives.
    • Ethical issues regarding food labeling and safety.

15.8 Biotech Barnyards

Transgenic Animals:

  • Used to study human diseases.
  • Knockout mice: Genes are inactivated to study their function.

Industrial and Medical Use:

  • Goats producing spider silk in milk.
  • Rabbits making human interleukin-2.
  • Pigs engineered for xenotransplantation (organ transplant across species).
  • Concern: Risk of animal viruses infecting humans.

15.9 Safety Issues

  • Initial fears included the creation of superpathogens or environmental disruption.
  • Regulations exist (e.g., NIH, USDA), but:
    • They are not globally consistent.
    • There is an ongoing risk of gene escape.
  • Scientists must adhere to ethical and safety standards.

15.10 Genetically Modified Humans

Gene Therapy:

  • Involves inserting a functional gene to fix or treat a disease.
  • Uses viral vectors or lipid nanoparticles.
  • Example: SCID-X1 (Severe Combined Immunodeficiency)
    • Gene therapy cured 18 out of 20 boys.
    • However, 5 developed leukemia, and 1 died.
  • Ethical Dilemmas:
    • Unintended effects (e.g., cancer, immune reaction).
    • Possibility of "designer babies" (e.g., height, intelligence).
    • Risks of eugenics and social inequality.