Mutations and Biotechnology

Official Syllabus and Success Criteria: Mutations and Biotechnology

Mutation Sources: Mutations in genes and chromosomes result from errors in DNA replication, cell division, or damage caused by mutagens.
Biotechnology Advancements: Developments such as polymerase chain reaction (PCR), gel electrophoresis, and DNA sequencing have increased access to genetic information and provide evidence for evolution. Note: The process of obtaining a DNA sequence is not required for the syllabus.
Success Criteria:
- Classify mutations based on heritability (somatic vs. germline), effect (missense, nonsense, neutral, silent), and extent of change (gene vs. chromosome).
- Describe point mutations (insertions, substitutions, deletions) and chromosomal mutations (duplication, deletion, inversion, translocation, non-disjunction).
- Explain how mutations arise from DNA replication or cell division errors.
- Describe common mutagens.

Introduction to Mutations

Definition: Mutations are permanent changes in DNA.
Role in Evolution: They form a critical part of human evolution by providing genetic diversity and introducing new traits. These traits can be helpful, harmful, or neutral.
Heritability Classification:
- Somatic Mutations:
  - Occur in all body cells except gametes.
  - Affect only certain cells in the individual.
  - Not inherited by offspring.
  - Example: Diseases such as cancer.
- Germline Mutations:
  - Occur in gametes only (found in the zygote/offspring formation).
  - May be inherited by offspring (maintained within the gene pool).
  - Will affect all cells in the body of the offspring.
  - Often do not affect the individual who possesses the initial mutation.
  - Example: Diseases like Huntington’s disease and cystic fibrosis.

Nature and Extent of Mutations

Gene Mutations (Loci Specific):
- Affects only one gene.
- Can involve a single base pair or thousands of base pairs.
- Types:
  - Insertion: A nucleotide is added.
  - Deletion: A nucleotide is removed.
  - Substitution: An existing nucleotide is replaced with a new one.
- Point Mutations: Specific gene mutations affecting only a single nucleotide.
- Frameshift Mutations: Caused by insertions or deletions; they result in a shift of the reading frame so that every amino acid after the mutation point is changed due to new codons being formed.
- Named Examples:
  - Sickle cell anaemia: Substitution.
  - Cystic fibrosis: Deletion.
  - Crohn's disease: Insertion.
Effect-Based Classification:
- Silent: Substitution of a base pair results in a codon that codes for the same amino acid (no change to protein).
- Neutral: Base sequence and amino acid change, but the protein function remains unchanged.
- Missense: Replacement of a single nucleotide leads to an incorrect amino acid, which may produce a malfunctioning protein.
- Nonsense: Base change results in a stop codon being reached too early, terminating the protein prematurely.
Chromosomal Mutations:
- Affect more than one gene, up to a whole chromosome.
- Types:
  - Duplication (Insertion): A section of a chromosome occurs twice.
  - Deletion: A piece of DNA is removed.
  - Inversion: A chromosome breaks and reattaches the wrong way around.
  - Translocation: A chromosome breaks and reattaches to the wrong (non-homologous) chromosome.
  - Non-disjunction: Chromosome pairs do not separate during meiosis, leading to aneuploidy.
- Named Examples:
  - Cri-du Chat: Deletion.
  - Charcot-Marie-Tooth Disease: Duplication.
  - Haemophilia A: Inversion.
  - Robertsonian Translocation: Translocation.
  - Down Syndrome/Turner Syndrome: Non-disjunction (Aneuploidy).

Causes and Timing of Mutations

DNA Replication: Occurs when nucleotides are mismatched during the copying process (complementary base pairing rules: AT and CG). Incorrect addition or removal leads to gene/point mutations.
Cell Division: Chromosomal mutations occur when chromosomes fail to separate or break apart improperly. Specific instances include crossing over (breaking and reattaching incorrectly) and non-disjunction.
Mutagens: Factors that increase the rate of mutation. Examples include:
- Radiation (e.g., UV rays).
- Viruses and microorganisms.
- Environmental poisons and irritants.
- Alcohol and dietary factors.

Biotechnology Tool: Polymerase Chain Reaction (PCR)

Definition and Purpose: PCR uses cycles of heating and cooling and DNA polymerase to rapidly replicate (amplify) small amounts of DNA for study.
Evolution Application: Used to amplify degraded DNA from French farmers ( $3-7$ thousand years ago) to track the lactase persistence gene mutation.
Required Reagents:
- Template DNA: Contains the target sample.
- DNA Primers: Two single-stranded sequences (forward and reverse) that target the specific sequence for copying.
- DNA Polymerase (Taq polymerase): Joins dNTPs to the template DNA.
- dNTPs: Free nucleotides for building new DNA strands.
- Buffer solution: Maintains pH for optimal enzyme function.
The Three-Step Process:
1. Denaturation ( $96\,^{\circ}\text{C}$ ): DNA heats up to separate double strands into single strands.
2. Annealing ( $55-65\,^{\circ}\text{C}$ ): Cools down to allow primers to bind to complementary sequences on the template.
3. Extension ( $72\,^{\circ}\text{C}$ ): Taq polymerase adds free nucleotides to extend the primers, producing two identical double-stranded DNA molecules.
Exponential Growth Data:
- Cycle $1$ : $1$ strand
- Cycle $10$ : $512$ strands
- Cycle $20$ : $524\,288$ strands
- Cycle $25$ : $16\,777\,216$ strands

Biotechnology Tool: Gel Electrophoresis (GEP)

Purpose: Separates DNA (or RNA/Proteins) based on size to create DNA profiles.
Preparation: Requires restriction enzymes to cut DNA into specific lengths before running GEP.
Process Detail:
- Gel Composition: Agarose dissolved in a buffer (containing ions for electrical current and pH stability). Higher agarose concentration means smaller pores.
- Loading: DNA samples are mixed with loading dye (increases density to sink into wells) and visualizing dye (fluorescent dye for UV light visibility).
- Movement: DNA fragments (negatively charged) move toward the positive electrode. Speed is inversely proportional to size: smaller fragments move faster.
- Documentation: Results are compared to a DNA ladder (molecular weight reference marker).

Biotechnology Tool: DNA Sequencing

Definition: Determining the exact order of nucleotides in a DNA molecule.
Medical Use: Diagnoses genetic diseases (Cystic fibrosis, Sickle cell, Huntington's) and identifies cancer risks.
Evolutionary Use: Identifies genetic variations, establishes similarity between species, and aids molecular clocks to determine shared ancestry timing.

Case Study: Antibiotic Resistance and Biotechnology Lab

The Outbreak: An $E. coli$ outbreak infected $42$ people. Patients were resistant to imipenem (carbapenem class) but responded to colistin. Resistance was caused by the $bla_{NDM-1}$ gene (New Delhi metallo-beta-lactamase 1) on a plasmid.
The Laboratory Context: Soil samples from Apple Point Farm and Barrow Creek Farm were tested via PCR to identify the presence of the $700$ base pair fragment of the $bla_{NDM-1}$ gene.
Controls: A $400$ base pair region of the $16S$ ribosomal RNA gene served as a PCR control to ensure DNA was present and amplification was successful.

Evidence for Evolution via Comparative Biochemistry

Comparative Genomics: Comparing complete DNA sets. High similarity suggests a common ancestor.
Endogenous Retroviruses (ERVs): Viral sequences inserted into non-coding DNA. If two species share ERVs at the same loci, they are closely related. Humans and chimpanzees share $16$ ERVs at identical locations.
Mitochondrial DNA (mtDNA):
- Small circular molecules of $16\,500$ base pairs.
- Inherited only from the mother.
- High mutation rate (substitution mutations).
- Used to trace human migration and define genetic relationships.
Protein Sequences: Comparing amino acids in ubiquitous proteins (e.g., cytochrome c). More similarity indicates closer relatedness.
- Example: Human beta-haemoglobin chain ( $146$ amino acids) compared to chimpanzees ( $0$ differences), gorillas ( $1$ difference), and chickens ( $45$ differences).
Molecular Clock Theory: If the protein evolution rate is constant, the amount of change can date evolutionary divergence.
- Example: $20$ substitutions in $400$ million years (mya) indicates a rate of $5$ substitutions per $100\,my$ .

Bioinformatics and Phylogenetic Trees

Bioinformatics: A discipline combining computer science and genetics to store and analyze huge datasets (the human genome contains approximately $2.9 \times 10^9$ base pairs).
Phylogenetic Trees: Diagrams showing evolutionary hypotheses.
- Branching patterns reflect evolution from common ancestors.
- Proximity of the most recent common ancestor determines the level of relatedness.
- Tree rotation at branch points does not change the core information.

Questions & Discussion

Question (PCR vs GEP): Contrast the purpose, process, and products.
- Answer: PCR purpose is amplification; GEP purpose is separation by size. PCR process uses a thermocycler with heat cycles; GEP uses an electric current in agarose. PCR end product is large amounts of copied DNA; GEP end product is a visual representation of DNA bands.
Question (Mutation Comparison): Compare mutations passed to future generations versus those that are not.
- Answer: Germline mutations occur in gametes, are involved in zygote formation, and stay in the gene pool. Somatic mutations occur in body cells and are removed from the gene pool when the individual dies.
Question (Point Mutations types): Define frameshift and two other point mutations.
- Answer: Frameshift results from adding/removing nucleotides causing new codons. Silent mutation codes for the same amino acid (due to insertion/deletion or substitution mentioned in context). Missense changes the code to a different amino acid via substitution. Nonsense forms a new stop codon via substitution.