single-stranded extensions that can be joined by complementary base-pairing and the activity of the enzyme DNA ligase that catalyzes the formation of phosphodiester bonds. Another cloning method known as recombinational cloning does not utilize restriction endonucleases or DNA ligase for insertion of target DNA into a vector but, rather, exploits a system used by some viruses to integrate into the host genome via recombination at specific attachment sequences.
Cloned DNA is introduced into host cells, often bacterial cells that are competent to take up exogenous DNA, a process known as transformation. Vectors that carry the target DNA into the host cell are usually derived from natural bacterial plasmids that have been genetically engineered with several unique endonuclease recognition sequences (multiple-cloning sites) to facilitate cloning. A vector can be propagated in a host cell if it possesses a DNA sequence (origin of replication) that enables it to replicate in the host. Transformation is generally inefficient; however, transformed cells may be distinguished from nontransformed cells by testing for the activity of genes that are present on the vector, including genes for resistance to antibiotics or synthesis of colored products.
To clone and express genes that encode eukaryotic proteins in a bacterial host, the introns must first be removed. Purified mRNA, which does not contain introns, is used as a template for the synthesis of cDNA by the enzyme reverse transcriptase. Oligonucleotide primers can be designed to target a specific mRNA for cDNA synthesis or to anneal to the poly(A) tails present on most eukaryotic mRNAs to generate a cDNA library that contains all of the protein coding sequences expressed by a source eukaryote under a given set of conditions. Construction of a genomic DNA library from a prokaryote is more straightforward and entails cleaving the DNA to obtain overlapping fragments for cloning. Libraries are screened by a variety of methods to identify clones with a particular sequence or that produce a target protein.
The CRISPR-Cas system is used to edit intact genomes in vivo. In this system, an sgRNA that contains a 20-nucleotide sequence complementary to the target site in the genome is introduced into the host cell together with the endonuclease Cas9. The sgRNA guides Cas9 to the genomic target site, adjacent to a specific PAM sequence, which is cleaved by the endonuclease. Double-stranded DNA cleavage activates cellular DNA repair systems that results in nucleotide deletions that disrupt the target gene. If specific donor DNA is also introduced into the cell, it may be incorporated into the genome by recombination between the target sequence and homologous sequences flanking the donor DNA. The CRISPR-Cas technology can be used to disrupt, insert, alter the regulation of, or tag a gene in the genomes of microorganisms or multicellular organisms.
Amplification, synthesis, and sequencing of DNA are also fundamental tools of molecular biotechnology. PCR is a powerful method for generating millions of copies of a specific sequence of DNA from very small amounts of starting material. Amplification is achieved in 30 or more successive cycles of template DNA denaturation, annealing of two oligonucleotide primers to complementary sequences flanking a target gene in the single-stranded DNA, and DNA synthesis extending from the primer by a thermostable DNA polymerase. Among innumerable applications, PCR can be used to detect or quantify a specific nucleotide sequence in a complex biological sample or to obtain large amounts of a particular DNA sequence either for cloning or for sequencing.
Oligonucleotides that are used as primers for PCR are produced in a stepwise method using phosphoramidites. To make double-stranded DNA molecules, two oligonucleotides with complementary sequences are synthesized separately then allowed to anneal. In addition to primers for PCR and DNA sequencing, oligonucleotides are used as adaptors to add specific sequences to the ends of DNA fragments, such as restriction endonuclease recognition sites for cloning, and to synthesize entire genes.
The nucleotide sequence of a gene can reveal useful information about the function, regulation, and evolution of the gene. The sequencing technologies currently used involve (i) addition of nucleotides by DNA polymerase to a primer based on complementarity to a template DNA fragment and (ii) detection and identification of the nucleotide(s) added. The dideoxynucleotide method has been used for several decades to sequence genes and whole genomes. This method relies on the incorporation of a synthetic dideoxynucleotide that lacks a 3′ hydroxyl group into a growing DNA strand, which terminates DNA synthesis. Conditions are optimized so that the dideoxynucleotides are incorporated randomly, producing DNA fragments of different lengths that terminate with one of the four dideoxynucleotides, each tagged with a different fluorescent dye. The fragments are separated according to their size by electrophoresis, and the sequence of fluorescent signals is determined and converted into a nucleotide sequence. Pyrosequencing entails correlating the release of pyrophosphate, which is recorded as the emission of light, with the incorporation of a particular nucleotide into a growing DNA strand. Sequencing using reversible chain terminators also reveals the sequence of a DNA fragment by detecting single-nucleotide extensions; however, in contrast to pyrosequencing, the four nucleotides are added to the reaction together in each cycle, and after the unincorporated nucleotides are washed away, the nucleotide incorporated by DNA polymerase is distinguished by its fluorescent signal. The fluorescent dye and a blocking group that prevents addition of more than one nucleotide during each cycle are chemically cleaved, and the cycle is repeated. In another method, sequences are obtained from single DNA molecules captured by a DNA polymerase attached to the bottom of a very low volume well. Emissions from fluorescently tagged nucleotides held in the polymerase’s active site are detected in real time by a narrowly focused laser, before the cleaved pyrophosphate and attached fluorophore diffuse away.
Next-generation sequencing technologies, together with cell-free methods to generate a library of genomic DNA sequencing templates in a dense array on a solid surface, have enabled rapid and inexpensive acquisition of genome sequences. Hundreds of millions of short nucleotide reads can be acquired simultaneously (massive parallelization) and assembled into contigs. Using these approaches, the genome sequences of thousands of organisms from all domains of life, including the metagenomes of entire communities of microorganisms in an environmental sample, have been completed or are in progress. The next steps are to annotate the sequence features and to determine the functions of the genes encoded in the genomes by investigating patterns of transcription (transcriptomics), protein synthesis (proteomics), and small-molecule production (metabolomics) using a variety of techniques such as DNA and protein microarray analysis, RNA sequencing, 2D PAGE, mass spectrometry, and NMR. Comparison of genome sequences can reveal the genetic basis of a disease, the mechanism of pathogenicity of a microbe, or the evolutionary relationships among organisms, while transcript, protein, and metabolite profiles can identify biomarkers for diagnosis and treatment of disease.
REFERENCES
Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, et al. 2008. Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 456:53−59.
Bondy-Denomy J, and Davidson AR. 2014. To acquire or resist: the complex biological effects of CRISPR-Cas systems. Trends Microbiol. 22:218−225.
Brasch, MA, Hartley JL, Vidal M. 2004. ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list. Genome Res. 14:2001−9.
Cello J, Paul PV, Wimmer E. 2002. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science. 297:1016−18.
Chen CS, Korobkova E, Chen H, Zhu J, Jian X, Tao SC, He C, Zhu H. 2008. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat Methods. 5:69−74.
Cohen SN, Chang ACY, Boyer HW, Helling RB. 1973. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA. 70:3240–44.
Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, Hsu PD, Wu XB, Jiang WY, Marraffini LA, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science. 339:819−23.
Czar MJ, Anderson JC, Bader JS, Peccoud J. 2008. Gene synthesis
