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