Biotechnology

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DNA ligase seals strands.

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DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.

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Slide 15

Cloning a Eukaryotic Gene in a Bacterial Plasmid

In gene cloning, the original plasmid is called a cloning vector

A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

Slide 16

Producing Clones of Cells Carrying Recombinant Plasmids

Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid:

The hummingbird genomic DNA and a bacterial plasmid are isolated

Both are digested with the same restriction enzyme

The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends

Animation: Cloning a Gene

Slide 17

Some recombinant plasmids now contain hummingbird DNA

The DNA mixture is added to bacteria that have been genetically engineered to accept it

The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids

This results in the cloning of many hummingbird DNA fragments, including the β-globin gene

Slide 18

Fig. 20-4-1

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restriction site

Sticky ends

ampR gene

TECHNIQUE

Slide 19

Fig. 20-4-2

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restriction site

Sticky ends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Slide 20

Fig. 20-4-3

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restriction site

Sticky ends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carrying plasmids

Slide 21

Fig. 20-4-4

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restriction site

Sticky ends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carrying plasmids

RESULTS

Colony carrying non- recombinant plasmid with intact lacZ gene

One of many bacterial

clones

Colony carrying recombinant plasmid with disrupted lacZ gene