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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