DNA analysis and nucleic acid probes
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What is a shotgun approach?
A mixture of fragments from the whole genome, it’s called a shotgun approach
What is a genomic library?
The set of 1000’s of recombinant clones, each carrying copies of a particular fragment from the initial genome is called a genomic library
What must be done to the genomic library to find the colony with the gene of interest?
A screening procedure
How else can DNA be cloned?
Phages
Baceteriophage lambda is one of the most widely used for several reasons. What are they?
• Molecular genetics well known • Can hold larger amounts of DNA than plasmids • More efficient than transformation - transfer by transduction • Recombinant DNA is packaged in phage heads in vitro • Phage infects bacteria • Plaques form where bacteria have lysed
The plaques formed from bacteriophages can be screened for the gene of interest using what process?
Hybridisation
What is important to remember when comes to restriction enzymes?
They don’t respect gene boundaries - so some genes in the library may be spread over two or more clones
Why is a cDNA library important?
Because it will be limited to the genes that are being transcribed in the starting cells
cDNA libraries are important for studying gene expression in particular types of cells e.g?
Brain and liver
What does BAC stand for?
Bacterial artificial chromosome
Can a BAC be used as a vector?
Yes
What is a BAC?
A BAC is a large plasmid that has been trimmed down and can carry a large DNA insert (100-300 kb)
A large insert size minimises what?
Large insert size minimises number of clones needed to make up genomic library
BAC are usually stored in what?
Usually stored in multiwell plates, one clone per well
A more limited kind of gene library can be developed by starting with what?
mRNA extracted from cells
The process of a retrovirus requires what to make a gene library?
Primer Reverse transcriptase derived from the retrovirus
dsDNA is called what?
complementary DNA or cDNA
What do you need to do to the cDNA to make create a library?
cDNA is modified by adding restriction sites to each end and then inserting into vector DNA
If you are only interested in the coding sequence of a gene do you use cDNA or mRNA and why?
cDNA there are no introns
cDNA is good for studying what?
Studying specialised functions of cells and studying gene expression at different stages of development of an organism
What is nucleic acid hybridisation?
Nucleic acid hybridisation consists of base pairing between the gene and a complementary sequence on another nucleic acid molecule – nucleic acid probe This short ss nucleic acid can either be RNA or DNA If we know at least part of the sequence of our gene of interest can then synthesise a probe complementary to it Probe can be traced by radioactive labelling or fluorescent marker The probe will bond specifically to complementary ssDNA of the gene of interest
Denaturation is important to do what?
Separate the DNA strands to ssDNA
How is denaturation done?
Chemicals or heat
Where does hybridisation take place?
Special filter - probe molecules are incubated with the filter