Cloning

The word “cloning” has several different meanings. For example, it is possible to clone cells, that is, to cause cells to reproduce themselves so as to make a population of identical cells. In molecular biology, any piece of DNA can be cloned—inserted into a vector that replicates in a host to produce many copies of the same recombinant vector. It is also possible to clone genes. Gene cloning is the process of identifying and isolating a specific gene of interest. Cloning genes is a major focus of molecular biology, and the ability to clone genes has revolutionized biology. Once a gene is identified and cloned into an appropriate vector, it can be manipulated in many different ways. Cloning dramatically amplifies the DNA so that it can be sequenced. A cloned gene inserted into an expression system creates an organism that produces up to 25% of its total protein or more as the gene product, allowing large-scale production of the protein (1). A cloned gene can be mutated, and the mutated form inserted back into an organism that lacks a functional copy of the gene (2). This allows structure-function analysis of the gene product. A cloned gene or set of genes can be introduced into a new host to create a new metabolic pathway or to modify an existing pathway (3).

In many cases, gene cloning is carried out by fragmenting the genomic DNA from the organism containing the gene of interest and inserting the fragments into self-replicating DNA molecules) vectors) using recombinant DNA techniques; transforming the resultant population of recombinant molecules, which comprise a DNA library, into a host organism, screening or selecting the transformants to identify cells that contain the desired DNA, and isolating the vector DNA.

1. Preparation of DNA for Cloning

 Genomic DNA must be fragmented before cloning into a suitable vector. It is important to have fragments that contain the gene intact. One way to ensure this is to use a restriction enzyme that makes frequent cuts in the DNA under conditions of partial digestion (so that only some of the sites

are cleaved) so as to obtain fragments of the desired size. The restriction enzyme Sau3A is often used because it has a four-base recognition sequence and therefore cuts DNA at many sites in the genome. Additionally, it produces cohesive, sticky ends that ligate with BamHI, a site often present in polylinkers. Mechanical shearing also fragments the DNA, but sheared DNA cannot be cloned directly. First, the ragged ends must be filled in and polylinkers added.

 Because small fragments of DNA ligate to a vector more readily than large fragments, the DNA to be cloned is often fractionated by sucrose density gradient centrifugation  to obtain DNA of the desired size (pp. 2.85–2.86 of Ref. 4). It is important to set up the conditions for ligation so that most of the recombinant molecules produced contain only a single DNA fragment. This means that nearly equal numbers of vector molecules and fragment molecules should be present and that the total DNA concentration should not be too high (~40 µg of vector DNA per mL).

 It is necessary to produce a large enough number of transformants or recombinant bacteriophage to have a high probability that a fragment carrying the intact gene is present in the population. The exact number depends on the size of the genome from which the gene is being cloned (the larger the genome, the more transformants need to be screened) and the size of the inserts (the larger the insert size, the fewer transformants need to be screened). A major problem in cloning is restriction enzymes in the host, which can degrade foreign DNA. Mutant strains of Escherichia coli are available that lack restriction enzymes, and these are best for constructing large libraries for screening or selection. Once a gene is cloned, restriction enzymes in a host are not usually a problem because large amounts of DNA overcome the restriction barrier (5).

Prokaryotic genes are usually cloned from genomic DNA, whereas eukaryotic genes are often cloned from complementary DNA (cDNA), which is prepared by copying messenger RNA with reverse transcriptase. Most eukaryotic genes contain introns, which prevent them from being expressed in prokaryotes, but the introns are not in the cDNA. The cDNA clone is often used as a probe to isolate the gene from a genomic library. The genomic clone can be sequenced to identify the nature and location of the introns and the upstream and downstream sequences of the gene.

2. Cloning Vectors

There are four general types of vectors that are used in E. coli: plasmids, phage, cosmids or bacterial artificial chromosomes (BACs):

1. Plasmids have the advantage that they are smaller than the other vectors and usually give larger amounts of vector DNA from a given volume of culture because of their high copy number.

2. Phage vectors allow larger sized inserts of foreign DNA, so that a smaller number of plaques are needed to give a high probability that the entire genome of the organism is present in the library that is being screened. Furthermore, phage systems give more plaques per weight of DNA than plasmids give colonies, and plaques are easier to screen than colonies because they are smaller and more uniform. Phage T4 vectors allow cloning inserts as large as 120 kbp (6), whereas phage P1 vectors allow 80-kbp inserts (7), and lambda phage vectors allow up to 40-kbp inserts (pp. 2.2 to 2.125 of Ref. 4). Once a positive plaque is identified, the insert DNA is subcloned into a plasmid vector to take advantage of the greater amplification and ease of DNA preparation associated with plasmid vectors.

3. Cosmid vectors combine features of the preceding two classes because they can be packaged into phage but also replicate as plasmids. They also allow inserts up to 40 kbp (8).

4. Bacterial artificial chromosomes are based on the single-copy F factor plasmid and accept >300-kbp DNA fragments (9). Another important system for cloning very large DNA pieces is yeast artificial chromosomes (YAC). YACs accept fragments over 200 kbp and are used in genome sequencing and positional cloning of genes (10). Although yeast is not as easy to work with as E. coli, it is useful for studying eukaryotic genes that cannot be expressed in E. coli.

3. Identifying the Gene of Interest

Screening is usually the most difficult step, and there are many different approaches available. The simplest method is to complement a mutant gene in a host organism, which allows direct selection of the desired clone (11). A powerful example of direct selection is cloning a transposon-tagged gene where selection is for antibiotic resistance encoded in the transposon.

Another approach is to screen transformant colonies for the gene product, either with specific antibodies  or by an enzymatic assay (12). This requires a sensitive, specific assay because foreign genes are often expressed at a low level, and enzymes from the host organism must not interfere with the assay. In addition, the gene to be cloned needs to be expressed in the host organism.

 The most general screening approach is to transfer the colonies to a filter, lyse the cells, denature the DNA on the filter and hybridize a labeled nucleic acid probe to the filter. The probe pairs with complementary sequences in the DNA on the filter, and the colony to which the probe hybridizes is visualized by the label. There are various methods to design or obtain an oligonucleotide probe that specifically hybridizes to the desired gene.

If one wants to clone the gene coding for a known protein, it is possible to sequence the protein, usually at its N-terminus, and to use reverse translation to determine the probe sequence. However, sequences from internal peptides produced by specific cleavage of the polypeptide chain (eg, by an arginine-specific proteinase or by cyanogen bromide cleavage at methionine residues) are also used if the N-terminus is blocked or if the N-terminal sequence is unsuitable for reverse translation.

Another method is using a segment of the DNA from the gene of a closely related organism as the hybridization probe (13). A modification of this approach is looking at homologous sequences from a set of organisms to identify highly conserved regions that are used to design an oligonucleotide probe specific for the desired gene. It is important to choose organisms with DNA base compositions close to that of the organism from which the gene is to be cloned.

With the large amount of genomic and cDNA sequence information  becoming available in computer databases, genes of interest are frequently identified during database searches. Hybridization probes are designed from the sequence information and used to screen a library or to amplify the gene directly.

An ingenious way of selecting for a full-length clone in a phage vector, when a partial clone is available, is inserting the sequence next to the supF gene in a plasmid, infect recA+ cells carrying the plasmid with the phage library, and plate the resulting phage on an E. coli strain, where only phage containing supF grows (14). Recombination between the complementary sequences introduces the supF only into the desired phage.

4. Positional Cloning of a Gene Identified by its Mutant Phenotype

Often genes are identified because they have an interesting phenotype when mutated. The mutation is mapped by using DNA-based markers. The most closely linked marker is used as a hybridization probe to screen a YAC, BAC, or cosmid library, thus initiating a chromosome walk to the gene (15) . Once a library clone is identified that is deemed to contain the gene of interest, it is subcloned into a vector used to transform the mutant. The subclone containing the gene is identified by its ability to complement the mutant phenotype.

4.1. Potential Problems with Library-Based Cloning 

One problem with direct selection is potential contamination of the library with host DNA, which leads to cloning a host gene, rather than the gene from the original organism. Therefore it is necessary to show that genes isolated by complementation are from the desired organism, which can be done by a Southern blot. Other potential cloning problems are that some DNA fragments cannot be cloned because they contain a poison sequence. DNA fragments containing repeated DNA sequences are often unstable, because they recombine easily. So recA mutant strains of E. coli are often used as a host for cloning to minimize recombination (16).

 A major problem in cloning a DNA fragment that has identical ends (cohesive or blunt) is recircularization of vector molecules that do not contain an insert because the two ends of the cut vector are also identical. This gives a high background of transformants or plaques lacking an insert. One method of minimizing this problem is treating the cut vector with calf alkaline phosphatase to remove its 5′ phosphate groups. Dephosphorylated vector molecules cannot circularize, but they ligate to the 5′-phosphate groups on the DNA fragments to be cloned. The resulting recombinant molecule circularizes, producing a circular molecule with a single-stranded nick in each strand. These molecules give transformants. It is necessary to completely inactivate the alkaline phosphatase before the DNA to be cloned is added to the vector. The dephosphorylation reaction must be run under conditions that remove most 5′-phosphate groups but do not inactivate the plasmid, so the reaction has to be monitored carefully.

Another method of dealing with the problem of vector molecules lacking inserts is blue-white screening. Many E. coli vectors contain an N-terminal portion of the beta-galactosidase gene that codes for the a-fragment that complements the inactive b-fragment. A polylinker is introduced into the b-galactosidase gene so that the a-fragment is inactivated when a DNA molecule is ligated into the polylinker. The ligation mixture is transformed into an E. coli host that produces the b-fragment, and the transformants are plated on selective plates. The colonies that contain an insert are white, whereas colonies without an insert are blue.

When a probe that hybridizes to the gene to be cloned is available, the cloning process is simplified by using the probe to identify an appropriately sized restriction fragment that contains the gene by using Southern blots run on digests of a number of restriction enzymes. The appropriately sized fragments are eluted from a preparative agarose gel electrophoresis run on genomic DNA digested with the chosen restriction enzyme. The fragments are ligated into a vector cut with a compatible restriction enzyme, and the resulting transformants are screened with the probe. This process significantly reduces the number of colonies or plaques that need to be screened (1 of 40 positive vs. 1 of 1,000 for a Thermomonospora fusca gene) (17).

5. PCR to Clone Genes

Whenever sequence information is available for a gene, it is possible to design PCR primers that amplify all or part of a gene directly from DNA isolated from the desired organism. The PCR product is used as a probe to screen for the clone, or the PCR product is cloned.

6. Insertional Mutagenesis

 Transposons (see Transposon Tagging) and retroviruses are used as insertional mutagens to “tag” genes. If a previously identified transposon or retrovirus inserts into a gene, the sequence of the transposon or retrovirus is used as a tag to identify the host DNA flanking the insertion. Techniques, such as IPCR, plasmid rescue, and library screening (using the element as a probe) are all successful in identifying flanking DNA from a “tagged” individual.

7. Cloning Tissue- or Treatment-Specific Genes

 In some cases, researchers do not want to identify a specific gene, but instead they are interested in a certain class of gene, for example, liver-specific genes or genes induced by a hormone. There are numerous methods for identifying such specific types of gene, including subtractive hybridization, differential display, database analysis, and analysis of microarrays.

8. Immunochemical Methods

Another method for cloning a gene, where the protein has been identified previously, is using an antibody that recognizes the protein to immunoprecipitate polysomes making the protein and then to isolate its mRNA. The mRNA produces a cDNA copy that is cloned. The cDNA clone is also used   as a probe to isolate a genomic clone.

9. Conclusion

There are a large number of ways to clone a gene or set of genes of interest. The method used depends on what materials are available, such as protein sequence, antibodies to the protein coded for by the gene, gene sequence information, useful libraries for screening, or a “tagged” or “untagged” mutation in the gene of interest. The purpose of cloning a gene is to generate large amounts of DNA for further analysis, such as sequencing or mutational analysis. A cloned gene is also useful for producing the protein or for antisense studies, and in some cases it is used to disrupt the endogenous gene by homologous recombination. A cloned gene is also useful as a probe in gene expression studies. Cloning a gene is one of the first steps to understanding its function.

References

1. C. Gellissen and L. P. Hollenberg (1997) Gene 190, 87–97. 

2. S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease (1989) Gene 77, 51–59. 

3. R. A. Dixon, C. J. Lamb, S. Masoud, V. J. H. Sewalt, and N. L. Paiva (1996) Gene 179, 61–71. 

4.J. Sambrook, E. F. Fritsch, and T. Maniatis (1989) Molecular Cloning; a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

5.G. G. Wilson and N. E. Murray (1991) Annu. Rev. Genet. 25, 585–562.  6. V. B. Rao, V. Thaker, and L. W. Black (1992) Gene 113, 25–33. 

7. J. C. Pierce and N. L. Sternberg (1992) Methods Enzymology 216, 549–574. 

8. N. Fairweather (1997) Methods Mol. Biol. 68, 137–148. 

9. B. A. C. Shizuya, B. Birren, U.-J. Kim, V. Mancino, T. Slepak, Y. Tarhiri, and M. Simon (1992) Proc. Natl. Acad. Sci. USA 89, 8794–8797.

10. D. T. Burke, G. F. Carle, and M. V. Olson (1987) Science 236, 806–812. 

11. D. Mazel, E. Loic, S. Blanchard, W. Savrin, and P. Marliere (1997) J. Mol. Biol. 266, 939–949. 

12. R. M. Teather and P. J. Wood (1982) Appl. Environ. Microbiol. 43, 777–780. 

13. J. Agnan, C. Korch, and C. Selitrennikoff (1997) Fungal Genet. Biol. 21, 292–301. 

14. A. J. Hanzlik, M. M. Osemlak-Hanzlik, and D. M. Kurnit (1992) Gene 122, 171–174. 

15. S. D. Tanksley, M. W. Ganal, and G. B. Martin (1995) Trends Genet. 11, 63–68. 

16. A. C. M. Radding (1982) Annu. Rev. Genet. 16, 405–437. 

17. S. Zhang, G. Lao, and D. B. Wilson (1995) Biochemistry 34, 3386–3395.