DNA Technology Notes

DNA Technology: An Overview

  • Dolly: The first cloned mammal, created as an exact copy of its mother.
  • DNA technology involves manipulating DNA to:
    • Copy and sequence DNA.
    • Turn genes on and off.
    • Locate specific DNA pieces.
    • Transfer DNA between species.

Applications of DNA Technology

  • DNA technology is used to:
    • Study evolution.
    • Cure diseases.
    • Enforce laws.
  • Tools of DNA Technology:
    • Recombinant DNA
    • DNA sequencing
    • Polymerase chain reaction (PCR)
    • DNA profiling
  • Applications in Research:
    • Stem cell research
    • Cloning
  • Applications in Medicine:
    • Genetic testing
    • Gene therapy

Transgenic Organisms

  • Transgenic organism: An organism whose genome contains recombinant DNA (genetic material from another species).
  • Uses:
    • Manufacturing pharmaceuticals.
    • Engineering hardy crops.
    • Testing human genetic diseases in mice.
  • Examples of Genetically Modified Foods:
    • Corn
    • Soy
    • Cotton
    • Papaya
    • Rice
    • Rapeseed (Canola)
    • Potatoes
    • Tomatoes
    • Dairy products
    • Peas

Creating Transgenic Organisms

  1. Obtain DNA: Isolate the DNA containing the gene of interest.
  2. Prepare a Plasmid: Use a plasmid (circular DNA) as a vector for cloning the new DNA.
  3. Create Recombinant DNA:
    • Enzymes cut the plasmid and the DNA containing the gene of interest (e.g., GFP gene).
    • DNA ligase joins the gene of interest with the plasmid DNA.
  4. Insert Recombinant DNA into Cells: Introduce the recombinant DNA into cells (e.g., by electroporation or heat shock).
  5. Transgenic Cells: Cells produce proteins encoded by the new genes because all organisms share the same genetic code.

DNA Sequencing

  • DNA sequencing reveals the exact sequence of bases in a DNA molecule.

  • Applications:

    • Defines protein sequences.
    • Determines evolutionary relationships among species.

  • How Biotechnologists Sequence DNA:

    1. Allow the Unknown DNA Sequence to Replicate: Replication occurs in a tube with all necessary ingredients.
    2. Add Terminator Nucleotides: These nucleotides halt DNA replication when incorporated into the strand.
      • Replication stops when a terminator nucleotide is added.
      • Results in DNA strands of various sizes.
    3. Transfer DNA Strands to a Sequencing Machine:
      • Capillary gel electrophoresis separates fragments by size.
    4. Gel Reveals the Order of Nucleotides:
      • The sequence is complementary to the original DNA sequence.

  • Fluorescent labels and computers simplify the DNA sequencing process.

Human Genome Project

  • The Human Genome Project sequenced all the DNA in a human and mapped approximately 30,000 genes.
  • Chromosome 1 contains the most genes (3168), while the Y chromosome has the fewest (344).
  • Chromosome 15
    • Hypertension, essential, susceptibility to
    • CLL/lymphoma, B-cell
    • Lymphoma, diffuse large cell
    • Necdin
    • Prader-Willi syndrome
    • Angelman syndrome
    • Hair color, brown
    • Spastic paraplegia
    • Limb deformity
    • Schizophrenia, neurophysiologic defect in
    • Isovalericacidemia
    • Spherocytosis, hereditary, Japanese type
    • Bartter syndrome
    • Amytrophic lateral sclerosis, juvenile recessive
    • Dyserythropoietic anemia, congenital, type III
    • Griscelli syndrome
    • Deafness, autosomal recessive
    • Hepatic lipase deficiency
    • Marfan syndrome
    • Shprintzen-Goldberg syndrome
    • Ectopia lentis, familial
    • Leukemia, acute promyelocytic, PML/RARA type
    • Cardiomyopathy, familial hypertrophic
    • Enhanced S-cone syndrome
    • Glutaricaciduria, type IIA
    • Epilepsy, nocturnal frontal lobe, type 2
    • PAPA syndrome
    • Diabetes mellitus, insulin-dependent
    • 100 million base pairs
    • Prader-Willi/Angelman syndrome (paternally imprinted)
    • Eye color, brown
    • Human coronavirus sensitivity
    • Albinism, oculocutaneous, type II and ocular
    • Andermann syndrome
    • Cardiomyopathy, dilated and familial hypertrophic
    • Epilepsy, juvenile myoclonic
    • Spinocerebellar ataxia
    • Microcephaly, primary autosomal recessive
    • Dyserythropoietic anemia, congenital, type I
    • Muscular dystrophy, limb-girdle, type 2A
    • Dyslexia
    • Amyloidosis, hemodialysis-related
    • Ceroid-lipofuscinosis, neuronal, late infantile
    • Gynecomastia, familial
    • Virilization, maternal and fetal
    • Colorectal cancer
    • Carbohydrate-deficient glycoprotein syndrome, type lb
    • Bardet-Biedl syndrome
    • Tay-Sachs disease
    • GM2-gangliosidosis
    • Tyrosinemia, type I
    • Mental retardation, severe
    • Hypercholesterolemia, familial, autosomal recessive
    • Retinitis pigmentosa, autosomal recessive
    • Otosclerosis
    • Bloom syndrome

Polymerase Chain Reaction (PCR)

  • Polymerase chain reaction (PCR): Uses enzymes to replicate small strands of DNA repeatedly, creating millions of copies in minutes.

  • Process:

    • Amplifies specific DNA sequences.
    • DNA polymerase enzyme makes DNA.
    • Thermus aquaticus: DNA polymerase enzyme tolerates high temperatures.
    • Primers: specific DNA segment to be amplified (only target DNA).
    • Cycles repeatedly doubling the amount of DNA at each cycle.

  • Steps:

    1. Sample is heated to denature DNA into single strands.
    2. DNA is cooled to allow annealing of primers.
    3. DNA is heated to 72∘C72^\circ C, the optimal temperature for Taq DNA polymerase to extend primers.

  • Cycle 2: 4 Copies

  • Cycle 3: 8 Copies

Question on PCR

  • If you start with one piece of DNA in a reaction tube, after five rounds of PCR, how many pieces of DNA will be in your reaction tube?
    • Answer: B. 32
    • Explanation: The formula for calculating the number of DNA pieces after nn rounds of PCR is 2n2^n. So, after 5 rounds, you would have 25=322^5 = 32 pieces of DNA.

DNA Profiling

  • Short Tandem Repeats (STRs): Short, repeated sequences of DNA unique to each individual.

  • Due to DNA profiling, the number of DNA exonerations has increased in the U.S. over the last couple of decades.

  • Steps for DNA Profiling:

    1. Extract DNA from a diploid cell from each of the three suspects.
    2. Amplify STR sequences using PCR.
    3. Sequence the amplified DNA to determine the number of repeats and generate each man's profile.

Stem Cells

  • Embryonic stem cells give rise to all cell types in the body.
  • Stem cells are important tools for biological research:
    • Potential to differentiate into other cell types.
  • Adult stem cells differentiate into a limited number of cell types:
    • Stem cells in bone marrow differentiate into all blood cell types.

Cloning

  • Two types of Cloning:

    • Reproductive cloning: reproduce exact copy of whole organism.
    • Molecular cloning: reproduce copies of genes or short fragments of DNA.

  • Cloning is common in bacteria

  • Cloning is less common in multicellular organisms.

  • Somatic Cell Nuclear Transfer:

  • Dolly: the first cloned mammal

Potential Uses of Stem Cells

  • Stroke
  • Baldness
  • Traumatic brain injury
  • Learning defects
  • Blindness
  • Alzheimer's disease
  • Deafness
  • Parkinson's disease
  • Missing teeth
  • Wound healing
  • Bone marrow transplantation (currently established)
  • Amyotrophic lateral sclerosis
  • Myocardial infarction
  • Muscular dystrophy
  • Diabetes
  • Multiple sites:
    • Spinal cord injury
    • Osteoarthritis
    • Rheumatoid arthritis
    • Crohn's disease
    • Cancers

Cloning Male Mammals

  • Question: Is it possible to create a clone of a male mammal?
  • Yes, the DNA inserted into an egg cell can be from a male or a female.

DNA Probes

  • DNA probe is a single-stranded sequence that is complementary to a known region of DNA.
    • test for cystic fibrosis allele probe is labeled with a fluorescent tag
    • detect the allele using the wavelength emitted by the probe

Genetic Diagnosis

  • Preimplantation genetic diagnosis:
    • DNA probes to reduce the odds of having a child with a genetic disease.
    • DNA from fertilized eggs (in vitro) is extracted and screened for harmful alleles.
    • Embryos with no diseases are candidates to be implanted in the women’s uterus.
  • Genetic Testing Can Detect Existing Diseases
    • DNA probes used to test for a genetic disease in a child or adult.

Gene Therapy

  • Gene therapy : replace a mutated gene in a person’s cells with a healthy gene
  • cystic fibrosis caused by a faulty gene that makes abnormal protein in lungs
  • healthy versions of the gene are placed inside of viruses:
    • patient inhales virus
    • viruses inject genetic material into lung cells
    • lung cells can now produce normal proteins

Question About Gene therapy

  • Suppose that Jan receives gene therapy for cystic fibrosis, via an inhaled virus. Could she pass the “repaired” DNA to her children?
  • C. No, the “repaired” DNA does not affect her eggs.

Gene Silencing with CRISPR

  • After the Cas9 enzyme is guided to the right location in the genome and cuts the DNA, the cell's natural repair mechanisms take over. The cut is fixed by non-homologous end joining.
  • This process is error-prone and does not perfectly replace the cut DNA, often resulting in an insertion or deletion mutation which silences the gene.

Gene Insertion with CRISPR

  • To insert a gene, the new gene is added into the original CRISPR plasmid. It is designed to line up perfectly with the cut DNA strands, so the cell uses a different technique, homology directed repair, to incorporate a new stretch of DNA into the genome.