BIOTECHNOLOGICAL TOOLS & TECHNIQUES

1. BIOTECHNOLOGICAL TOOLS & TECHNIQUES Part One Section 6.1 Page 278

2. What is biotechnology? • Applied biology • genetics; molecular biology; microbiology; biochemistry • Uses living organisms and their components to create “bio-products” • industry, agriculture, medicine • Involves manipulation of DNA

3. Manipulating DNA • RecombinantDNA – a fragment of DNA composed of sequences from at least two different sources

4. Biotechnological tools and techniques • Restriction endonucleases • Methylases • Ligase • Gel electrophoresis

5. Imagine joining two DNA sequences: You would need tools: • Scissors to cut the fragments out of their sources • Glue to join the fragments together Biotechnology uses tools that are already existing within biological systems

6. Restriction endonucleases (RE) • aka restriction enzymes • “molecular scissors” What do they do? • recognize specific base-pair sequences in DNA, and then cut the double-stranded DNA at those sites http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

7. Function: • Crude immune system in bacteria • Cleaves virus DNA into fragment • Host DNA is methylated – RE knows not to cleave it

8. Recognition site • Recognition site: the sequence recognized by the enzyme Characteristics: • Specific to each different RE (there are over 2500) • 4-8 bp in length • Usually palindromic

9. What is a palindrome? Example 1: MADAM I'M ADAM Example 2: Recognition site of restriction enzyme EcoRI: 5’-GAATC -3’ 3’-CTTAG-5’

11. Ends produced by RE cleavage • Stickyends: Cleavage produces an overhang • Depending on where the RE cuts, it an be a 5’ or a 3’ overhang • Bluntends: No overhang

13. Frequency of recognition sites • The probability of encountering a recognition site depends on the number of bases in the site Probability = 1/4n where n = the number of bases in the recognition site

14. Example: • EcoRI has a 6-bp recognition site • The probability of finding this sequence in a strand of DNA is: 1/46 = 1/4096, or every 4096 base pairs Longer recognition site  Less frequent cleavage

15. For biotechnology, sticky ends are more useful • If two fragments are cut with the same RE, they will have complementary sticky ends • These fragments can be joined (“glued” together)

16. Naming restriction enzymes BamHI

17. HindII

18. Page 281 practice SmaI recognition sequence: CCCGGG Cuts between the C and the G • Location of cuts? • How many fragments? • What type of ends? 5’-AATTCGCCCGGGATATTACGGATTATGCATTATCCGCCCGGGATATTTTAGCA-3’ 3’-TTAAGCGGGCCCTATAATGCCTAATACGTAATAGGCGGGCCCTATAAAATCGT-5’

19. HindIII recognition sequence: AAGCTT • Cleaves between the two A’s What type of ends are produced? 5’-AAGCTT-3’

20. Page 281 practice Calculate the number of expected cuts in a DNA sequence of 75 000 base pairs, by a restriction enzyme that has a six-base pair recognition site. Steps: a. Determine the frequency of cuts b. Divide

21. Methylases • Methylases are enzymes • Add a methyl group to the recognition site • Prevents RE from cleaving the DNA • Function: Protect host DNA from own RE’s • As a biotechnological tool: • Allow protection of fragments/specific sequences

22. Ligases • Where have you seen this enzyme before?? • DNA replication • Joins sugar/phosphate backbones of DNA fragments • Can be used to join fragments that have complementary ends • Phosphodiester bond • Most frequently used: T4 DNA ligase

23. Overview: Producing recombinant DNA

24. Gel electrophoresis • Method of separating DNA fragments • Used in genetic engineering to isolate desired fragments

25. RE may cut at several sites. • Want to make sure the correct fragment is isolated.

26. Useful properties of DNA • DNA is negatively-charged (phosphate groups) • Charge-to-mass ratio of all nucleotides is consistent • because molar mass of each nucleotide is consistent

27. Principle of electrophoresis • Separates DNA fragments based on their sizes • Involves forcing DNA fragments through a gel matrix • Matrix acts like a sieve – has pores through which DNA can travel

28. Separation of fragments Fragments will migrate through the gel at a rate that is inversely proportional to logarithm of their size • Smaller fragments will migrate faster • Larger fragments will migrate more slowly Animation: http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

29. Procedure • DNA is cleaved into smaller fragments. Depending on the cut sites, the fragments will be different sizes. • The sample of DNA is loaded into small wells within the gel matrix. • A charge is applied across the gel: Negative at the sample end; positive at the opposite. • DNA fragments will migrate towards positive pole. Depending on fragment size, migration rates will vary

30. Wells/indents within gel

31. Sizing the bands • A “ladder” of fragments of known sizes is run alongside samples • Compare samples to bands of known size

32. Gel • Polyacrylamide • Agarose

33. Visualizing the DNA • Stain with ethidium bromide • Ethidium bromide inserts itself into the DNA backbone • Fluoresces under UV light

34. Obtaining the desired fragment • Literally cut the band out of the gel • Purify to obtain the fragment

35. Homework • Pg. 282 #9, 10 • Pg. 284 #11-14 • Pg. 291 #2, 3, 6, 8, 14-17