Biotechnology

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Film over blot

Probe detection

Hybridization with radioactive probe

Fragment from sickle-cell -globin allele

Fragment from normal -globin allele

Probe base-pairs with fragments

Nitrocellulose blot

1

4

5

3

2

Slide 56

Fig. 20-11a

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

DNA + restriction enzyme

III Heterozygote

II Sickle-cell allele

I Normal -globin allele

1

3

2

Slide 57

Fig. 20-11b

I II III

I II III

Film over blot

Probe detection

Hybridization with radioactive probe

Fragment from sickle-cell -globin allele

Fragment from normal -globin allele

Probe base-pairs with fragments

Nitrocellulose blot

4

5

Radioactively labeled probe for -globin gene

Slide 58

DNA Sequencing

Relatively short DNA fragments can be sequenced by the dideoxy chain termination method

Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths

Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment

The DNA sequence can be read from the resulting spectrogram

Slide 59

Fig. 20-12

DNA (template strand)

TECHNIQUE

RESULTS

DNA (template strand)

DNA polymerase

Primer

Deoxyribonucleotides

Shortest

Dideoxyribonucleotides (fluorescently tagged)

Labeled strands

Longest

Shortest labeled strand

Longest labeled strand

Laser

Direction of movement of strands

Detector

Last base of longest labeled strand

Last base of shortest labeled strand

dATP

dCTP

dTTP

dGTP

ddATP

ddCTP

ddTTP

ddGTP

Slide 60

Fig. 20-12a

DNA (template strand)

TECHNIQUE

DNA polymerase

Primer

Deoxyribonucleotides

Dideoxyribonucleotides (fluorescently tagged)

dATP

dCTP

dTTP

dGTP

ddATP

ddCTP

ddTTP

ddGTP

Slide 61

Fig. 20-12b

TECHNIQUE

RESULTS

DNA (template strand)

Shortest

Labeled strands

Longest

Shortest labeled strand

Longest labeled strand

Laser