Chapter 12:Biotechnology

2. Recombinant DNA Applications

1. Recombinant DNA

1. Recombinant DNA

What is “Recombinant DNA”?The joining of DNA from different sources.

This can happen in nature (in vivo)…• the transfer of DNA involving bacteria or viruses

…or in the laboratory (in vitro)• the cutting & splicing of DNA fragments by

molecular biologists

The term “recombinant DNA” generally refers tolaboratory kind…

Recombinant DNA in Nature

Bacteria can acquire DNA from an outside source bya process known astransformation

• chemical treatment of bacterialcells followed by a brief heattreatment can induce the cellsto internalize & retain the DNA

PlasmidsBacteria are transformedmore commonly by small,circular DNA fragmentscalled plasmids

• taken up from outside ortransferred from onebacterium to another

Restriction enzymerecognition sequence

G A A T T CC T T A A G

DNA1

2

3

4

C T T A AA AT TC

A AT TCG

C T T A A

Addition of a DNAfragment fromanother source

Two (or more)fragments sticktogether bybase-pairing

G A AT T CC T TA A G

G A AT T CC T TA A G

5

DNA ligasePastes the strand

Restriction enzymecuts the DNA intofragments

Recombinant DNA molecule

GG

Sticky end

G

The cutting and splicing of DNA in vitro involves the use of restriction enzymes (RE’s):

e.g.EcoR I cuts at:

..GAATTC..

..CTTAAG..

Recombinant DNA in the Laboratory

Hind III cuts at:..AAGCTT....TTCGAA..

There are many different RE’s, each cutting a different sequence

E.coli

Plasmid

Isolate DNAfrom two sources

Cut both DNAswith the samerestriction enzyme

Human cell

DNA

2

1

3

4

5

6

Gene V

Sticky ends

Mix the DNAs;they join bybase-pairing

Add DNA ligaseto bond the DNA covalently

Recombinant DNAplasmid Gene V

Put plasmid into bacteriumby transformation

Recombinant bacterium

Clone the bacterium

Bacterial clone carrying manycopies of the human gene

“Cloning” a GeneDNA “sequence of interest” is inserted into a plasmid & “carried” inside bacteria

1) cut plasmid and DNA to be cloned with same RE

2) ligate fragments togetherusing DNA ligase enzyme

3) transform bacteria, select for “clones” with plasmid

**In this form the “DNA of interest” can be easily multiplied and purified**

PCR AmplificationThe creation of recombinant DNA molecules in the laboratory requires large amounts of the DNA fragments to be combined:

• the PCR (PolymeraseChain Reaction) technique is routinely used to generatehuge numbers of identicalDNA fragments

• involves in vitro DNAreplication to amplify desired DNA sequencesexponentially

2. Recombinant DNAApplications

Useful Applications InvolvingRecombinant DNA Technology

DNA sequencing, fingerprinting• involves the separation of DNA fragments by size

using gel electrophoresis

Commercial Production• using recombinant bacteria, yeast to make “lots” of

a protein (e.g., insulin)

Gene therapy• replacing defective genes (still experimental)

Transgenic organisms (GMO’s)• putting “desirable” genes into animals, plants

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Powersource

Gel

Mixture of DNAmolecules ofdifferent sizes

Longermolecules

Shortermolecules

Completed gel

Gel Electrophoresis

DNA fragments of different lengths generated by RE’s or other methods are separated by size by “running a gel”

• used in DNA sequencing, DNA fingerprinting and numerous other techniques

TransgenicOrganisms

Potato plant containing pest resistance gene

Salmon containing extra growth hormone gene

**Transgenicorganisms contain

a foreign gene**

Key Terms for Chapter 12

Relevant Review Questions: 2-4, 6

• plasmid

• recombinant DNA

• transformation

• restriction enzymes• PCR• gel electrophoresis• transgenic