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Detection
and
Selection
of
Clones
Genes -
Having discussed enzymes and cloning vehicles, let us now see how these two can be employed to prepare recombinant DNA molecules. A collection of fragments obtained by digestion with a restriction enzyme can be made to anneal with a cleaved vector molecule, yielding a large number of hybrid vectors containing different fragments of foreign DNA.
In one method if the DNA of interest is known to De contained in a particular restriction fragment, that fragment can be isolated from a gel after electrophoresis and joined to an appropriate vector. However, eukaryotic cells contain about a million cleavage sites for a typical restriction enzyme; so direct isolation of a eukaryotic gene from a mixture of fragments separated by electrophoresis is not feasible.
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Another technique for selecting a particular DNA molecule depends on an unusual polymerase, reverse transcriptase, which can use a single stranded RNA molecule (such as mRNA) as a template and synthesize a double stranded DNA copy, called complementary DNA or cDNA. If the template RNA molecule is an mRNA molecule, the corresponding full length cDNA will contain an uninterrupted coding sequence. These DNA copies are selected to be introduced into a vector.
Foreign DNA can be cut into segments by a restriction enzyme that also makes one cut in the plasmid vehicle. In solution the two DNAs can come together to produce a larger circular DNA held together by hydrogen bonding of the complementary ends. In the presence of the enzyme polynucleotide ligase, the single stranded gaps in the sugar phosphate backbones are sealed and the structure is stabilized. The result is a recombinant DNA molecule.
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The mRNA fraction is copied into single-stranded DNA using reverse transcriptase. The enzyme can only add residues to a 3' -OH group of an existing primer, which is base-paired with the template. Oligo dT is hybridized to the poly (A) tail in order to supply this primer. The RNA strand of the resultant RNA-DNA hybrid is destroyed by alkaline hydrolysis prior to second-strand synthesis. The second strand synthesis reaction is carried out using either DNA polymerase I or reverse transcriptase and is self priming.
The single stranded cDNA has a transitory hairpin structure at the 3' -end which is stabilized by second strand synthesis. The hairpin loop and any single stranded overhang at the other end are then digested away with the single strand specific SI nuclease. The final product is a population of double stranded, blunt ended DNA molecules complementary to the original mRNA fraction.
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The double stranded DNA is now ready for insertion into the plasmid. Homopolymer tailing is probably the most commonly used, but not the only method of inserting cDNA into a plasmid.
A string of cytosine residues is added to the cDNA, using the enzyme terminal transferase, to form oligo dC tails on the 3' -ends. Similarly, a plasmid is cut open at a unique restriction endonuclease site and tailed with oligo-dG. The homopolymer tails of the cDNA and plasmid then pair to form a circular recombinant plasmid carrying a cDNA insert.
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Small plasmids are usually used for cDNA cloning. They accommodate only a relatively small piece of foreign DNA to be included into it. Large plasmids do not respond favourably in cloning experiments. It is generally desirable to clone larger pieces of genomic DNA since large clone is more likely to contain an intact copy of a gene I plus some of the flanking sequences. Cloning large pieces also brings the number of different clones needed to cover the entire genome down to a manageable number.
Genomic cloning is often carried out by using the bacteriophage lambda as a vector. The phage genome is double-stranded and approximately 50 kbp in length. Since its first use as a cloning vector in 1974, many different vectors have been constructed which enable replacement of part of the lambda genome with foreign DNA. Part of the central portion is nonessential and so can be replaced without detriment to phage reproduction; this type of construct is termed a replacement vector.
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High molecular weight genomic DNA is partially digested with a restriction enzyme and then size fractionated to about 20 kbp. The nonessential region of the phage genome is removed with the same enzyme and the vector arms purified. The vector arms and the genomic fragments are annealed together; because the genome has cohesive ends it can as well anneal to the foreign DNA, to form a concatamer.
During in vitro packaging endonucleolytic cleavage occurs at the cohesive ends (the "cos" site) to reduce the DNA to unit lengths. The phage particles are then used to infect E. coli for amplification of the DNA. Once inside the host, the DNA replicates as a plasmid because the phage genes required for replication are not present. These vectors are called cosmids and recombinants can be selected on the basis of drug resistant plasmid.
Once recombinant DNA has been produced, hybridization techniques similar to those used for cDNA clones can be applied to identify individual clones. To facilitate isolation of a vector containing a particular gene, some means is needed to ensure, first, that a vector does possess an inserted DNA fragment and second, that it is the DNA segment of interest.
Before the recombinant DNA can be bulked up it must be taken up by a suitable "competent" bacterial host cell. These recombinant plasmids are used to transform bacteria. The host bacterial cell must accept the plasmid with the foreign gene, get it incorporated into its genome and start transcribing that gene.
Bacterial cells treated with calcium chloride will take up plasmid molecules from the surrounding medium and the host cell will repair any gaps in the recombinant plasmids; now the bacterium is said to be transformed. Usually a strain of E. coli which lacks a restriction system to degrade foreign DNA is selected.
The selection of transformed cells usually depends on their resistance to an antibiotic. So it is important to incubate the cells in a medium without antibiotic for about an hour, to allow the plasmid antibiotic resistant genes to be expressed. If the plasmid has been chosen carefully, it is possible to select transformed from non transformed bacteria on the basis of antibiotic resistance. The cells can be plated on a solid medium containing antibiotic for selection of colonies with recombinant DNA.
Only cells in which a plasmid has become established will form a colony. In the case of pBR 322, cloning into the unique Pst 1 site destroys ampicillin resistance but leaves tetracycline resistance intact. Bacteria transformed with a recombinant plasmid will be sensitive to ampicillin but resistant to tetracycline. Selection of transformed colonies is done using the colony hybridization technique with radioactive probes.
Depending on the specificity of the probe, this can lead to rapid identification of one colony among many thousands. A nitrocellulose filter disk is placed on the surface of an appropriate agar plate and the colonies are transferred to this disk. A replicate agar plate is retained as a reference set.
The colonies are grown and the nitrocellulose disk carrying the colonies then removed. The bacteria are lyzed and the DNA is denatured by treating the disk with alkali. The DNA will adhere to the nitrocellulose while protein is removed and debris washed away. DNA is firmly fixed in position by backing at 80°C
A radiolabeled probe is hybridized to the DNA on the filter. The DNA probe can be labeled by "nick" translation, a process in which nicks are introduced into a DNA sequence and the enzyme DNA polymerase then digests away from the nick while replacing the strand as it proceeds.
A labeled nucleotide is incorporated in the replacement strand. Any colony carrying a sequence complementary to the probe will, during hybridization, become radiolabeled and can be identified by autoradiography. Any clone showing a positive result can be picked from the master plate and grown to provide DNA for further analysis. After selecting colonies with cloned genes, they can be multiplied to obtain the desired results.
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