Part One BIOTECHNOLOGICAL TOOLS & TECHNIQUES

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 Do YOU HAVE AN OPINION ON genetic research & biotechnology?

Biotechnology Most forms of biotechnology involve the formation of recombinant DNA, fragments of DNA composed of sequences originating from at least two different sources This is the basis of genetic engineering The following slides explain how a gene is isolated, purified, and cloned

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

Biotechnological tools and techniques 1. Restriction endonucleases 2. Methylases 3. Ligase 4. 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

Function: Crude immune system in bacteria Cleaves virus DNA into fragment Host DNA is methylated – R.E. 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

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

Ends produced by R.E. 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

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

Example: EcoRI has a 6-bp recognition site The probability of finding this sequence in a strand of DNA is: 1/4 6 = 1/4096, or every 4096 base pairs Longer recognition site  Less frequent cleavage

For biotechnology, sticky ends are useful but can be limiting If two fragments are cut with the same R.E., they will have complementary sticky ends These fragments can be joined (“glued” together)

Naming restriction enzymes Bgenus Bacillus amspecies amyloliquefaciens Hstrain Ifirst endonuclease isolated from this strain BamHI

Hgenus Haemophilus inspecies influenzae dstrain Rd IIsecond endonuclease isolated from this strain HindII

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’

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

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

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

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 1. DNA is negatively-charged (phosphate groups) 2. Charge-to-mass ratio of all nucleotides is consistent because molar mass of each nucleotide 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: trophoresis.html trophoresis.html

Procedure 1. DNA is cleaved into smaller fragments. Depending on the cut sites, the fragments will be different sizes. 2. The sample of DNA is loaded into small wells within the gel matrix. 3. A charge is applied across the gel: Negative at the sample end; positive at the opposite. 4. 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

Gel Polyacrylamide Agarose

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