CHAPTER 22

22. 1 RECOMBINANT DNA TECHNOLOGY

CHAPTER REVIEW
Recombinant DNA Technology Recombinant dna tecnology

Application of Recombinant DNA Technology

rDNA technology
Tools RE & Palindromic sequence

Methods in gene cloning

Agriculture
Production of insulin Environment

Types Cloning vector & characteristics

Steps in gene PCR cloning

Host cell (bacteria) & characteristics Modifying enzyme & functions

Describe the characteristics of cloning vector Describe host cell (bacteria) and its characteristics Define modifying enzyme and its function

CLONING VECTOR

 Vector is a DNA molecule derived from a

Cloning vector...

plasmid/ bacteriophage/cosmids that carries gene of target / DNA molecule into the host cell
After restriction enzyme splits the

potential donor DNA into many fragments - the next step is to construct a recombinant DNA molecules by combining the isolated DNA fragment with DNA from another source

Characteristics

DNA cloning vectors should have : Ability to accept foreign DNA in multiple cloning sites (MSC) Ability to replicate freely in the host cell Posses selectable marker (resistance to antibiotics ampR) Ability to express cloned genes Contain an origin of replication/Ori)

HOST CELL

Host Cell...

organism that is used to receive a recombinant DNA (rDNA) for cloning purposes

the organism that can act for this function is bacteria (e.g : E. coli) and yeast.

Host Cell...
Characteristics

A host cell must be able to :

Receive DNA recombinant through

the transformation process Maintain the structure of the DNA recombinant from one generation to another Amplify the gene product from DNA recombinant.

Host Cell...

Example :

E. Coli bacteria - can be used as a host cell. A recombinant plasmid are taken into a E. Coli cells under certain conditions.

Host Cell...

How is the recombinant plasmid inserted into host cell?

Host Cell...
Host cell Recombinant plasmid Calcium chloride

To make the bacterial cell wall permeable to plasmid

heat
Treat the host cell with heat and presence of CaCl2 make the bacterial cell wall permeable to plasmid

MODIFYING ENZYME

What is modifying enzyme? Enzyme that join the target DNA fragment with the vectors.
Eg : DNA Ligase and Taq Polymerase

Modifying enzyme
DNA Ligase

Join the separate DNA strands together by catalyzing the reformation of phosphodiester bonds between them.
Involve in DNA replication & rDNA formation

Modifying enzyme
Taq Polymerase

A heat stable type of DNA polymerase Add along in PCR process Copy the rest of the fragment in DNA replication, beginning from the primer Isolated from bacteria living in hot springs