Shotgun cloning
Shotgun cloning is a method used in molecular biology to randomly insert a diverse array of DNA fragments into cloning vectors, which are typically plasmids. This technique generates numerous recombinant DNA molecules that are then introduced into host cells, such as bacteria, where they can be amplified. The primary advantage of shotgun cloning is the increased probability of isolating a clone that contains a fragment of interest, facilitating the study of specific DNA sequences.
The process begins with isolating DNA from the organism of interest and digesting it with restriction enzymes, which cut the DNA at specific sites. The resulting fragments are mixed with a cloning vector, allowing them to join randomly. Once sealed with DNA ligase, these recombinant molecules are introduced into bacterial cells. By selecting for specific marker genes within the vectors, researchers can identify and grow colonies that carry the desired recombinant DNA, ultimately creating a genomic library.
While shotgun cloning remains a valuable technique, advancements in DNA sequencing technologies have begun to limit its use, as newer methods allow for the cloning of longer gene sequences more efficiently. Alternatives like size-selection through gel electrophoresis and the creation of complementary DNA (cDNA) libraries are also employed to streamline cloning processes.
Shotgun cloning
SIGNIFICANCE: Shotgun cloning is the random insertion of a large number of different DNA fragments into cloning vectors. A large number of different recombinant DNA molecules are generated, which are then introduced into host cells, often bacteria, and amplified. Because a large number of different recombinant DNAs are generated, there is a high likelihood one of the clones contains a fragment of DNA of interest.
Recombinant DNA Cloning and Shotgun Cloning
Before the development of recombinant DNA cloning, it was very difficult to study DNA sequences. Cloning a DNA fragment allows a researcher to obtain large amounts of that specific DNA sequence to analyze without from the presence of other DNA sequences. There are many uses for a cloned DNA fragment. For example, a DNA fragment can be sequenced to determine the order of its nucleotides. This information can be used to determine the location of a gene and the sequence of the gene’s protein product. Cloned pieces of DNA are also useful as DNA probes. Because DNA is made of two strands that are complementary to each other, a cloned piece of DNA can be used to for copies of the same or similar DNA sequences in other samples. A cloned gene can also be inserted into an where it will produce the gene’s protein product.
Shotgun cloning begins with the isolation of DNA from the organism of interest. In separate test tubes, the DNA to be cloned and the DNA are digested (cut) with a restriction that cuts the in just one location and the many times. Many restriction endonucleases create single-stranded ends that are complementary, so the end of any DNA molecule cut with that endonuclease can join to the end of any other DNA cut with the same endonuclease. When the digested vector and foreign DNA are mixed, they join randomly and are then sealed using DNA ligase, an that seals the small gap between two pieces of DNA. This creates recombinant DNA molecules composed of a copy of the vector and a random copy of foreign DNA. The recombinant DNA molecules are then introduced into host cells where the cloning vector can replicate each time the cell divides, which is approximately every twenty minutes in the case ofEscherichia coli. The resulting collection of clones, each containing a potentially different fragment of foreign DNA, is called a genomic library. If a large collection of clones is produced, it is likely that every part of the genome from which the DNA came will be represented somewhere in the genomic library.
The presence of the cloning vector in host cells is determined by selecting for a marker gene in the cloning vector. Most vectors have two marker genes, and often both are different resistance genes. A common example is the plasmid pBR322, which has a tetracycline and an ampicillin resistance gene. A cuts once somewhere in the tetracycline resistance gene, and if a foreign DNA fragment becomes incorporated, the resulting recombinant will have a nonfunctional tetracycline resistance gene. A bacterial cell transformed with a recombinant plasmid will therefore be resistant to ampicillin but will be sensitive to tetracycline. Many plasmids will not incorporate any foreign DNA and will be nonrecombinant. Cells that are transformed with a nonrecombinant plasmid will be resistant to both tetracycline and ampicillin. After the bacterial cells have been transformed, they are grown on a medium with ampicillin. The only cells that will survive will be those that have received a plasmid vector. To determine which cells have received a recombinant plasmid, the colonies are carefully transferred onto a new medium that has both ampicillin and tetracycline. On this medium, only cells with nonrecombinant plasmids will survive. Thus, colonies that grew on the first medium, but not on the second, contain recombinant plasmids. Cells from these colonies are collected and grown, each in a separate tube, and these constitute a genomic library.
Once a has been produced, the DNA fragments contained in it can be screened and analyzed in various ways. Using the right techniques, specific genes can be found, which can then be used in future analyses and experiments.
Alternatives to Shotgun Cloning
In shotgun cloning, many different DNA fragments from an organism are cloned, and then the specific DNA clone of interest is identified. The number of clones can be reduced, making the search easier, if the DNA of interest is known to be in a restriction endonuclease fragment of a specific size. DNA can be size-selected before cloning using gel electrophoresis, in which an electric current carries DNA fragments through the pores or openings of an gel. DNA migrates through the gel based on DNA fragment size, with the smaller fragments traveling more rapidly than the larger fragments. DNA of a specific size range can be isolated from the gel and then used for cloning. Finally, to clone a piece of DNA known to code for a protein, scientists can use an enzyme called reverse transcriptase to make DNA copies (called a complementary DNA or cDNA) of isolated messenger RNA (mRNA). The cDNA is then cloned, in a similar manner to that already discussed, to produce what is called a cDNA library. One of the advantages of this approach is that the number of clones is greatly reduced.
Although the process of shotgun cloning was still used in the 2020s, technological advancements allowing for the production of longer gene sequences had begun to limit its use.
Key Terms
- cloning vectora plasmid or virus into which foreign DNA can be inserted to amplify the number of copies of the foreign DNA
- markera gene that encodes an easily detected product that is used to indicate that foreign DNA is in an organism
- recombinant DNAa novel DNA molecule formed by the joining of DNAs from different sources
- restriction endonucleasean enzyme that recognizes a specific nucleotide sequence in a piece of DNA and causes cleavage of the DNA; often simply called a restriction enzyme
Bibliography
Glick, Bernard R. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 4th ed. Washington: ASM, 2010. Print.
Godiska, Ronald, et al. “Beyond pUC: Vectors for Cloning Unstable DNA.” DNA Sequencing: Optimizing the Process and Analysis. Ed. Jan Kieleczawa. Sudbury: Jones, 2005. Print.
Graham, Colin A., and Alison J. M. Hill, eds. DNA Sequencing Protocols. 2nd ed. Totowa: Humana, 2001. Print.
Green, Eric. "Shotgun Sequencing." National Human Genome Research Institute, 9 Sept. 2024, www.genome.gov/genetics-glossary/Shotgun-Sequencing. Accessed 9 Sept. 2024.
Kreuzer, Helen, and Adrianne Massey. Recombinant DNA and Biotechnology: A Guide for Students. 3rd ed. Washington: ASM, 2008. Print.
Kreuzer, Helen, and Adrianne Massey. Recombinant DNA and Biotechnology: A Guide for Teachers. 3rd ed. Washington: ASM, 2008. Print.
Micklos, David, Greg A. Freyer, and David A. Crotty. DNA Science: A First Course. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2003. Print.
Sambrook, Joseph, and David W. Russell. “DNA Sequencing.” Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2012. Print.
Watson, James D., et al. The Molecular Biology of the Gene. 7th ed. San Francisco: Pearson, 2014. Print.
"What Is Shotgun Sequencing?" Yourgenome.org, 2024, www.yourgenome.org/theme/what-is-shotgun-sequencing/. Accessed 9 Sept. 2024.