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Chapter 4 Cell-based DNA Cloning I. Basics of DNA technology: Current DNA technology is based on two different approaches: 1. DNA cloning: DNA fragment is selectively amplified 2. Molecular Hybridization: detecting the location of a specific gene using complementarity rules of DNA:DNA, DNA:RNA and RNA:RNA hybrid molecules Two different DNA cloning approaches: • Cell-based DNA cloning. This is an in vivo method where a DNA fragment is cloned in a vector then transformed into a host. The host then propagates and while doing so amplifies the DNA fragment along. • Cell-free DNA cloning. Using PCR II. Principles of Cell-Based DNA Cloning: • Cell-based cloning DNA cloning requires 4 steps: 1. Construction of recombinant DNA molecules 2. Transformation 3. Selective propagation of cell clones 4. Isolation of recombinant DNA clones • Propagation of a DNA fragment cloned in a vector requires that the vector contains extrachromosomal replicons. Examples are 1. Plasmids (plasmids containing F factor and drug resistance genes) 2. Bacteriophages • Type II Endonuclease Restriction enzymes, a must in DNA cloning that cut double stranded DNA sequences at palindromic sequences (sites where the sequence of bases is the same on both strands when read in the 5’ ----> 3’ direction. Two types of endonuclease restriction enzymes based on how they cut DNA: 1. Blunt-ended 2. Sticky ends or Cohesive termini • DNA ligase catalyzes the formation of a phosphodiester bond in a DNA strand by connecting a 5’ phosphate of a nucleotide and the 3’ -OH group of a neighboring nucleotide. This is the cloning enzyme that links a DNA fragment (gene) into a cloning vector creating a recombinant vector. • Plasmid vectors that have been linearized by cutting with a restriction enzyme can always re-circularize before ligating the foreign DNA fragment. Two things can be done to prevent this from happening: 1. Cutting vectors with two different restriction enzymes. 2. Vector dephosphorylation (removal of the 5’ phosphate from both ends of the linearized plasmid DNA). • Upon ligation and creating a recombinant vector, transformation of a bacterial cell (or tansfection of an animal cell) is needed to propagate the recombinant vector. • DNA libraries are a comprehensive set of clones (bacterial or phages) that represent the full chromosome complement of an organism or the expressed genes in a particular developmental stage (complementary DNA library) Two type of libraries: 1. Genomic DNA library: the complexity of the library is the number of independent DNA clones and is defined by genome equivalents (GE). A GE value of 1 is reached when number of independent clones = genome size/average size insert For a human genomic DNA library of 40 kb average insert size 1 GE = 3000 Mb/40 kb = 75,000 independent clones (1-fold library) To have a high chance of recovering a specific gene the GE should higher than one (e.g. 4 GE) 2. cDNA libraries: mRNA expressed from protein-coding genes is purified from a specific tissue or developmental stage then reverse transcribed into its complementary DNA (cDNA). Then latter only contains the exons of the expressed protein-coding genes and is cloned in a suitable vector. • For successful cell-based DNA cloning two points have to be met: 1. A method to select the host cells containing the recombinant molecule. This is achieved by: - antibiotic resistance genes in the vector or - -galactosidase gene compolementation where the vector provides a functional -galactosidase gene which is missing from the host chromosome. 2. Then a second level of selection that follows and that identifies the host cells containing a recombinant vector (with the desired cloned gene). This is achieved by: - by using insertional inactivation of the marker gene. E.g. -galactosidase gene. - by insertional inactivation of a suppressor tRNA genes III. Vector Systems for Cloning different sizes DNA Fragments: • Plasmid Vectors: - insertion of a multiple cloning site polylinker - insertion of an antibiotic resistance gene - insertion of a selection system for screening recombinants. - disadvantage of a plasmid vector is that it can only accommodate 5-10 kb inserts. • Replacement vectors: - can accommodate DNA fragments of up to 23 kb long • Insertion vectors: - used for cDNA libraries and can accommodate cDNA <5 kb long • Cosmid vectors: - contain cos sequences inserted into a plasmid vector - can accommodate large fragments (30-44 kb) • Large DNA fragments can be cloned in bacterial cells using - F factor plasmids (low copy number 1-2 copies per cell) can accommodate 300 kb fragments - Bacteriophage P1 vectors & PACs: P1 can accommodate up to 100 kb DNA fragments while PAC can accommodate up to 122 kb • Yeast Artificial Chromosomes (YACs) - contains 3 important sequences: centromeres, telomeres, and autonomous replicating sequences (ARS) - can accommodate up to 2 Mb DNA fragments • cDNA expression libraries are used for immunological screening