Biotechnology

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This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size

A current is applied that causes charged molecules to move through the gel

Molecules are sorted into “bands” by their size

Video: Biotechnology Lab

Slide 49

Fig. 20-9

Mixture of DNA mol- ecules of different sizes

Power source

Power source

Longer molecules

Shorter molecules

Gel

Anode

Cathode

TECHNIQUE

RESULTS

1

2

+

+

–

–

Slide 50

Fig. 20-9a

Mixture of DNA mol- ecules of different sizes

Power source

Longer molecules

Shorter molecules

Gel

Anode

Cathode

TECHNIQUE

1

2

Power source

–

+

+

–

Slide 51

Fig. 20-9b

RESULTS

Slide 52

In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

The procedure is also used to prepare pure samples of individual fragments

Slide 53

Fig. 20-10

Normal allele

Sickle-cell allele

Large fragment

(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

201 bp 175 bp

376 bp

(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene

Normal -globin allele

Sickle-cell mutant -globin allele

DdeI

Large fragment

Large fragment

376 bp

201 bp

175 bp

DdeI

DdeI

DdeI

DdeI

DdeI

DdeI

Slide 54

A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization

Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

Slide 55

Fig. 20-11

TECHNIQUE

Nitrocellulose membrane (blot)

Restriction fragments

Alkaline solution

DNA transfer (blotting)

Sponge

Gel

Heavy weight

Paper towels

Preparation of restriction fragments

Gel electrophoresis

I II III

I II III

I II III

Radioactively labeled probe for -globin gene

DNA + restriction enzyme

III Heterozygote

II Sickle-cell allele

I Normal -globin allele