Biotechnology

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Fig. 20-6-5

DNA in nucleus

mRNAs in cytoplasm

Reverse transcriptase

Poly-A tail

DNA strand

Primer

mRNA

Degraded mRNA

DNA polymerase

cDNA

Slide 33

Screening a Library for Clones Carrying a Gene of Interest

A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene

This process is called nucleic acid hybridization

Slide 34

A probe can be synthesized that is complementary to the gene of interest

For example, if the desired gene is

– Then we would synthesize this probe

G

5

3

…

…

G

G

C

C

C

T

T

T

A

A

A

C

3

5

C

C

G

G

G

A

A

A

T

T

T

Slide 35

The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest

Once identified, the clone carrying the gene of interest can be cultured

Slide 36

Fig. 20-7

Probe DNA

Radioactively labeled probe molecules

Film

Nylon membrane

Multiwell plates holding library

clones

Location of DNA with the complementary sequence

Gene of interest

Single-stranded DNA from cell

Nylon membrane

TECHNIQUE

Slide 37

Expressing Cloned Eukaryotic Genes

After a gene has been cloned, its protein product can be produced in larger amounts for research

Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

Slide 38

Bacterial Expression Systems

Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells

To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

Slide 39

Eukaryotic Cloning and Expression Systems

The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems

YACs behave normally in mitosis and can carry more DNA than a plasmid

Eukaryotic hosts can provide the post-translational modifications that many proteins require