Download Introduction to molecular cloning

Technique involving the insertion of a
fragment of foreign DNA into a vector
capable of replicating autonomously in a
host cell (usually Escherichia coli). Growing
the host cell allows the production of
multiple copies of the inserted DNA for use
in a variety of purposes.
 Foreign DNA
 Host organism
 Vector DNA for cloning
 Means of inserting foreign DNA into the vector
 Method of placing the in vitro modified DNA
into the host cell
 Methods for selecting and/or screening cells that
carry the inserted foreign DNA
Restriction Endonucleases
Polymerases
DNA Polymerase – catalyzes the polymerization of
deoxyribonucleotides along the template strand
DNA-dependent RNA Polymerase
Nucleases
Enzymes capable of cleaving the phosphodiester bonds
between nucleotide subunits of nucleic acids
Other Modifying Enzymes
Ligases
forms phosphodiester bonds to join two pieces of DNA
utilizes ATP in the presence of Mg++
Kinases
transfers phosphate groups from donor molecules
phosphorylase
Phosphatases
catalyzes the removal of 5’-phosphate residues
 Foreign DNA
PCR product
genomic DNA
complementary DNA (cDNA)
 Host organism
bacterial host – E. coli
eukaryotic host – yeast (Saccharomyces cerevisiae)
other hosts – other yeasts, insect cells, etc.
 Vector DNA
DNA molecule that functions as a “molecular carrier”
that carry the DNA of interest into the host cell & facilitates
its replication.
Plasmids – used in cloning small segments of DNA (10-15 kb)
Bacteriophage λ – used in cloning larger segments of DNA
(~20 kb)
Cosmids – plasmids containing DNA sequences (cos) from
bacteriophage λ used to clone larger fragments
of up to 45 Kb
• small circular dsDNA that autonomously replicates
apart from the chromosome of the host cell
• “molecular parasites”
• carry one or more genes some of which confer
resistance to certain antibiotics
• origin of replication (ORI) --- a region of DNA that
allows multiplication of the plasmid within the
host
Desirable properties of plasmids:
 small size
 known DNA sequence
 high copy number
 a selectable marker
 a second selectable gene
 large number of unique restriction sites
 Means of inserting foreign DNA into the vector
Ligation of the DNA into the linearized vector
Requirements for a ligation reaction:
• two or more fragments of DNA (blunt/cohesive)
• buffer containing ATP
• T4 DNA ligase
 Method of placing the in vitro modified DNA
into the host cell
Transformation into the host cell
• bacterial cells take up naked DNA molecules
• cells are made “competent”
• cells treated with ice-cold CaCl2 then heat-shocked
• efficiency of 107 to 108 transformed colonies/μg DNA
• maximum transformation frequency of 10-3
• “electric field-mediated membrane
permeabilization”
• high strength electric field in the
presence of DNA
• protocols differ for various species
• efficiencies of 109 per μg DNA (3 kb)
& 106 (136 kb)
http://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpg
Electroporation of the DNA into the host cell
 Methods for selecting and/or screening cells that
carry the inserted foreign DNA
Selection refers to application of conditions that
favors the growth of cells or phages that
carry the vector or vector and insert.
• antibiotic resistance
• nutrient requirements
Screening allows all cells to grow, but tests the
resulting clones for the presence of the insert
in the vector.
• antibiotic resistance/sensitivity
• nutrient requirements
• blue-white selection (β-galactosidase)
• specific (hybridization, antibodies, PCR)
Positive selection
disruption of the lacZα-ccdB gene
permits growth of positive recombinants
 DNA isolation for:
making probes
restriction mapping
sequencing
reintroduction into organism
 Establishment of collections: DNA Libraries
 Further molecular studies: production of special
proteins
http://www.bio.indiana.edu/~chenlab/potocols/MolecularClonging.htm
http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html
http://www.protocol-online.org/forums/index.php?showforum=30
http://bioresearch.ac.uk/browse/mesh/D003001.html
http://www.jax.org/~jcs/techniques/techniques.html
http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/clones.html
http://opbs.okstate.edu/~Melcher/MG/MGW4/MG428.html
http://www-micro.msb.le.ac.uk/109/GeneticEngineering.html
http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer
/cloningvectors.html