methods for dna transfer bt 201 biotechnology techniques i
TRANSCRIPT
Methods for DNA Transfer
BT 201
Biotechnology Techniques I
Transferring Genes
• Vectors are used to move genes around
• Plasmids, Bacteriophage, Cosmids, YACs, BACs, Viruses are used
• E. coli often used to express genes that have been transferred
• Transformation is a common method for gene transfer
Transformation
• Recipient cells take up foreign DNA from surrounding media
• Often accomplished using plasmid vectors
• Artificially induced in laboratories
• Allows introduction of unrelated genes into bacterial expression systems
• Products of interest can be produced, extracted, and purified for use
Bacterial Plasmids• Circular pieces of
DNA that can be replicated outside the bacterial chromosome
• Occur in varying sizes• Capable of carrying
varying sizes and types of genes
• May produce several hundred copies in a single cell
Vector Creation
• Restriction enzymes are used to cut DNA to be inserted into small fragments
• Plasmids are cut open with REs so DNA fragments may be inserted
• Plasmids, DNA fragments, and DNA ligase are mixed to put it all back together: cloning
• Ready for transformation now
Restriction enzymerecognition sequence
Restriction enzymecuts the DNA intofragments
Addition of a DNAfragment fromanother source
DNA G CAAT T
T
T
CG
GC
C
CGG
AA
AA
AATTTTAA
T
T
Sticky end
Two (or more)fragments sticktogether bybase-pairing
DNA ligasepastes the strand
Recombinant DNA molecule
GG
GG C
CC
CAAAA
AATT
TTTT
T TAA
CT TAAG
Vector Creation
Transformation• Transformation was
discovered in 1928 by Frederick Griffith using S. pneumoniae in mice: “Transforming Principle”
• In 1944 a genetic basis was for process was discovered by Avery, McLeod, and McCarty– They named the process
“Transformation”
Transformation of E. coli
• Cells capable of taking up foreign DNA are competent– Some cells are naturally competent, some cells have
to be made competent – E. coli not naturally competent
• Artificial competence induced using cold and cationic solutions (cold CaCl2)
• Plasmid introduced into iced solution• Cells heat shocked to force plasmid uptake• Solution allowed to return to ambient
temperature, pores close
Isolate DNAfrom two sources
Cut both DNAswith the samerestriction enzyme
Mix the DNAs;they join bybase-pairing
Add DNA ligaseto bond the DNA covalently
Recombinant DNAplasmid
E. coli
Plasmid
Human cell
Sticky endsGene V
DNA
Gene V
Put plasmid into bacteriumby transformation
Recombinantbacterium
Clone the bacterium
Bacterial clone carrying manycopies of the human gene
Detecting Transformation
• Many plasmids carry antibiotic resistance genes: R factors
• Used to select transformants• pBestLuc plasmid contains
ampr and luc genes• Ampicillin resistance,
Luciferase production• Potential transformants plated
on ampicillin-containing media • Colonies exposed to luciferin
solution and observed for luminescence
Manipulating Genes for Biotechnology
• To make products• To improve crops• To improve livestock• To improve quality of
life• To treat disease
Biotechnology Applications• Recombinant pharmaceutical products
• Transgenic animals as pharmaceutical “factories” and organ sources
• Transgenic crops
Bacterialchromosome
Plasmid
BacteriumPlasmidisolated
Recombinant DNA(plasmid)
Recombinantbacterium
Copies of gene
Clone of cellsGene for pestresistanceinserted intoplants
Gene used to alter bacteriafor cleaning up toxic waste
Protein used to dissolve bloodclots in heart attack therapy
Copies of protein
Cell multiplies withgene of interest
Protein used tomake snowform at highertemperature
Plasmid put intobacterial cell
DNAGene ofinterest
Gene insertedinto plasmid
DNAisolated
Cell containing geneof interest
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