Bioinorganic Chemistry: Site-Directed Mutagenesis and Recombinant DNA Technology, Study notes of Inorganic Chemistry

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Bioinorganic Chemistry

CHEM-

Site-Directed

Site-Directed

Mutagenesis

Mutagenesis

Recombinant DNA

Recombinant DNA

Identification and

isolation of desired

segment in DNA

Restriction

endonucleases open

the DNA in the vector

DNA ligase inserts the

gene and closes the

DNA

Recombinant DNA

introduced into host

cells

Cell replication

produces recombinant

DNA and translated

proteins

1. Locate specific cleavage sites at the

beginning and end of the gene of interest.

2. Treat the vector and DNA with the same

nucleases to create a compatible opening.

3. Covalently couple the fragments using DNA

ligase.

4. Insert mutated DNA into cells to replicate

(transformation).

5. Isolate desired DNA segments and the desired

proteins for further studies and use.

Identification and

isolation of desired

segment in DNA

Restriction

endonucleases open

the DNA in the vector

DNA ligase inserts the

gene and closes the

DNA

Recombinant DNA

introduced into host

cells

Cell replication

produces recombinant

DNA and translated

proteins

DNA splicing

Restriction endonucleases are enzymes that

have become exceptional tools for cutting DNA

at specific nucleotide sequences.

These enzymes are able to cut both strands of

DNA at specific palindrome sequence across the

double helix.

5’-GGATCC-3’

3’-CCTAGG-5’

5’-RGATCY-3’

3’-YCTAGR-5’

where

R = A or G and

Y = C or T

BamHI BstYI EndonucleasesEndonucleases:

Palindrome Palindrome

Sequences Sequences

Restriction enzymes Restriction enzymes cleave DNA at specific sites.

DNA

splicing

Recognition site Recognition site

Use of nucleases to cleave

DNA at specific recognition

sites

An artificial self-

replicating circular

DNA vector

Contains a selectable

marker for

ampicilin ampicilin

resistance resistance.

Contains a

polycloning site for

gene insertion

Identification and

isolation of desired

segment in DNA

Restriction

endonucleases open

the DNA in the vector

DNA ligase inserts the

gene and closes the

DNA

Recombinant DNA

introduced into host

cells

Cell replication

produces recombinant

DNA and translated

proteins

Modified DNA

segment

Mutated protein

G

G

enetically

enetically

M

M

odified

odified

O

O

rganisms

rganisms

Once the gene for

human insulin was

identified, the

segment that codes

for insulin was cut

from the chromosome

and recombined with

a cut circular DNA

plasmid.

The plasmid is

inserted into a

suitable bacterium

and production starts

as the gene is

Human Human

Insulin Insulin

Recombinant DNA Technology Recombinant DNA Technology

There are may applications of

recombinant DNA technology.

For an overview of this technology

consult:

Recombinant RNA technology

For an overview of applications of this

technology consult:

Applications of Recombinant RNA Tech

nology

Antibiotic Resistance Antibiotic Resistance

It is not possible to have

100% of the bacteria

containing the recombinant

DNA.

In order to avoid competition

a specific antibiotic is used.

The recombinant DNA

besides having the desired

gene, it has a gene that

makes the bacteria

resistant to a specific

antibiotic.

Only colonies having the recombinant DNA

survive and reproduce.