Lecture 2 Flashcards
Gene cloning is a biological method of
Purification
Where does the gene of interest get inserted into
Vector
Give an example of a vector
Plasmid
Once placed into a vector where does it go next
Introduced into a host cell
When the vector replicates what does this allow
Allows large amounts of it to be isolated and used for multiple applications
Once a gene has been cloned what can be determined
Nucleotide sequence
How is a clone used to determine where the cloned gene is expressed
It is used as a probe
Why would you use a clone as a probe
Determine where and when the clones gene is expressed
How is gene function determined
Over expression or mutation of the cloned gene
What is the gene product
Protein
Gene product can be purified for what
Enzymes
Antibodies
Protein structure
What is transgenesis
Genes that are artificially expressed in other organisms
Name 6 things you would need in a gene cloning toolkit
Source of DNA
Polymerase chain reaction
Restriction endonucleases
DNA Ligase
Vector
Host cells
Source of DNA will include
Genomic DNA gDNA
Complementary DNA cDNA
Where does cDNA derive from
Reverse transcription of mRNA
What is a clone
DNA molecule that is identical to the DNA molecule from where it derived from
Does the mRNA have any introns
No
When can we only run pcr
Nucleotide sequence is known
Why is it good to use pcr
Produce lots of the desired DNA sequence
What is restriction endonucleases for
To cut DNA
Restriction endonucleases recognise what
4bp frequent
6bp intermediate
8bp infrequent
What is DNA Ligase for
Join together fragments
What is a vector
Where cloned DNA is inserted
What are the two ways of thinking of a gene clone
Method trying to identify a gene in the first place
As a way of manipulating the gene once it has been identified
What are the 3 approached for gene identification
Mapping
Homology
Functional complementation of mutants
What is a collection of gDNA fragments
gDNA library
How to make a gDNA library
Digest gDNA with restriction enzymes and Ligase it into a vector cut with the same enzyme
What are the 2 approaches for creating a gDNA
Complete digest and partial digest
What is complete digest
Is with one or several enzymes, incase the chosen gene contains a restriction site for the enzymes used
What is partial digest
Is with frequent cutting enzyme. This creates overlapping of fragments.
After the plasmids are inserted into the host what needs to happen next
Select the mutants with the plasmids
Screen for hosts that can grow on the medium lacking arginine
Isolate plasmid from the colony
Sequence the DNA fragment
When a plasmid has a fragment inserted what is this called
Recombinant DNA
What do we use the library for
Isolate genes
Identify a full sequence
Identify a close related species
How can we get a probe
It could be a fragment of the target gene
Or it could be a gene from a different species that share the same gene
After gene manipulation what do you do next
Analyse the cloned gene sequence
Analyse gene expression profile
Analyse gene function
Produce recombinant protein
Make transgenic organisms
Why do we analyse the gene expression profile
Determine where and when the gene is expressed
Why do we analyse gene function
Examine phenotype
Over express the gene
Mutation in the genes
Associate it with certain diseases
Why do we produce recombinant proteins
Raise antibodies for gene products
Why do we make transgenic organisms
So we can express the gene to give some desired trait
What do pcr primers do
Used to incorporate restriction enzyme sites into the gene
What must be used to produce a recombinant protein
Expression vector
What does pcr do
Amplifies a particular ba sequence for cloning
