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

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

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, 1 st r.e. isolated HindII  Haemophilus influenzae, strain Rd, 2 nd 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 BacteriaRestriction Enzyme Recognition Site Escherichia coliEcoRI5’-GAATTC-3’ 3’-CTTAAG-5’ Haemophilus parainfluenzae HindIII5’-AAGCTT-3’ 3’-TTCGAA-5’ Arthrobacter luteus AluI5’-AGCT-3’ 3’-TCGA-5’

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) BacteriaRestriction enzyme Recognition siteAfter digestion by restriction enzyme Escherichia coliEcoRI5’-GAATTC-3’ 3’-CTTAAG-5’ 5’-G AATTC-3’ 3’-CTTAA G-5’ Haemophilus parainfluenzae HindIII5’-AAGCTT-3’ 3’-TTCGAA-5’ Arthrobacter luteus AluI5’-AGCT-3’ 3’-TCGA-5’

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

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/Fingerprintin g/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/b ackground/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 http://www.answers.com/topic/agarosegel-jpg A = kilobase DNA ladder B = uncut plasmid DNA C = single digestion of the plasmid with EcoRI D = single digestion with XhoI E = double digestion - both EcoRI and XhoI. A B C D E

18. 2. Gel Electrophoresis: Visualizing DNA Fragments Gel ElectrophoresisGel Electrophoresis 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/pr otein_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