Biotechnology: Tools and Techniques

Transcription

1 Biotechnology Tools

2 The science of biotechnology is based on recombining the DNA of different organisms. That is, a gene from one organism is spliced into the 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 1. Restriction Endonucleases Also known as restriction enzymes, these are found in bacteria and act as 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? radarradar radar kayakkayak kayak madam madam madam

4 Table 1. List of Restriction Enzymes and Their Respective Recognition Sites. Microorganism of origin Enzyme Recognition Site After restriction enzyme digestion Escherichia coli EcoRI 5 -GAATTC CTTAAG G AATTC CTTAA G- 5 Serratia marcescens SmaI 5 -GGGCCC GGG CCC CCCGGG CCC GGG- 5 Arthobacter luteus AluI 5 -AGCT TCGA AG CT TC GA- 5 Streptomyces albus SalI 5 -GTCGAC CAGCTG G TCGAC CAGCT G- 5 Haemophilus parainfluenae HindIII 5 -AAGCTT TTCGAA A AGCTT TTCGA A- 5 Sticky ends short single stranded o v e r h a n g s c a n b e e a s i l y r e j o i n e d Blunt ends no single stranded overhangs makes the segments difficult to anneal.

5 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 kayak madam radar kayak madam

6 2. Methylases One of the roles of restriction enzymes in bacteria is to protect them from infection by viruses. The enzymes are able to add a methyl side group (-CH3) to specific recognition sites. This changes the shape of the site, Bacteriophage (virus) blocking the action of the restriction enzyme. E. coli bacterium restriction enzyme EcoRI viral DNA Bacterial DNA The virus injects its own DNA into the bacterium in order to reproduce.

7 2. Methylases One of the roles of restriction enzymes in bacteria is to protect them from infection by viruses. The enzymes are able to add a methyl side group (-CH3) to specific Bacteriophage lacking DNA recognition sites. This changes the shape of the site, blocking the action of the restriction enzyme. E. coli bacterium viral DNA fragments The restriction enzyme is able to cut up the viral DNA before it infects the bacterial DNA.

8 2. Methylases One of the roles of restriction enzymes in bacteria is to protect them from infection by viruses. The enzymes are able to add a methyl side group (-CH3) to specific recognition sites. This changes the shape of the site, blocking the action of the restriction enzyme. Why isn t the bacterium s own DNA damaged? A group of enzymes called methylases are able to protect the bacterial DNA.

9 3. DNA ligase DNA ligase is used to rejoin the phosphodiester bonds that were broken by the restriction enzyme. As a result they can take a gene which has been cut-out of one organism and splice it into the DNA of another. DNA ligase works best when sticky ends are available.

10 Restriction Enzyme EcoR1 Animation by: Drew Berry 2003 HHMI The Walter and Eliza Hall Institute Dolan DNA Learning Center

11 Read pgs Do Q#1-4 pg. 375