BIOTECHNOLOGICAL TOOLS & TECHNIQUES Section 6.1 Page 278

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

Manipulating DNA Recombinant DNA – a fragment of DNA composed of sequences from at least two different sources

Biotechnological tools and techniques Restriction endonucleases Methylases Ligase Gel electrophoresis

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

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

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

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

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’

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

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)

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

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

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?

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

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

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

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

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

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

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

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

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/gelelec trophoresis.html

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

Wells/indents within gel

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

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

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

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