Clone identification Flashcards
What is the problem with identifying clones in genomic or cDNA libraries?
They are very large and it can be hard to identify the gene of interest amongst many other colonies/plaques.
What are the two strategies that can be used to identify the clone?
Direct selection or clone identification via shotgun cloning.
How does direct selection work?
Set up the process so that only the desired clone can survive. The selection for the desired clone occurs at the plating out stage.
How does shotgun identification work?
This involves cloning all of or most genes in the cell and identification needs to happen through analysis of individual clones to identify the correct one.
What genes can direct selection be used for?
It works with some prokaryotic (bacterial) genes as bacterial genes have similar regulatory regions but cannot be used with eukaryotic genes.
Give an example of direct selection.
Cloning the KanR gene from plasmid R6-5 into pBR322. The plasmid is cut using EcoRI and then these fragments are plated out on kanamycin agar after being ligated into pBR322. Only the KanR recombinants can grow.
What is marker rescue?
A variant on direct selection which uses an E.coli mutant that is deficient in the gene you want to clone (auxotrophic mutant) the gene compliments the mutant and “rescues” the genetic marker mutation in the E.coli host strain.
What does marker rescue work best for?
Bacterial genes coding for enzymes in metabolic pathways, as these enzymes are often essential for growth on particular substrates.
What is an example of marker rescue?
Cloning for the enzymes to make tryptophan (enzyme = trpA). trpA on the recombinant plasmids complements the trpA mutation in the host and rescues the marker. This means that on minimal medium, only trpA recombinants can survive.
Why can eukaryotic genes not normally be identified directly?
They are not normally expressed in bacteria.
What is the first step in identifying a eukaryotic gene from clones?
Searching literature to see if the gene of interest has already been cloned from another organism in another lab - due to common ancestors there will be some degree of similarity.
What can be done if the first step is successful for identifying eukaryotic genes from clones?
Ask for a clone of the gene and sign and Material Transfer Agreement form and wait for the clone to arrive. Nucleic acid hybridisation can be completed.
What is nucleic acid hybridisation?
Uses sequence complementarity as a basis to identify the DNA of interest from a variety of clones, depending on how well the DNA hybridises to the known gene.
What are the steps in nucleic acid hybridisation?
The DNA is heated or treated with alkali to break the hydrogen bonds (denaturation). Under some conditions the DNA will try to base pair chain dependent on sequence complementarity between the two strands. The degree of hybridisation between the two strands can be determined by the difficulty to re-separate the strands and is called stringency.
What is stringency affected by?
Temperature and salt concentration.
How can stringency be controlled so the clone can be identified?
Using different combinations of temperature and salt so that molecules below a certain percentage similarity will separate and those above will not.
What is heterologous probing?
Using similarities between the same gene from different organisms to find either gene.
What is the process for heterologous probing?
Immobilise library B onto a nylon membrane and denature the DNA to separate the strands. Make the gene from A radioactive and denature the gene probe. Hybridise gene A with library B and wash off non-specifically bound probes. Expose the membrane on an X-ray film and pick out the correct clone from library B where the black spots are.
What preparation of the library is needed in heterologous probing?
Must be transferred to the nylon membrane and denatured and immobilised on the membrane. The sugar phosphate backbone must be stuck to the membrane.
What is produced when the hybrids are exposed to an X-ray film?
An autoradiograph.
What is DNA often radioactively labelled with?
A radioactive isotope of phosphorus 32 due to phosphate being in DNA. It is is the alpha phosphate that is labelled as the beta and gamma are lost in the formation of the phosphodiester bond.
What is random priming?
A method used to label DNA with 32P.
How does random priming work?
The DNA is denatured and random hexamer oligonucleotides are annealed. These serve as primers. Add Klenow polymerase from DNA pol I, dNTPs and radioactively labelled dATP. Synthesise the labelled DNA, denature the probe and hybridise it to the library. The Klenow Pol will synthesise a complementary strand with the radioactive phosphorus combined.
What else can heterologous probing be used for?
To identify similar sequences between species and within species.
