6.1 Biotechnological Tools and Techniques

1. 6.1 Biotechnological Tools and Techniques Recombinant DNA & Gel electrophoresis

2. Recombinant DNA • Cutting DNA fragments from different sources and recombining them together • Cutting DNA fragments from different sources and recombining them together • Purpose • To investigate genetic disorders • Production of drugs (ie. insulin)

3. What complications do you foresee? • Consider: • The size of DNA • Where to cut? • How to put back together?

4. 1. Restriction Endonucleases • Also known as restriction enzymes • Essentially are molecular scissors • Recognize a specific DNA sequence and cuts the strands at a particular position or “recognition site” • Isolated and purified only from bacteria • Name reflects which bacteria the enzyme originates • ie. EcoRI  Escherichia coli, strain R, 1st r.e. isolated HindII  Haemophilus influenzae, strain Rd, 2nd r.e.

5. 1. Restriction Endonucleases: Recognition site • Each restriction endonuclease recognizes its own specific recognition site (specific DNA sequence) • Usually 4-8 base pairs long, characterized by a complementary palindromic sequence

6. 1. Restriction Endonucleases: Function • Scans DNA and binds to its specific recognition sequence • Disrupts the phosphodiester bonds between particular nucleotides through a hydrolysis reaction • Hydrogen bonds of the complementary base pairs in between the cuts are disrupted • Result: 2 DNA fragments http://www.scq.ubc.ca/?p=249

7. 1. Restriction Endonucleases: DNA Fragment Ends • Different DNA fragment ends are produced after digestion by different restriction enzymes • Sticky ends: DNA fragment ends with short single-stranded overhangs (ie. EcoRI, HindIII) • Blunt ends: DNA fragment ends are fully base paired (ie. AluI)

8. 1. Restriction Endonucleases: DNA Fragment Ends (continued) Restriction site Animation Palindrome Fragment 2 Fragment 1 http://www.bio-rad.com/LifeScience/docs/Official_Crime_Scene_PowerPoint_Spring_2005_rev_B.ppt

9. How do we control the snips? • Consider: • What about the organisms own DNA? • Frequency of recognition sequences within the DNA sequence

10. 1. Restriction Endonucleases: Length of recognition sites • Longer recognition sites result in lower frequency of cuts • EcoRI  5’-GAATTC-3’ = ¼×¼×¼×¼×¼×¼ = 1/4096 • AluI  5’-AGCT-3’ = ¼ ×¼ ×¼ ×¼ = 1/256 • Higher frequency of cuts – may cut gene into several fragments • Lower frequency of cuts – may produce large fragments than desired

11. 1. Restriction Endonucleases: Methylases • Enzymes that add a methyl group to a nucleotide in a recognition site to prevent restriction endonuclease from cutting DNA • Distinguishing between foreign (viral) DNA and bacteria’s own DNA

12. 1. Restriction Endonucleases: DNA Ligase • Enzyme that rejoins cut strands of DNA together by reforming a phosphodiester bond • DNA ligase joins sticky ends • T4 DNA ligase (from T4 bacteriophage) joins blunt ends

13. How do we sort out the DNA • DNA is chopped into many pieces • How to differentiate one piece from other

14. 2. Gel Electrophoresis • Technique used to separate charged molecules based on their size • Acts like a molecular sieve http://www.biotech.iastate.edu/ppt_presentations/html/Fingerprinting/StudentInstruction-gel/images/image08.jpg http://www.solve.csiro.au/1105/img/sieve-bloke.jpg

15. 2. Gel Electrophoresis: DNA Preparation • Restriction enzymes digest DNA into smaller fragments of different lengths • Different DNA samples are loaded into wells of the gel (agarose or polyacrylamide) http://www.oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg

16. 2. Gel Electrophoresis: Attraction Migration • Negatively charged electrode at the end where wells are located • Positively charged electrode at opposite end • Negatively charged DNA migrate towards positive end due to attraction

17. 2. Gel Electrophoresis: Rate of Migration • Shorter/smaller DNA fragments migrate through gel faster since they can move through the pores in the gel more easily • Longer/larger DNA fragments migrate through gel slower • Rate of migration = 1/log(size) • Different DNA fragment lengths are separated A B C D E A = kilobase DNA ladder B = uncut plasmid DNA C = single digestion of the plasmid with EcoRI D = single digestionwith XhoI E = double digestion - both EcoRI and XhoI. http://www.answers.com/topic/agarosegel-jpg

18. 2. Gel Electrophoresis: Visualizing DNA Fragments • Ethidium bromide is a fluorescent dye that makes DNA fragments visible by staining the gel • DNA fragments can then be isolated and purified http://www.answers.com/topic/agarosegel-jpg

19. 2. Gel Electrophoresis: Proteins too! • Gel electrophoresis can also be used to separate proteins, usually using polyacrylamide gels http://www.biotechlearn.org.nz/var/biotech/storage/images/multimedia/images/protein_electrophoresis/48251-4-eng-GB/protein_electrophoresis_medium.jpg http://www.bio-link.org/vlab/Graphics/Tools/ProteinGel2.jpg

20. 3. Plasmids • Small, circular double-stranded DNA that can enter and exit bacterial cells • Lack a protein coat • Independent of bacterial chromosome • 1000-200,000 base pairs

21. 3. Plasmids: Endosymbiosis • Use host bacterial enzymes and ribosomes to replicate and express plasmid DNA • Carry genes that express proteins to protect bacteria against antibiotics and heavy metals

22. 3. Plasmids • Foreign genes (ie. insulin) can be inserted into plasmids, so bacteria can express gene and make its respective protein • Higher copy number of plasmids (number of individual plasmids) in bacteria • results in larger number of gene copies, thus more of its respective protein is synthesized

23. 3. Plasmids • Restriction endonucleases splice foreign genes into plasmids • DNA ligase reforms phosphodiester bond between the fragments, resulting in recombinant DNA http://www.accessexcellence.org/RC/VL/GG/inserting.html

24. 4. Transformation • Introduction of foreign DNA (usually a plasmid) into a bacterium • Plasmids can be used as a vector (vehicle that DNA can be introduced to host cells) to carry a specific gene into a host cell http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Siegenthaler/fig2.gif

25. 4. Transformation: Competence • Competent cell - Bacterium that readily takes up foreign DNA (ie. able to undergo transformation) • Most cells are not naturally competent, but can be chemically induced to become competent • Calcium ion in calcium chloride stabilizes negatively charged phosphates on bacterial membrane

26. 4. Transformation: Competence