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Strand 3: Interactions of Life

Genetic Engineering

Genetic engineering techniques, DNA profiling, PCR, gel electrophoresis, sequencing, and ethical issues.

3.3 Genetic Engineering

In brief:Genetic engineering involves modifying an organism's DNA to introduce new traits. Modern biotechnology has enormous applications but raises ethical questions.

Recombinant DNA

Recombinant plasmid formation using restriction enzymes and ligase

Wikimedia Commons (CC)

Key Points

  • 1Genetic engineering steps: DNA isolation → cutting (restriction enzymes) → ligation → transformation → expression.
  • 2Restriction enzymes: cut DNA at specific base sequences, creating 'sticky ends'.
  • 3Ligase: enzyme that joins DNA fragments together.
  • 4Plasmids/vectors: used to carry the gene of interest into a host organism.
  • 5PCR (Polymerase Chain Reaction): makes millions of copies of a DNA sample quickly.
  • 6Gel electrophoresis: separates DNA fragments by size using an electric field.
  • 7DNA profiling (fingerprinting): unique pattern of DNA fragments used in forensics, paternity.
  • 8DNA sequencing: reading the order of bases in DNA.
  • 9Uses: agriculture (GM crops), health (gene therapy), forensics, reproduction (genetic screening).
  • 10Stem cells: undifferentiated cells that can become any cell type (therapeutic uses, cloning).

Learning Outcomes

  • Outline the concept of genetic engineering and its applications
  • Outline what is meant by DNA profiling and its potential uses
  • Model the steps involved in generating a DNA profile
  • Outline the principle of DNA sequencing
  • Use a genome database to search for alleles that are known to cause (or be responsible for) specific genetic diseases
  • Investigate patterns using a DNA profile, use primary or secondary data to support conclusions
  • Discuss the ethical issues arising from advancements in genetic technologies

2. DNA Profiling, Gel Electrophoresis and Sequencing

In brief:A DNA profile is a banding pattern unique to an individual; sequencing reads the bases themselves.

DNA profiling (genetic fingerprinting) produces a pattern of bands unique to an individual, apart from identical twins. It does not read the whole genome. It looks at short repeating sequences in the non-coding DNA, where the number of repeats varies a lot from person to person.

The steps

  1. Collect a sample - blood, saliva, hair root, semen or skin cells.
  2. Extract the DNA from the cells: break the cell and nuclear membranes with detergent, add salt and protease, then add cold ethanol so the DNA precipitates out as white strands.
  3. Amplify the chosen regions with PCR, so even a trace sample gives enough DNA to work with.
  4. Cut the DNA into fragments using restriction enzymes, which cut at specific base sequences.
  5. Separate the fragments by gel electrophoresis.
  6. Compare the band patterns of the samples.

Gel electrophoresis

The DNA fragments are loaded into wells at one end of an agarose gel and a voltage is applied. DNA is negatively charged because of its phosphate groups, so all fragments move towards the positive electrode. Short fragments travel through the gel mesh faster and end up furthest from the wells; long fragments lag behind. A stain or fluorescent dye makes the bands visible. Two samples from the same person give identical band patterns.

Uses

Forensic investigation, identifying human remains, paternity and family testing, immigration cases, checking pedigree in animal breeding, detecting disease-causing alleles, and comparing species in evolutionary studies.

DNA sequencing and genome databases

Sequencing determines the actual order of bases in a piece of DNA. Fragments of every possible length are generated, each labelled according to its final base; separating them by size lets the sequence be read off in order. Modern high-throughput machines sequence a whole human genome in about a day, and the cost has collapsed from billions to a few hundred euro.

Sequences are deposited in public genome databases such as GenBank and Ensembl. Using one, you can choose an organism, search a gene or disease name, and find the alleles known to cause a condition, their chromosome location and their effect on the protein. This is how a diagnostic laboratory checks a patient's sequence against the reference to confirm a diagnosis.

Ethical issues

Who owns and controls genetic data? Could insurers or employers use it to discriminate? Should national DNA databases hold profiles of people never convicted? Consent, privacy and secure storage matter, and most sequenced genomes still come from a narrow range of populations, which limits how well the results apply to everyone.

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DNA profiling and gel electrophoresis

Generating a DNA profile 1. Collect sample 2. Extract DNA 3. Amplify by PCR 4. Cut with restriction enzymes 5. Separate by electrophoresis 6. Compare band patterns Reading the gel Crime scene Suspect 1 Suspect 2 Fragments carry a negative charge, so they move towards the positive electrode. Short fragments travel furthest. Suspect 2's bands match the crime scene.

Key Points

  • 1A DNA profile compares repeat sequences in non-coding DNA, not the whole genome
  • 2Steps: collect, extract, amplify by PCR, cut with restriction enzymes, separate by electrophoresis, compare
  • 3DNA is negatively charged so it moves to the positive electrode; short fragments travel furthest
  • 4Sequencing reads the actual base order; results are stored in databases such as GenBank and Ensembl
  • 5Ethical issues include consent, privacy, discrimination and who controls genetic data

Learning Outcomes

  • Outline what is meant by DNA profiling and its potential uses
  • Model the steps involved in generating a DNA profile
  • Outline the principle of DNA sequencing
  • Use a genome database to search for alleles known to cause specific genetic diseases
  • Discuss the ethical issues arising from advancements in genetic technologies