Gene Splicing and Restriction Maps

Friday, June 12, 15

Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site.

Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site.

Restriction enzyme must be specially chosen to cut on either side of

insulin geneHuman cell

DNA

insulin gene

Friday, June 12, 15

Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site.

Researchers expose plasmids to different restriction enzymes and then measure the length of the pieces produced. They use this data to construct a restriction map for the plasmid. Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and BamH1

• expose Plasmid X to only EcoR1 (single digestion)• expose Plasmid X to only BamH1 (single digestion)• expose Plasmid X to both EcoR1 and BamH1(double digestion)

Plasmid X

The length of pieces are determined using gel electrophoresis Here are the results!

Friday, June 12, 15

Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site.

Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and BamH1

undigested (bp)digested with EcoR1

(bp)digested with BamH1

(bp)digested with EcoR1

and BamH1 (bp)

1400 1400600 800

100600700

Table 1: Results of Plasmid X Digestion with EcoR1 and BamH1

700 bp 600 bp 100 bp

Friday, June 12, 15

Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site.

Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and BamH1

700 bp 600 bp 100 bp

Plasmid X1400bp

EcoR1

Plasmid X

600 bp

BamH1

800 bp

BamH1

Friday, June 12, 15

If the recognition site for EcoR1 was 100 bp counterclockwise from the BamH1 recognition site, you would expect the following fragments.

Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site.

Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and BamH1

700 bp 600 bp 100 bp

BamH1

EcoR1

digested with EcoR1 and BamH1 (bp)

100600700

digested with EcoR1 and BamH1 (bp)

100600700

actual results expected results

100 bp

600 bp

700 bp

BamH1

Friday, June 12, 15