Lecture outline

DNA Technology

• gene cloning- preparation of well-defined, gene-sized DNA in multiple identical copies
• recombinant DNA- DNA in which genes from 2 sources are linked
• plasmids- small circular DNA molecules that replicate in bacterial cells
• restriction enzymes- enzymes that cut DNA molecules at a specific nucleotide sequence
• restriction site- recognition sequence for a specific restriction enzyme
• DNA ligase- enzyme that joins together DNA molecules
• construction of a DNA library
  • genomic library- complete set of thousands of recombinant clones each carrying a copy of a particular segment from the initial genome
  • cDNA library- library of DNA molecules complementary to mRNA molecules produced by a cell
  • can use plasmids or bacteriophage as cloning vectors
  • identification of a specific gene or segment of DNA in a library- use a radioactively labeled piece of DNA (probe) with a sequence similar to the one you're looking for
• PCR- polymerase chain reaction- technique by which any piece of DNA can be amplified without using cells
• analysis of DNA
  • gel electrophoresis- method to separate molecules based on size
  • Southern blotting- can be used to detect a specific DNA among a mixture of DNA molecules or to detect differences in DNA between individuals
  • DNA sequencing- determination of sequence of nucleotides in a DNA molecule
• Practical applications of DNA technology
  • diagnosis of disease
    • use of PCR to amplify HIV DNA
    • genetic test for hemophilia, cystic fibrosis
    • DNA repair gene involved in some cancers
    • genes active in prostate tumors
    • gene involved in inherited blindness
  • gene therapy - replace or supplement defective allele with functional, normal allele
  • pharmaceutical products- use recombinant DNA to obtain large amount of protein product of a gene
  • forensic uses of DNA technology- RFLP analysis, analysis of variation in length of satellite DNA
  • environmental uses of DNA technology- engineering of microorganisms that can degrade harmful compound into non-toxic forms
  • agriculture- genetic engineering of plants- pesticide resistance, control of ripening

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