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Biotechnology & DNA Technology

Chapter 9

BIO 220

Biotechnology

• Is the use of microorganisms, cells, or cell

components to make a product

• By inserting, deleting, or modifying genes with

recombinant DNA (rDNA) technology

microbes are being used as “factories” to

produce chemicals they would not ordinarily

Recombinant DNA procedure

Fig. 9.1

Vectors

• Vehicle (DNA molecule) that transports

foreign DNA into a cell

• i.e. plasmid or viral genome

• Must self-replicate (needs origin of

replication)

• Selectable marker (i.e. antibiotic resistance)

• Restriction enzyme sites

Fig. 9.3

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Restriction enzymes

Fig. 9.2

How to make recombinant DNA

Fig. 9.1

Obtaining DNA

Where does the gene of interest come from?

Genomic libraries

• Genomic DNA digested

• RFs spliced into vectors

• Vectors introduced into

bacterial cells

• One clone for every gene

Fig. 9.8

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Obtaining DNA

Where does the gene of interest come from?

Synthetic DNA

• Must know gene sequence

Fig. 9.10

Polymerase Chain Reaction (PCR)

Fig. 9.4

Denature

Anneal

Elongation

Some applications of PCR

• Detection of pathogens (i.e. Mycobacterium

tuberculosis)

• Mycology and parasitology

• Viral characterization

• Dentistry

• Environmental microbiology

We made the recombinant plasmid, now . . .

Fig. 9.1

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Inserting foreign DNA into cells

• Conjugation

– Plasmids transferred between closely related

microbes

• Transformation

– Cells must be made “competent” in order to take

up plasmids

• Electroporation

– Cell walls must usually be digested (protoplasts)

– Form pores in plasma membrane

Gene gun

Fig. 9.6

Microinjection

Fig. 9.7

Big picture

• There are a number of ways to get foreign

DNA into a variety of cell types. However, the

foreign DNA will only survive if it is (1)

incorporated into a self-replicating vector, or

(2) incorporated into one of the cell’s

chromosomes by recombination.

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The rDNA is in the cell, but . . .

Fig. 9.1

How do we find the cells with the gene of

interest?

• Blue-white screening

Colony hybridization – Is it really the gene

of interest?

Fig. 9.12

Now what?

Fig. 9.1

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We have made the gene product . . .