Clone identification

Question 1

What is the problem with identifying clones in genomic or cDNA libraries?

Answer 1

They are very large and it can be hard to identify the gene of interest amongst many other colonies/plaques.

Question 2

What are the two strategies that can be used to identify the clone?

Answer 2

Direct selection or clone identification via shotgun cloning.

Question 3

How does direct selection work?

Answer 3

Set up the process so that only the desired clone can survive. The selection for the desired clone occurs at the plating out stage.

Question 4

How does shotgun identification work?

Answer 4

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.

Question 5

What genes can direct selection be used for?

Answer 5

It works with some prokaryotic (bacterial) genes as bacterial genes have similar regulatory regions but cannot be used with eukaryotic genes.

Question 6

Give an example of direct selection.

Answer 6

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.

Question 7

What is marker rescue?

Answer 7

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.

Question 8

What does marker rescue work best for?

Answer 8

Bacterial genes coding for enzymes in metabolic pathways, as these enzymes are often essential for growth on particular substrates.

Question 9

What is an example of marker rescue?

Answer 9

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.

Question 10

Why can eukaryotic genes not normally be identified directly?

Answer 10

They are not normally expressed in bacteria.

Question 11

What is the first step in identifying a eukaryotic gene from clones?

Answer 11

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.

Question 12

What can be done if the first step is successful for identifying eukaryotic genes from clones?

Answer 12

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.

Question 13

What is nucleic acid hybridisation?

Answer 13

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.

Question 14

What are the steps in nucleic acid hybridisation?

Answer 14

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.

Question 15

What is stringency affected by?

Answer 15

Temperature and salt concentration.

Question 16

How can stringency be controlled so the clone can be identified?

Answer 16

Using different combinations of temperature and salt so that molecules below a certain percentage similarity will separate and those above will not.

Question 17

What is heterologous probing?

Answer 17

Using similarities between the same gene from different organisms to find either gene.

Question 18

What is the process for heterologous probing?

Answer 18

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.

Question 19

What preparation of the library is needed in heterologous probing?

Answer 19

Must be transferred to the nylon membrane and denatured and immobilised on the membrane. The sugar phosphate backbone must be stuck to the membrane.

Question 20

What is produced when the hybrids are exposed to an X-ray film?

Answer 20

An autoradiograph.

Question 21

What is DNA often radioactively labelled with?

Answer 21

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.

Question 22

What is random priming?

Answer 22

A method used to label DNA with 32P.

Question 23

How does random priming work?

Answer 23

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.

Question 24

What else can heterologous probing be used for?

Answer 24

To identify similar sequences between species and within species.

Question 25

What should you do if you don’t have a heterologous clone from a colleague?

Answer 25

You will need to use other methods. Usually, your interest in the gene is due to the interest in the enzyme which you will already have a lot of data on, so can be easier to find clone.

Question 26

What method should be used to find a gene without a heterologous clone?

Answer 26

Synthesise an oligonucleotide or raise an antibody to it and use these to identify your clone.

Question 27

How can the amino acid sequence of the desired gene be used to find the clone?

Answer 27

Use the genetic code to reverse translate the amino acids to nucleotides and design an oligonucleotide probe (a short, synthetic single-stranded DNA sequence). Radio label the oligonucleotide and use it to probe the library.

Question 28

What must be taken into account when synthesising the oligonucleotide?

Answer 28

The genetic code is degenerate so this must be taken into account and all possible sequences must be synthesised.

Question 29

What are the olignucleotides labelled with?

Answer 29

Gamma labelled dNTP (32 phosphate) at the end, the alpha phosphate does not need to be labelled.

Question 30

How can the oligonucleotides be used to find the clone?

Answer 30

Hybridise them to the library and immobilise them on the nylon membrane.

Question 31

How can a second oligonucleotide be used to identify false positives?

Answer 31

Using another oligo from another area of the sequence, as the first oligo might bind to other things so a second oligo can eliminate these false positives.

Question 32

How can antibodies be prepared for clone identification?

Answer 32

By raising a polyclonal antibody by injecting purified protein into a rabbit (“your” protein), taking the blood and purifying the antibody from the blood. This can then be used to identify clones in a cDNA library that are constructed in certain insertion vectors like lambdagt11.

Question 33

How can the antibodies be used to identify the clone?

Answer 33

1/6 of the cDNA inserts into the EcoRI site in the lacZ gene will express a fusion protein with part of the amino acid sequence of your protein. The antibody will bind to this and it can be marked with a radioactive tag or a secondary antibody with a visual tag to identify it.

Question 34

What is IPTG?

Answer 34

An artificial inducer of the LacZ gene.

Question 35

What is the fusion protein?

Answer 35

Made up of part beta galactosidase and part of whatever the cDNA encodes.