1. BIOTECHNOLOGICAL TOOLS & TECHNIQUES
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
Recombinant DNA – 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

7. Function: Host DNA is methylated – RE knows not to cleave it
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. 5’-GAATC -3’ 3’-CTTAG-5’ 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
Sticky ends: Cleavage produces an overhang
Depending on where the RE cuts, it an be a 5’ or a 3’ overhang
Blunt ends: No overhang

13. 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)

14. Naming restriction enzymes
BamHI B
genus Bacillus am
species amyloliquefaciens H
strain I
first endonuclease isolated from this strain

15. HindII H genus Haemophilus in species influenzae d strain Rd II
second endonuclease isolated from this strain

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

17. 5’-AAGCTT-3’ HindIII recognition sequence: AAGCTT
Cleaves between the two A’s
What type of ends are produced?
5’-AAGCTT-3’

18. 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

19. 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

20. Overview: Producing recombinant DNA
So what enzymes act as the scissors and glue???

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

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

23. Useful properties of DNA
DNA is negatively-charged (phosphate groups)
Charge-to-mass ratio of all nucleotides is consistent

24. 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

25. 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: trophoresis.html

26. Depending on fragment size, migration rates will vary
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

27. Wells/indents within gel

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

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

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

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