16.2 In vevo gene cloning

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What happens after fragments of DNA have been obtained?
They need to be cloned.
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What is in vivo cloning?
Transferring the fragments to a host cell using a vector.
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What is in vitro cloning?
Using the polymerase chain reactions.
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Why are 'sticky ends' important in gene cloning?
If the same restriction endonuclease which targets the same 6bp sequence is used, then the two strands of DNA should be complimentary to each other. This means the single strand fragment can be joined to any other 'sticky end'.
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What does DNA ligase do?
It joins the phosphate sugar framework of the 2 sections of DNA and so unite them as one.
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Once an appropriate fragment of DNA has been cut,what is the next step?
To join it to a carrying unit called a VECTOR.
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What is a vector?
A carrying unit used to carry fragments of DNA before being inserted into the host cell.
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What is the most commonly used vector?
The plasmid of a bacterial cell.
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Why are plasmids commonly used as vectors?
Because plasmids almost ALWAYS contain the gene for antibiotic resistance, and so restriction endonucleases can be used at one of these resistant genes to break the loop.
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What is DNA ligase used for?
To join the fragment of DNA to the plasmid.
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What is transformation?
When the plasmids are reintroduced into bacterial cells.
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What happens in transformation?
The plasmids are mixed with bacteria in a medium containing Ca+ ions. The Ca+ ions and a change in temperature make the bacterial cells permeable to the plasmids.
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Why may some bacterial cells take up the DNA fragments? (2 reasons)
1. Only a few bacterial cells actually take up the plasmids when they are mixed. 2. Some plasmids will have closed up again without incorporating the DNA fragment.
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Card 2

Front

What is in vivo cloning?

Back

Transferring the fragments to a host cell using a vector.

Card 3

Front

What is in vitro cloning?

Back

Preview of the front of card 3

Card 4

Front

Why are 'sticky ends' important in gene cloning?

Back

Preview of the front of card 4

Card 5

Front

What does DNA ligase do?

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