What are restriction enendonuclease enzymes are used for

These are used to cut out the gene of interested

Where are the enzymes cut

These enzymes cut at recognition sites leaving to sticky ends

What happens to the DNA fragments

The DNA fragments must be modified to ensure transcription of these genes occur

A promoter region must be added-

At the start of the DNA fragment this is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur

A Terminator region must be added-

This is added at the end of the gene. It causes RNA polymerase to detach and stop transcription so only one gene at a time is copied into mRNA

Something to carry the isolated DNA fragment into the host cell

What do circular DNA do

Circular DNA seperate from main bacterial genome which only contains a few genes

How is DNA inserted into a vector process 1 `

The plasmid is cut open using the same restriction enzyme endonuclease This creates the same sticky ends Therefore the DNA fragment sticky ends are complemntary to the sticky ends on the plasmid

The DNA fragment and cut plasmid are combined and the enzyme ligase sticks them together Ligase catalyses reaction to form phosphodiester bonds between nucleotides

How are they transformed

The vector next needs to be inserted into the host cell where the genes will be expressed to create the protein required

How would you increase the permeablity

The host cells are mixed with Ca 2+ and heat shocked This enables sthe vector to enter the host cells cytoplasm

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Topic 8

In vivo cloning

Term	Definition
What are restriction enendonuclease enzymes are used for	These are used to cut out the gene of interested
Where are the enzymes cut	These enzymes cut at recognition sites leaving to sticky ends
What happens to the DNA fragments	The DNA fragments must be modified to ensure transcription of these genes occur
A promoter region must be added-	At the start of the DNA fragment this is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur
A Terminator region must be added-	This is added at the end of the gene. It causes RNA polymerase to detach and stop transcription so only one gene at a time is copied into mRNA
What is a vector	Something to carry the isolated DNA fragment into the host cell
What are the most common vectors	Plasmids
What do circular DNA do	Circular DNA seperate from main bacterial genome which only contains a few genes
How is DNA inserted into a vector process 1 `	The plasmid is cut open using the same restriction enzyme endonuclease This creates the same sticky ends Therefore the DNA fragment sticky ends are complemntary to the sticky ends on the plasmid
Process 2	The DNA fragment and cut plasmid are combined and the enzyme ligase sticks them together Ligase catalyses reaction to form phosphodiester bonds between nucleotides
How are they transformed	The vector next needs to be inserted into the host cell where the genes will be expressed to create the protein required
To do this the host cell membrane	Must be permeable
How would you increase the permeablity	The host cells are mixed with Ca 2+ and heat shocked This enables sthe vector to enter the host cells cytoplasm

Created by: yHya

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