happy monday! checking off: notes on ch 20.1, 20.2 with your group, discuss what you know about...
TRANSCRIPT
Happy Monday!
Checking off: Notes on Ch 20.1, 20.2
With your group, discuss what you know about these:– Methylation and Acetylation– Genetic Engineering/Biotechnology– Restriction enzymes– Gel Electrophoresis
Methylation and Acetylation
2005-2006
The BIG Questions…• How can we use our knowledge of DNA to:– diagnose disease or defect?– cure disease or defect?– change/improve organisms?
• What are the techniques & applications of biotechnology?– direct manipulation of genes for practical purposes
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Biotechnology Today• Genetic Engineering– manipulation of DNA– if you are going to engineer DNA & genes &
organisms, then you need a set of tools to work with
– this “unit” is a survey of those tools…
Our tool kit…
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Bioengineering Tool kit• Basic Tools– restriction enzymes– ligase– plasmids / cloning– DNA libraries / probes
• Advanced Tools– PCR – DNA sequencing – gel electrophoresis – Southern blotting– microarrays
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Cut, Paste, Copy, Find…• Word processing metaphor…– cut• restriction enzymes
– paste• ligase
– copy• plasmids
– Bacteria transformation• PCR
– find• Southern blotting / probes
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Cut DNA• Restriction enzymes– restriction endonucleases– discovered in 1960s– evolved in bacteria to cut up foreign DNA
(“restriction”) • protection against viruses & other bacteria
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What do you notice about these phrases?
radarracecarMadam I’m AdamAble was I ere I saw Elbaa man, a plan, a canal, PanamaWas it a bar or a bat I saw?
Restriction Enzymes
Also known as restriction endonucleases, are enzymes that cut a DNA molecule at a particular place. They are essential tools for recombinant DNA technology. The enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to six nucleotides.
2005-2006
Restriction Enzymes, c’td• Action of enzyme – cut DNA at specific sequences• restriction site
– symmetrical “palindrome”– produces protruding ends• sticky ends
• Many different enzymes– named after organism they are found in• EcoRI, HindIII, BamHI, SmaI
Madam I’m Adam
CTGAATTCCGGACTTAAGGC
CTG|AATTCCGGACTTAA|GGC
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AATTC
AATTC
AATTC
GAATTC
G
G
GG
G
GAATTCCTTAAG
GAATTCCTTAAG
CTTAA
CTTAA
CTTAAG
DNA ligasejoins the strands.
DNA
Sticky ends (complementarysingle-stranded DNA tails)
Recombinant DNA molecule
AATTCGG
CTTAA
How restriction enzymes are used
Restriction enzymecuts the DNA
Add DNA from another source cut with same restriction enzyme
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Paste DNA• Sticky ends allow:– H bonds between
complementary bases to anneal
• Ligase– enzyme “seals”
strands • bonds sugar-
phosphate bonds• covalent bond of
DNA backbone
Then what?
• DNA, cut at restriction sites, will be different lengths and will contain genes within those lengths.
• To analyze, mix with solutions that will allow them to travel through a medium based on size, shape, and charge.
• Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.
Homework
• Complete Pre-lab activity for Transformation Lab– Get to Know Bacterial Transformation– Flowchart all steps