Lecture Notes on Mutations and Biotechnology Tools

Mutations

• Germline Cells:
  • These are the cells that give rise to an individual's eggs or sperm.
  • A mutation in a gamete (egg or sperm) is present in all gametes of that individual and is passed on to the next generation.
• Somatic Cells:
  • These are all cells in the body that are not part of the germline.
  • Somatic cells differentiate along different lines to become specialized cells.
  • A mutation in a somatic cell will be passed on to daughter cells when the cell divides, but it will not be inherited by offspring.

Effects of Mutation

• Silent Mutation:
  • This is a base substitution that alters a codon.
  • The altered codon codes for the same amino acid, so there is no change in the protein.
• Missense Mutation:
  • This is a base substitution that alters the codon.
  • The altered codon codes for a different amino acid, resulting in a change in the protein sequence.
• Nonsense Mutation:
  • This is a base substitution that alters the codon.
  • The altered codon becomes a stop codon, leading to a shorter polypeptide chain.
  • This can result in a non-functional protein if the full amino acid sequence is not made.

Factors That Increase the Rate of Mutation

• Ionizing Radiation:
  • This includes high-energy radiation like X-rays and UV light.
  • UV light (e.g., from the sun) creates bonds between DNA bases that don't usually exist, disrupting transcription and translation.
  • X-rays create ions that break the sugar-phosphate backbone of DNA and base pairs.
• Mutagenic Chemicals:
  • These chemicals can change the chemical structure of a base.
  • They can disrupt cell division, damage DNA, and break chromosomes.
• Viruses:
  • When viruses infect host cells, they may cause a change to the host cell DNA.
  • This can lead to substitutions, deletions, or insertions in the host cell's DNA.

Biotechnology Tools and Techniques

• Polymerase Chain Reaction (PCR)
  • Requirements:
    • DNA Sample
    • Primers
    • Nucleotides
    • Taq polymerase
    • Mixed buffer
    • PCR tube
  • Process:
    • Denaturation: The DNA strand is separated by heating it up to 95 degrees Celsius.
    • Annealing: Primers bind to the template DNA at approximately 55 degrees Celsius.
    • Extension: Taq polymerase synthesizes a new DNA strand at 72 degrees Celsius.
• DNA Sequencing
  • Determines the actual sequence of DNA bases in a specific gene segment or an entire genome of an organism.
  • Process:
    • Collect the DNA template to be sequenced.
    • PCR creates copies of the DNA to be sequenced.
    • Fluorescently tagged terminator nucleotides induce chain termination while also marking the terminating base
    • This results in an array of DNA fragments of a single base difference because they have been terminated at a different place along the DNA template.
    • Newly made DNA fragments undergo capillary electrophoresis, which identifies the terminated nucleotide of each fragment.
    • The DNA sequence is produced from an electropherogram, which is a picture and a graph.
    • This allows the bases to be determined.
• Genetic Engineering
  • Applications:
    • Agriculture: Nutrient-rich food, pest-resistant crops, and disease-resistant crops.
    • Medical Fields: Insulin production and treatment of genetic diseases.
• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
  • Allows for precise editing of DNA.
  • Process:
    • Guide RNA (gRNA): A short sequence of RNA that is synthetically made to be complementary to a specific section of DNA.
    • The gRNA binds to the DNA and guides the Cas9 enzyme to the correct sequence of DNA.
    • Cas9 Enzyme: Acts as a pair of scissors that cuts the double-stranded DNA of the host cell at a specific location.