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

2. 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.

3. 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

4. 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

5. 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

6. 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.

7. 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

8. 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

9. 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

10. Vector DNA - Plasmids

11. Vector DNA - Plasmids

12. 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

13. TOPO TA cloning kits

14. 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

15. 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)

16. 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

17. 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)

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

19. 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

20. TOPO TA cloning kit

21. References