Restriction Enzymes, Endonucleases, Recombinases, Thermostable DNA Polymerases, DNA Synthesizers & DNA Ligase Are Used to Engineer & Prepare Chimeric DNA Molecules
المؤلف:
Peter J. Kennelly, Kathleen M. Botham, Owen P. McGuinness, Victor W. Rodwell, P. Anthony Weil
المصدر:
Harpers Illustrated Biochemistry
الجزء والصفحة:
32nd edition.p446-447
2025-10-23
48
Sticky, or complementary cohesive-end ligation of DNA fragments is technically easy, but some special techniques are often required to overcome problems inherent in this approach. Sticky ends of a vector may reconnect with themselves, with no net gain of DNA. Sticky ends of fragments also anneal so that heterogeneous tandem inserts can form. Also, sticky-end sites may not be available or in a convenient position. To alleviate these problems, an enzyme that generates blunt ends can be used. Blunt ends can be ligated directly; however, ligation is not directional. To circumvent this problem new DNA ends of specific sequence can be added by direct blunt-end ligation using the bacteriophage T4 enzyme DNA ligase. Alternatively, convenient RE recognition sites can be added to a DNA fragment through the use of polymerase chain reaction (PCR) amplification via thermostable DNA polymerases or, alter natively through direct DNA synthesis on a DNA synthesizer (see Figure 1).
Fig1. The polymerase chain reaction technique is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes two strands complementary to the original two. This cycle is repeated 30 or more times, giving an amplified product of defined length and sequence. Note that the four dXTPs and the two primers are present in excess to minimize the possibility that these components are limiting for polymerization/amplification. It is important to note though that as cycle number increases incorporation rates can drop, and mutation/ error rates can increase.
As an adjunct to the use of restriction endonucleases to combine and engineer DNA fragments, scientists have begun utilizing recombinases such as bacterial lox P sites, which are recognized by the CRE recombinase, bacteriophage λ att sites recognized by the λ phage encoded INT protein or yeast FRT sites recognized by the yeast Flp recombinase. These recombinase systems all catalyze specific incorporation of two DNA fragments that carry the appropriate recognition sequences and carry out homologous recombination between the relevant recognition sites. A novel DNA editing/ gene regulatory system termed CRISPR-Cas9 (clustered regularly interspersed short palindromic repeats–associated gene 9) first discovered in 2012, has revolutionized genomic DNA studies. The CRISPR system, found in many bacteria, represents a form of acquired, or adaptive immunity to prevent reinfection of a bacterium by specific bacteriophages. CRISPR complements the system of restriction endonucleases and methylases described earlier. CRISPR uses RNA-based targeting to bring the Cas9 nuclease to foreign (or any complementary) DNA. Within bacteria this CRISPR-RNA-Cas9 complex then degrades and inactivates the targeted DNA. The CRISPR system has been adapted for use in eukaryotic cells, including human cells, where it has been shown to be an RNA-directed site-specific nuclease just as it is in bacteria. Variations on the use of CRISPR allow for gene deletion, gene editing, gene visualization, and even modulation of gene transcription. Thus, CRISPR has added an exciting new, highly efficient, and very specific technology to the toolbox of methods for the manipulation of DNA and genetic analysis of mammalian cells. The basic aspects of CRISPR-Cas9 function are outlined in Figure2.
Fig2. Overview of CRISPR-Cas9.Shown is the two-domain CRISPR-Cas9 nuclease protein bound to target genomic DNA (red, blue) and specific guide RNA (green), which through base complementarity (20 nts) locates its genomic target, which is adjacent to a short protospacer adjacent motif, or PAM. The guide RNA binding, and nuclease domains are labeled. Once specifically localized, the two distinct Cas9 nuclease active centers cleave both strands of the targeted genomic DNA (cleavage; arrows) immediately downstream of the PAM, which results in DNA double-strand break. Subsequent DNA repair by cellular activities can introduce mutations thereby inactivating the targeted gene. Variations on the use of CRISPR-Cas9 are numerous and allow for the sculpting of the structure and expression of genomic DNA.
The similarities of the CRISPR-Cas RNA-directed targeting and gene inactivation method and mi/siRNA-mediated repression of expression in higher eukaryotes are notable. Both methodologies are being actively pursued for experimental and therapeutic purposes. Interestingly, a variant of the CRISPR-Cas system, C2c2, has been shown to site-specifically cleave RNA. This discovery paves the way for potential specific alteration of mRNA/ncRNA levels in cells absent the ethical and technical challenges inherent in genome editing with the CRISPR-Cas9 system.
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