Application of genetic III

Recombinant DNA technology
The principal:
genetic code for protein production is universal, gene inserted into foreign host cell can still express.

Basic steps:

1. locate specific gene/use DNA probe
2. isolate this gene
3. multiply this gene by polymerase chain reaction
4. insert the gene in vector
5. introduce the gene into host cell in which gene express strongly

limii

1.Locating
--DNA probe

• It is a short strand of DNA that carry part of the base sequence of wanted.
• It will attach to the complementary base sequence in the gene.
• It is radioactive or fluorescent to act as marker.

--Reverse transcription

• mRNA act as a template.
• after complementary DNA is formed from mRNA, mRNA is removed and double strand DNA is made by adding enzyme polymerase and DNA nucleotide
• e.g. retrovirus(HIV e.t.c.)

--Artificial DNA synthesis

• human use DNA polymerase to join nucleotides
• useful for small protein which have short base sequence

2.Isolation
Enzyme/detergent break down cell walls and membranes
Cell debris removed by filtering or cetrifugation
Obtained DNA treated with protease to get pure protein

Enzymes:
Restriction endonucleases
--Cut DNA strands
DNA ligase
--join DNA strands

3.Transferring

• sorting
• multiplication
• insertion
• introduction

Sorting:
gel electrophoresis
--fragment move toward anode
--larger the fragment, more slowly they move

Multiplication
polymerase chain reaction
--automated, rapid and efficient

Insertion
Insert gene a vector which is for transporting DNA into host cell
Types of vector:

1. plasmid= circular ring of DNA found in bacteria
   
2. phages= virus
   
3. cosmid= hybrid of phages
   

Most commonly used is plasmid.
It contain gene for antibiotic resistance where restriction endonucleases are used to break the plasmid loop.
Introduction
plasmids re-introduce into bacterial cell= transformation
Antibiotics are used to identify the cell process the desired gene.

Advantage of recombinant DNA tech

1. Product is pure, work more efficient and less likely to be rejected
2. Large quantity, less expansive
3. Flexible process, made as wanted

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Application of genetic engineering

• microorganism used to control pollution
• GM plant
• transgenic animal
• gene therapy
• genetic fingerprinting

Implications of Genetic engineering

• unpredictable ecology consequence