ANSC 4050 Lecture Notes - Lecture 3: Ethidium Bromide, Herbert Boyer, Agarose Gel Electrophoresis
25 Sep 2018
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Department
Course
Professor
Lecture 3
→ Recombinant DNA
Isolation of DNA (gene of interest) →
Modification (manipulation of the DNA construct or fragments) →
Transfection (deliver the transgene into the desired host) →
Detection of expression (mRNA, protein, activity)
→ Basic Knowledge and Technology Required
• DNA library and screening
• polymerase chain reaction
• sequencing
• restriction endonucleases
• vectors construction and DNA manipulation
• transfection and detection of gene expression
Herbert Boyer (University of California) & Stanley Cohen (Stanford University):
-the pioneers who made the foundation of recombinant DNA
• DNA molecules from one organism can be precisely targeted and manipulated for insertion into
the DNA of another organism
• living organisms can serve as carriers for genes from another organism
restriction endonucleases (restriction enzymes) – enzymes that recognize specific double stranded DNA
sequences and cleave the DNA at these sequences
• proteins made in bacteria
• the recognition site are often palindromes
palindrome – sequence that is read the same from either direction
• named based on the bacterium that produces it
EcoR I GAATTC
CTTAAG
• restriction enzymes can generate either sticky (protruding) ends or blunt ends
EcoR I GIAATTC
G AATTC Sticky ends
CTTAAIG CTTAA G
Hpa I GTTIAAC GTT AAC Blunt ends
CAAITTG CAA TTG
• compatible sticky ends can be joined to other compatible sticky ends
• blunt ends can be joined to other blunt ends
→ Separation of DNA
• DNA is negatively charged
• apply an electric field
• separate through agarose gel
• separate DNA fragments by their size
• visualise DNA fragments via. ethidium bromide staining
vector –
• capable of accepting a foreign DNA fragment
-allows us to put the gene of interest inside
• can replicate itself inside the host
most common vectors: plasmid, bacteriophage, cosmid
plasmid (small), bacteriophage (medium), cosmid (large) – in terms of carrying capacity
plasmids –
1. circular DNA
2. replicate independently of chromosome
3. can be multiple copies per bacterial cell
4. manipulatable (add or remove a piece of DNA)
5. can be isolated relatively easily from bacteria
→ Major Components and Functions of Cloning Plasmid Vectors
• origin of replication
-region of specific DNA that allow the plasmid to start replicating
• multiple cloning sites
• selectable marker gene (antibiotic resistant gene)
→ Transformation and Selection
• introduction of the recombinant plasmid into bacterial host
• selection of bacteria containing recombinant DNA
• antibiotic resistance
• “blue-white” screening
-widely used for cloning of PCR products
Document Summary
Modification (manipulation of the dna construct or fragments) . Transfection (deliver the transgene into the desired host) . Basic knowledge and technology required: dna library and screening, polymerase chain reaction, vectors construction and dna manipulation transfection and detection of gene expression sequencing restriction endonucleases. Herbert boyer (university of california) & stanley cohen (stanford university): Cttaag restriction enzymes can generate either sticky (protruding) ends or blunt ends. Ttg compatible sticky ends can be joined to other compatible sticky ends: blunt ends can be joined to other blunt ends. Separation of dna: dna is negatively charged, apply an electric field, visualise dna fragments via. ethidium bromide staining separate dna fragments by their size separate through agarose gel vector capable of accepting a foreign dna fragment. Major components and functions of cloning plasmid vectors: origin of replication. Region of specific dna that allow the plasmid to start replicating: multiple cloning sites selectable marker gene (antibiotic resistant gene)
