DNA Cloning

  1. The use of DNA cloning and duplication is useful in forensics, in amplifying certain genes, and in duplicating rare pieces of DNA.
  2. To create recombinant DNA, restriction enzymes are used.
    • Restriction enzymes sever the phosphodiester bonds of a piece of DNA at specific sequences known as restriction sites.
    • Restriction fragments are the results of restriction enzyme processing and are the many pieces of "cut-up" DNA.
    • In many cases of restriction enzyme processing, sticky ends emerge, which are small extensions of nucleotides on both ends of a restriction fragment. This allows for the formation of hydrogen bonds to complementary segments of DNA.
    • DNA ligase is normally an enzyme used in DNA repair. In genetic engineering, it is used to seal the sticky ends to an actual DNA strand to form recombinant DNA. It catalyzes the formation of phosphodiester bonds.
    • The last vital component needed to form recombinant DNA is a cloning vector, which is a movable piece of DNA that can be inserted into the cell. Vectors can be bacterial plasmids, artificial chromosomes, or viruses.
  3. The generalized five steps to cloning DNA:
    1. Isolate a gene of interest.
    2. Process a vector, which is normally a plasmid, and the desired gene with restriction enzymes to produce sticky ends. Then allow DNA ligase to attach the restriction fragments with the gene of interest into the plasmid.
    3. Introduce the vector into the cells by making them competent, or able to accept the vector.
    4. Culture the cells and allow the cells to divide.
    5. Identify which cells contain the desired gene through the use of fluorescent tags. (Nucleic acid hybridization, which is the use of a nucleic acid probe that is complementary to the desired gene sequence to locate the desired gene.)
  4. Other techniques and methods in DNA cloning.
    1. The use of expression vectors, which hold the desired gene, but instead of cloning the entire sequence, it undergoes massive translation to produce the desired protein product.
      • This normally requires the removal of introns.
      • Sometimes, a fully processed mRNA molecule will be duplicated by reverse transcriptase to form complementary DNA or cDNA that bacteria can use.
    2. Instead of using bacterial cells, single-celled fungi or yeast cells may be used.
      • Eukaryotic cell use is advantageous because the mRNA can be translated normally and in the event that the protein product requires further chemical changes, the cell can modify the protein for it to function.
    3. The use of Yeast Artificial Chromosomes, which are vectors that can hold more DNA (longer strands).
    4. Better DNA insertion techniques such as electroporation and a DNA gun or microscopically thin needles.
  5. Genomic libraries of cloned DNA can be made to map a genome.
    • cDNA libraries allow further research into gene functions.
  6. The Polymerase Chain Reaction or PCR is a technique that can rapidly clone small segments of DNA.
    • It is done in a pool of nucleotides in a heated solution with a special enzyme known as Tac Polymerase, which can withstand the heat.
    • It is helpful to generate mass copies of a sequence that is rare or limited.
    • However, the amount of good copies are limited.

DNA Analysisf_s02gelelect.gif

  1. Gel-electrophoresis is a procedure that separates restriction fragments of DNA, molecules of protein, or nucleic acids by physical properties like length, size, charge, etc.
    • Southern Blotting is an additional process that helps identify the presence of a specific gene or sequence. This technique is also used to analyze genetic markers known as restriction fragment length polymorphisms, which are differences in DNA sequences on loci on specific chromosomes. These polymorphisms can then be used to generate a linkage map.c7_20_10_blotting.jpg
  2. Genomes are now being sequenced. The Human Genome Project was a major research effort that sequenced the entire human genome.
    • This information can be used to identify gene functions and identify the source of some genetic disorders.
    • Other genomes are also being mapped now of different species.
  3. Mapping the genome:
    • Linkage mapping can be used through genetic markers like restriction fragment length polymorphisms.
    • Physical mapping involves the ordering of restriction fragments through their lengths and overlaps to determine the original sequence. Chromosomal walking, which is the use of nucleic acid probes to identify known sequences along a chromosome is often used.
    • Two methods for DNA sequencing are used.
      1. The Sangar Method
      2. Whole Genome Shotgun Approach
  1. Gene expression can be studied using DNA microassays, or the "gene chip". This involves the use of inserting DNA onto a silicon chip and fluorescently tagged probes to identify if a gene is expressed. http://www.bio.davidson.edu/Courses/genomics/chip/chip.html
  2. Gene function can be studied through approaches like:
    1. In vitro mutagenesis, which is the introduction of mutations or changes on a target gene sequence to identify the resulting protein that is affected.
    2. RNA interference (RNAi) which is the introduction of double-stranded RNA molecules into a cell to disrupt a specific mRNA transcript and cause it to degrade to halt expression.
  3. New advances in research include:
    1. Proteomics- the study of protein sets
    2. Bioinformatics- the use of math and computer science to analyze biological/genetic data. The DNA microassay falls under this field.

Practical Applications of DNA Technology

  1. The genetic data that is gathered can be used to diagnose and identify genetic disorders.
  2. Some genetic disorders can even be treated through gene therapy, which is the correction of deformed genes in the bone marrow stem cells of affected patients. However, this technique has yet to be proven effective.
  3. Genetic engineering has allowed for the mass production of certain proteins that can be used to treat certain disorders like diabetes (insulin). Some new advancements may even bring about proteins that block off the receptors of viruses.
  4. DNA technology can be used in forensics.
    • The use of gel electrophoresis and Southern blotting detects differences in each individual's DNA. When DNA is processed via gel electrophoresis, the bands that emerge from a person's DNA is their DNA fingerprint.
      • Every persons' DNA is different due to single nucleotide polymorphisms, or simply mutations.
      • Another distinguishing feature are simple tandem repeats, which are simply repetitive sequences that occur throughout a chromosome at different amounts depending on the person.
    • Applications include:
      1. Criminal Evidence
      2. Parent-Child Connection
      3. Family Tree Identification/Evidence
  5. Environmentally, genetically modified organisms can be used to treat and clean-up wastes. Also, some can even aid in the extraction of certain compounds, which can be helpful for mining.
  6. In agriculture:
    1. Farm animals are being genetically modified to enhance their performance in terms of production. They may become transgenic organisms, which are organisms that contain DNA from a foreign species.
    2. Crops may become transgenic as well through the addition of genes such as pesticide resistance and more nutritious products using primarily a plasmid known as the Ti plasmid, which is taken from the Agrobacterium tumefaciens.
    3. Plants may be modified to be able to grow in any condition, such as in nitrogen poor soil.
  7. The potential dangers and problems of genetic engineering are threatening. Aside from ethical issues, which involve the debate between "messing" with nature and advancing human technology, biological dangers can emerge. Some genetically modified organisms may undergo some mutations and introducing a new disease in their products. Bacteria which are enhanced can escape and cause a plague. "Superweeds" (weeds that can resist pesticides, herbicides, etc.) can also break out if we are not careful.