Introduction to Molecular Cloning Pierriden A. Perez ABE Workshop Leeward Community College June 18, 2007

What is 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.

Requirements for Molecular Cloning 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

Enzymes used in molecular cloning 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

Enzymes used in molecular cloning 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

Requirements for Molecular Cloning 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.

Requirements for Molecular Cloning 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

Vector DNA - Plasmids 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

Vector DNA - Plasmids 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

Vector DNA - Plasmids

Vector DNA - Plasmids

Inserting the DNA into the vector 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

TOPO TA cloning kits

Transfer of DNA into the host cell 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

Transfer of DNA into the host cell Electroporation of the DNA into the host cell “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

Selecting/screening recombinants 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

Selecting/screening recombinants 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)

TOPO TA cloning kit Positive selection disruption of the lacZα-ccdB gene permits growth of positive recombinants

Why do we have to clone? DNA isolation for: making probes restriction mapping sequencing reintroduction into organism Establishment of collections: DNA Libraries Further molecular studies: production of special proteins

TOPO TA cloning kit

References 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