Gene Cloning and Analysis

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Overview

Gene cloning is a common practice in molecular biology labs that is used by researchers to create copies of a particular gene for downstream applications, such as sequencing, mutagenesis, genotyping or heterologous expression of a protein. The traditional technique for gene cloning involves the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element, such as a bacterial plasmid. This technique is commonly used today for isolating long or unstudied genes and protein expression. A more recent technique is the use of polymerase chain reaction (PCR) for amplifying a gene of interest. The advantage of using PCR over traditional gene cloning, as described above, is the decreased time needed for generating a pure sample of the gene of interest. However, gene isolation by PCR can only amplify genes with predetermined sequences. For this reason, many unstudied genes require initial gene cloning and sequencing before PCR can be performed for further analysis.

Related Topics: Gene Expression Analysis, Mutational Analysis, and Epigenetics and Chromatin Structure.

DNA Sequencing

DNA sequencing is typically the first step in understanding the genetic makeup of an organism, which helps to:

  • Locate regulatory and gene sequences
  • Compare homologous genes across species
  • Identify mutations

Sequencing uses biochemical methods to determine the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a DNA oligonucleotide. Knowing the sequence of a particular gene will assist in further analysis to understand the function of the gene. PCR is used to amplify the gene of interest before sequencing can be performed. Many biotechnology companies offer sequencing instruments, however, these instruments can be expensive. As a result, many researchers usually perform PCR in-house and then send out their samples to sequencing labs.

Site-Directed Mutagenesis

Site-directed mutagenesis is a widely used procedure for the study of the structure and function of proteins by modifying the encoding DNA. By using this method, mutations can be created at any specific site in a gene whose wild-type sequence is already known. Many techniques are available for performing site-directed mutagenesis. A classic method for introducing mutations, either single base pairs or larger insertions, deletions, or substitutions into a DNA sequence, is the Kunkel method.

The first step in any site-directed mutagenesis method is to clone the gene of interest. For the Kunkel method, the cloned plasmid is then transformed into a dut ung mutant of Escherichia coli. This E. coli strain lacks dUTPase and uracil deglycosidase, which will ensure that the plasmid containing the gene of interest will be converted to DNA that lacks Ts and contains Us instead.

The next step is to design a primer that contains the region of the gene which you wish to mutate, along with the mutation you want to introduce. PCR can then be used with the mutated primers to create hybrid plasmids; each plasmid will now contain one strand without the mutation and uracil bases, and another strand with the mutation and lacking uracil.

The final step is to isolate this hybrid plasmid and transform it into a different strain that does contain the uracil-DNA glycosylase (ung) gene. The uracil deglycosidase will destroy the strands that contain uracil, leaving only the strands with your mutation. When the bacteria replicate, the resulting plasmids will contain the mutation on both strands.

Genotyping

Genotyping is the process of determining the DNA sequence specific to an individual's genotype. This process can be accomplished by several techniques, such as high resolution melt (HRM) analysis, or any other mutation detection technique. All of these techniques will provide an insight into the individual's genotype, which can help determine specific sequences that can be manipulated and cloned for further analysis.

Heterologous Protein Expression

Heterologous protein expression uses gene cloning to express a protein of interest in a self-replicating genetic element, such as a bacterial plasmid. Heterologous expression is used to produce large amounts of a protein of interest for functional and biochemical analyses.

Further Reading

Araya-Garay JM et al. (2011). cDNA cloning of a novel gene codifying for the enzyme lycopene β-cyclase from Ficus carica and its expression in Escherichia coli. Appl Microbiol Biotechnol 92, 769–777. PMID: 21792589

Eckert C et al. (2006). DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 57, 14–23. PMID: 16291869

Handa P and Varshney U (1998). Rapid and reliable site-directed mutagenesis using Kunkel's approach. Indian J Biochem Biophys 35, 63–66. PMID: 9753863

Hanner M et al. (1996). Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci USA 93, 8072–8077. PMID: 8755605

Kerovuo J et al. (1998). Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol 64, 2079–2085. PMID: 9603817

Lauretti L et al. (1999). Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate. J Antimicrob Chemother 43, 1584–1590. PMID: 10390207

Morales G et al (2010). Resistance to linezolid is mediated by the cfr gene in the first report of an outbreak of linezolid-resistant Staphylococcus aureus. Clin Infect Dis 50, 821–825. PMID: 20144045

Naldini L et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267. PMID: 8602510

Riordan JR et al. (1999). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066–1073. PMID: 2475911

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