Molecular Genetics

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Techniques for accomplishing DNA manipulations for a variety of purposes are included in the term molecular cloning. Molecular cloning for modification of cells and organisms is covered elsewhere. This section concentrates on techniques for production of molecules (DNA, protein) for further molecular study.

Efficient molecular cloning has five requirements.

• A host organism is needed.
  • E. coli is a frequently used bacterial host because decades of research have made it a well understood and conveniently manipulable organism.
  • S. cerevisiae is a frequently used eucaryotic host that retains many of the convenient features of E. coli.
  • Other hosts include Picchia pastoris (another yeast) and cultured cells of Spodoptera frugiperda (an insect; for use with baculoviruses).
• A vector DNA carries the payload DNA into the host cell and facilitates its replication in that cell.
• A means of placing DNA within the vector such the the payload and vector DNAs are linked covalently. The method used depends on the application:
  • ligation of DNA fragments into restricted vector DNA;
  • exchange of engineered DNA into vector DNA by homologous recombination.
• A method of placing the in vitro modified (engineered) DNA into the host cell is needed. Most commonly used are:
  • transformation;
  • transfection, the in vitro packaging of the DNA into phage particles then used to infect the cells;
  • electroporation.
• Methods for selection and/or screening allow recovery of cells with the desired constructs.
  • Selection refers to applying conditions that allow the desired cells or phages (containing vector or vector and insert) to replicate while preventing others from replicating. Typical selections include:
    • antibiotic sensitivity and resistance;
    • nutrient requirements;
    • plaque formation.
  • Screening allows all cells to grow, but tests the resulting clones for particular properties. Often these are indicative of the presence of an insert in the vector. Screening properties used include:
    • antibiotic sensitivity and resistance;
    • nutrient requirements ;
    • plaque type;
    • blue-white selection (b-galactosidase activity);
    • specific (by nucleic acid hybridization or antibodies).
• • isolation of specific DNA for use as probes, for restriction mapping, for nucleotide sequencing and for reintroduction into organisms.
  • establishment of collections of cells or viruses each containing a different segment of a large DNA population and collectively containing a representative of almost every sequence in the population (libraries).
  • production of quantities of specific proteins for further study (expression of proteins).

Further information

• Transformation and transfection have different meanings when applied to mammalian cells in culture.
• There are many purposes for molecular cloning.