Friday, June 12, 15. Biotechnology Tools

Transcription

1 Biotechnology Tools

2 Biotechnology: Tools and Techniques Science of biotechnology is based on recombining DNA of different organisms of another organism. Gene from one organism spliced into genome of another organism Biotechnology relies on three (3) naturally occuring classes of molecules that cut and splice genes 1. Restriction Endonucleases 2. Methylases 3. DNA ligase

3 Biotechnology: Tools and Techniques 1. Restriction Endonucleases Also known as restriction enzymes, these are molecular scissors that can cut double stranded DNA at specific base-pair sequences Each restriction enzyme recognizes a specific sequence of nucleotides known as a recognition site Most recognition sites are 4-8 nucleotides in length and are usually complimentary palindromic sequences What is a palindrome? radar radar kayak kayak madam madam

4 Table 1. List of Restriction Enzymes and Respective Recognition Sites Microorganism of Origin Escherichia coli Serratia marcescens Arthobacter luteus Streptomyces albus Haemophilus parainfluenae Sticky ends Enzyme Recognition Site After Restriction Enzyme Digestion EcoRI 5 -GAATTC CTTAAG G AATTC CTTAA G- 5 SmaI 5 -GGGCCC CCCGGG GGG 3 -CCC AluI 5 -AGCT TCGA AG 3 -TC SalI 5 -GTCGAC CAGCTG G TCGAC CAGCT G- 5 HindIII 5 -AAGCTT TTCGAA A AGCTT TTCGA A- 5 short single stranded overhangs can be easily rejoined (annealled) Blunt ends CCC- 3 GGG- 5 CT- 3 GA- 5 no single stranded overhangs make segments difficult to anneal

5 Biotechnology: Tools and Techniques 2. Methylases One of roles of restriction enzymes in bacteria is to protect them from infection by viruses Bacteriophage (virus) viral DNA E. coli bacterium Virus injects its own DNA into bacterium in order to reproduce restriction enzyme EcoRI Bacterial DNA

6 Biotechnology: Tools and Techniques 2. Methylases One of roles of restriction enzymes in bacteria is to protect them from infection by viruses viral DNA fragments Bacteriophage lacking DNA E. coli bacterium Restriction enzyme is able to cut up viral DNA before it infects bacterial DNA

7 Biotechnology: Tools and Techniques 2. Methylases One of roles of restriction enzymes in bacteria is to protect them from infection by viruses Enzymes are able to add a methyl side group (-CH3) to specific recognition sites Site changes shape - blocking action of restriction enzyme Group of enzymes called methylases are able to protect bacterial DNA

8 Biotechnology: Tools and Techniques 3. DNA ligase DNA ligase is used to rejoin phosphodiester bonds that were broken by restriction enzyme They can take a gene which has been cut-out of one organism and splice it into DNA of another DNA ligase works best when sticky ends are available

9 Bacteria have a large circular chromosome as well as many smaller circular structures called plasmids Plasmids are an important tool in gene splicing 1 µm

10 Human cell Gene Splicing to Produce Insulin Restriction enzyme must be specially chosen to cut on either side of insulin gene Insulin gene DNA 1. Human DNA cut into fragments using restriction enzyme Many fragments made, but one will have insulin gene

11 Gene Splicing to Produce Insulin complimentary ends Using same restriction enzyme means that plasmids and fragments have complimentary ends Antibiotic resistance gene is important for a later process Antibiotic resistance gene 1. Human DNA cut into fragments using restriction enzyme Many fragments made, but one will have insulin gene 2. Plasmids are cut with same restriction enzyme as in step1 Plasmids also contain antibiotic resistance gene

12 Some recombinant plasmids will contain insulin gene, and some will not 3. Mix DNA fragments, cut plasmids and DNA ligase, to produce recombinant DNA plasmids

13 Gene Splicing to Produce Insulin 4. Through transformation, recombinant plasmids enter bacterial cells As bacteria divide, billions of copies of recombinant plasmids are made Some of these bacteria will make insulin

14 Gene Splicing to Produce Insulin Hybridization requires colonies to be antibiotic resistant 5. Process called hybridization is used to identify bacterial colonies that produce insulin These colonies will be isolated and given optimal conditions needed for producing large quantities of insulin