Biotechnology Tools

Friday, June 12, 15

Biotechnology: Tools and TechniquesScience of biotechnology is based on recombining DNA of different organisms of another organism.Gene from one organism spliced into genome of another organismBiotechnology relies on three (3) naturally occuring classes of molecules that cut and splice genes

1. Restriction Endonucleases

2. Methylases

3. DNA ligase

Friday, June 12, 15

Biotechnology: Tools and Techniques1. Restriction Endonucleases

What is a palindrome?

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Also known as restriction enzymes, these are “molecular scissors” that can cut double stranded DNA at specific base-pair sequencesEach restriction enzyme recognizes a specific sequence of nucleotides known as a recognition siteMost recognition sites are 4-8 nucleotides in length and are usually complimentary palindromic sequences

Friday, June 12, 15

5’ -GAATTC- 3’

3’ -CTTAAG- 5’

5’ -G AATTC- 3’3’ -CTTAA G- 5’

5’ -GGGCCC- 3’

3’ -CCCGGG- 5’ 3’ -CCC GGG- 5’5’ -GGG CCC- 3’

5’ -AGCT- 3’3’ -TCGA- 5’

5’ -AG CT- 3’3’ -TC GA- 5’

5’ -GTCGAC- 3’3’ -CAGCTG- 5’

5’ -G TCGAC- 3’3’ -CAGCT G- 5’

5’ -AAGCTT- 3’3’ -TTCGAA- 5’

5’ -A AGCTT- 3’3’ -TTCGA A- 5’

Microorganism of Origin Enzyme Recognition Site After Restriction Enzyme Digestion

Escherichia coli EcoRI

Serratia marcescens SmaI

Arthobacter luteus AluI

Streptomyces albus SalI

Haemophilus parainfluenae HindIII

Table 1. List of Restriction Enzymes and Respective Recognition Sites

“Sticky ends” “Blunt ends”short single stranded overhangs can be easily rejoined (annealled)

no single stranded overhangs make segments difficult to anneal

Friday, June 12, 15

Virus injects its own DNA into

bacterium in order to reproduce

2. Methylases

Biotechnology: Tools and Techniques

Bacteriophage (virus)

viral DNA

Bacterial DNArestriction enzymeEcoRI

E. coli bacterium

One of roles of restriction enzymes in bacteria is to protect them from infection by viruses

Friday, June 12, 15

One of roles of restriction enzymes in bacteria is to protect them from infection by viruses

Bacteriophage lacking DNA

2. Methylases

Biotechnology: Tools and Techniques

Restriction enzyme is able to “cut up” viral DNA before it infects bacterial

DNA

E. coli bacterium

viral DNAfragments

Friday, June 12, 15

Enzymes are able to add a methyl side group (-CH3) to specific recognition sites Site changes shape - blocking action of restriction enzyme

2. Methylases

Biotechnology: Tools and Techniques

Group of enzymes called methylases are able to protect bacterial DNA

One of roles of restriction enzymes in bacteria is to protect them from infection by viruses

Friday, June 12, 15

3. DNA ligase

Biotechnology: Tools and Techniques

DNA ligase is used to rejoin phosphodiester bonds that were broken by restriction enzyme

DNA ligase works best when “sticky ends are available”

They can take a gene which has been “cut-out” of one organism and “splice” it into DNA of another

Friday, June 12, 15

1 µm

Plasmids are an important tool in gene splicing

Bacteria have a large circular chromosome as well as many smaller circular structures called plasmids

Friday, June 12, 15

Gene Splicing to Produce Insulin

Many fragments made, but one will have insulin gene

Restriction enzyme must be specially

chosen to cut on either side of insulin gene

1. Human DNA cut into fragments using restriction enzyme

Human cell

DNA

Insulin gene

Friday, June 12, 15

Gene Splicing to Produce Insulin

Many fragments made, but one will have insulin gene1. Human DNA cut into fragments using restriction enzyme

Plasmids also contain antibiotic resistance gene

Using same restriction enzyme means that plasmids and fragments have complimentary ends

Antibiotic resistance gene is important for a later process

complimentaryends

Antibiotic resistance gene

2. Plasmids are cut with same restriction enzyme as in step1

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3. Mix DNA fragments, cut plasmids and DNA ligase, to produce recombinant DNA plasmids

Some recombinant plasmids will contain

insulin gene, and some will not

Friday, June 12, 15

Gene Splicing to Produce Insulin

4.

Some of these bacteria will make insulin

Through transformation, recombinant plasmids enter bacterial cellsAs bacteria divide, billions of copies of recombinant plasmids are made

Friday, June 12, 15

These colonies will be isolated and given optimal conditions needed for producing large quantities of insulin

Gene Splicing to Produce Insulin

5. Process called hybridization is used to identify bacterial colonies that produce insulin

Hybridization requires colonies to be

antibiotic resistant

Friday, June 12, 15