1. Lecture # 04
Cloning Vectors

2. Recombinant DNA Technology
Common General Cloning Strategy
Target DNA from donor organism extracted, cut with compatible restriction endonuclease and ligated into a cloning vector.
Recombinant construct transferred into host cell.
Host cells which do not take up construct are eliminated by selection protocol
Host cell library screened to identify desired clone.

3. Continue!!
Restriction
Endonuclease
DNA Ligase
transformation

4. Cloning Vectors
The molecular analysis of DNA has been made possible by the cloning of DNA.
The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a host (bacterial or yeast) cell and produces many copies of itself and the foreign DNA.

5. Essential features of all cloning vectors
Ideally they should be fairly small DNA molecule.
(to facilitate isolation and handling)
Origen of replication: sequences that permit the propagation of itself in host cell.
A cloning site to insert foreign DNA; The vectors contain a site that can be cut by unique restriction enzymes.
Some sort of selectable markers: to select host cell containing a vector with foreign DNA; usually accomplished by selectable markers for drug resistance

6. Types of Cloning Vectors
Plasmid - an extrachromosomal circular DNA molecule that autonomously replicates inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or kilobases (kb)
Phage - derivatives of bacteriophage lambda; linear DNA molecules, whose region can be replaced with foreign DNA without disrupting its life cycle; cloning limit: 8-20 kb
Cosmids - is a combination of plasmids and phage; cloning limit kb
Bacterial Artificial Chromosomes (BAC) - based on bacterial mini-F plasmids. cloning limit: kb (350kb in some books).
Yeast Artificial Chromosomes (YAC) - an artificial chromosome that contains telomeres, origin of replication, a yeast centromere, and a selectable marker for identification in yeast cells; cloning limit: kb (3000kb in some books)

7. General Steps of Cloning with Any Vector
prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends.
ligate the foreign DNA into the vector with the enzyme DNA ligase
introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation
select cells containing foreign DNA by screening for selectable markers (usually drug resistance)

8. Cloning Tools Restriction endonucleases Ligase Vectors Host
Methods for introducing DNA into a host cell

9. Cutting DNA Restriction endonucleases (restriction enzymes)
sticky ends
blunt ends
Nomenclature
EcoRI
E = genus (Escherichia)
co = species (coli)
R = strain
I = # of enzyme

10. Blunt & Sticky ends

11. Pasting DNA Complementary ends (sticky ends) H-bond
Ligase forms phosphodiester bond to seal strands together.

12. What determines the choice vector?
insert size
vector size
restriction sites
copy number
cloning efficiency
ability to screen for inserts

13. How to clone DNA (Summary)
Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest)
Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase
Introduction of cloning vector into cells (transformation by bacterial cells)
Cloning of cells (and foreign genes)
Identification of cell clones carrying the gene of interest

14. End
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