Download II. Principles of Cell

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