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NadiaA

Recombinant DNA
LO:
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Understand the principles that underlie gene cloning
Discuss recombinant DNA technology and genome analysis

Terminology
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Recombinant DNA- 2 or more DNA molecules from different origins united into a new
combination of info
Clone- multiple copies of the same genetic info
Plasmids:
 Origin of replication
 Selectable mark
 Transformable
Restriction enzymes:
 Very specific
 Identical overhangs
Ligase enzyme - joins two DNA strands or other molecules by a phosphate ester linkage
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Disc can be screened for correct colony
Isolate the plasmid
Isolate the fragment from the plasmid

Summary:
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Each colony has a plasmid
Plasmid on filter
Denature so it’s single stranded
Take probe
Label it
Single stranded so it floats around
Only binds where it finds a specific sequence (as single stranded probe and single stranded
DNA bind at specific sequence)
Expose to xray…
Identify a colony

Restriction enzymes- used to identify changes in DNA sequences
Southern Blot- Agarose gel electrophoresis of digested DNA to separate fragments
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Denature and transfer to filter
Take cloned gene fragment and radioactively label it
Add to filter to allow hybridisation
Lay on photographic film to visualise bands

Polymerase chain reaction
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Cloning without cells
Rapidly isolates DNA fragments
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NadiaA
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No radioactivity process
Based on previously determined DNA sequence, develop short oligonucleotides,
complimentary to sequences flanking the target DNA (approx

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