This involves the formation of phosphodiester bonds between adjacent 5'-phosphate and 3'-hydroxyl residues, which can be catalyzed by two different ligases: E. The latter is the preferred enzyme because it can also join blunt-ended DNA fragments. The efficiency of the ligation reaction depends on:. The ligation of blunt-ended fragments is less effective than that of sticky-ended ones.
Blunt-end ligation may be enhanced by:. Sambrook, J. Cloning Cloning Methods Cloning using restriction enzymes. The first three letters of a restriction enzyme's name are abbreviations of the bacterial species from which the enzyme has been isolated e. Roman numerals are also used as part of the name when more than one restriction enzyme has been isolated from the same bacterial strain. Today, scientists recognize three categories of restriction enzymes: type I, which recognize specific DNA sequences but make their cut at seemingly random sites that can be as far as 1, base pairs away from the recognition site; type II, which recognize and cut directly within the recognition site; and type III, which recognize specific sequences but make their cut at a different specific location that is usually within about 25 base pairs of the recognition site.
As originally postulated by Arber, all restriction enzymes serve the purpose of defense against invading viruses. Bacteria protect their DNA by modifying their own recognition sequences, usually by adding methyl CH 3 molecules to nucleotides in the recognition sequences and then relying on the restriction enzymes' capacity to recognize and cleave only unmethylated recognition sequences.
Also, as Arber suspected, bacteriophages that have previously replicated in a particular host bacterial strain and survived are similarly modified with methyl-labeled nucleotides and thereby protected from cleavage within that same strain.
Within just a few years of the initial discoveries of EcoB, EcoK, and HindII, scientists were already testing ways to use restriction enzymes. The first major application was as a tool for cutting DNA into fragments in ways that would make it easier to study and, in particular, identify and characterize genes. A second major use was as a device for recombining, or joining, DNA molecules from different genomes, usually with the goal of identifying and characterizing a gene or studying gene expression and regulation Heinrichs, Nathans and Danna then used the enzyme to cut, or digest, the DNA of the eukaryotic virus SV40 into 11 unique linear fragments.
Found in both monkeys and humans, SV40 has the capacity to cause tumors and was being intensively studied at the time for its cancer-causing potential. Finally, they separated the fragments using gel electrophoresis , a technique developed in the s and still commonly used as a way to sort nucleic acid molecules of different sizes Figure 1.
Clearly, he must have had a vision at the very beginning of this that just the simple idea of being able to separate the fragments of viral DNA into specific pieces would have enormous applications" Brownlee, Today, scientists still use restriction enzyme digestion, followed by electrophoresis , as a way to separate DNA fragments.
Many scientists also use what is known as a probe , or a DNA or RNA molecule with a base sequence that is complementary to a DNA sequence of interest, to identify where in the genome i. This basic procedure is outlined in Figure 2. After separating the DNA fragments through electrophoresis, the fragments are transferred from the gel to a solid medium, or membrane. When DNA fragments are separated and transferred in this manner, the process is known as Southern blotting , named after the scientist who developed the technique, Edwin Southern Southern, After transfer, the membrane is immersed in a solution of either radioactive or chemically labeled probes.
The probes bind to their complementary sequences on the membrane, if any are present. The membrane is then washed, leaving only bound probes that can be detected using autoradiography , if the probes are radioactive, or other means. At the time, scientists had identified the specific site and sequence of cleavage for only one restriction enzyme, HindII. With HindII, cleavage occurred in the middle of a six-base-pair recognition site, yielding what are known as blunt-end fragments see Figure 3, in which PvuII similarly produces blunt-end fragments.
Mertz and Davis discovered that another restriction enzyme, EcoR1, by contrast, cleaves its recognition site in a staggered way that generates fragments with single-stranded overhanging ends known as cohesive, or sticky, ends. After two fragments with complementary sticky ends are joined, the DNA backbone may be covalently sealed using another enzyme called DNA ligase.
This gives molecular biologists powerful tools to create nearly limitless combinations of recombinant DNA. Today, scientists are mixing and matching DNA fragments from different species in ways that continue not only to demonstrate the power of this method, but also to raise serious ethical and social questions. Arber, W. DNA modification and restriction.
Annual Review of Biochemistry 38 , — Brownlee, C. Danna and Nathans: Restriction enzymes and the boon to molecular biology. Proceedings of the National Academy of Sciences , Danna, K. Specific changes of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proceedings of the National Academy of Sciences 68 , — Heinrichs, A. Making the cut: Discovery of restriction enzymes. Nature Milestones.
Konforti, B. The servant with the scissors. Nature Structural Biology 7 , doi Luria, S. A nonhereditary, host-induced variation of bacterial viruses.
Journal of Bacteriology 64 , — Mertz, J. Proceedings of the National Academy of Sciences 69 , — Meselson, M. DNA restriction enzyme from E. Nature , — doi Smith, H. A restriction enzyme from Hemophilus influenzae. Base sequence of the recognition site. How, though, would a DNA-degrading enzyme distinguish between the two? Arber hypothesized that bacterial cells might express two types of enzymes: a restriction enzyme that recognizes and cuts up the foreign bacteriophage DNA and a modification enzyme that recognizes and modifies the bacterial DNA to protect it from the DNA-degrading activity of its very own restriction enzyme.
He predicted that the restriction enzyme and the modification enzyme act on the same DNA sequence, called a recognition sequence. In this way, the bacterial cell's own self-defense mechanism, which aggressively degrades invading bacteriophage DNA, would also protect its own DNA from degradation at the same time.
This prediction was confirmed in the late s by Stuart Linn and Arber when they isolated a modification enzyme called methylase and a restriction enzyme responsible for bacteriophage resistance in the bacterium Escherichia coli. The methylase enzyme added protective methyl groups to DNA, and the restriction enzyme cut unmethylated unprotected DNA at multiple locations along its length.
A few years later, in , Hamilton Smith not only independently verified Arber's hypothesis, but also elaborated on the initial discovery by Linn and Arber. He successfully purified a restriction enzyme from another bacterium, Haemophilus influenzae H.
Interestingly, he also showed that this enzyme did not cut at this very same DNA sequence when it occurred in H. Building on this result, a first glimpse of how restriction enzymes could be useful tools for scientific research emerged one year later in experiments carried out by Dan Nathans and Kathleen Danna. They used Smith's restriction enzyme to cut the 5, base-pair genome of the SV40 virus, which infects monkey and human cells, and identified eleven differently sized pieces of DNA.
Nathans's lab later showed that when the SV40 genome was digested with different combinations of restriction enzymes, the sizes of the resulting pieces of DNA could be used to deduce a physical map of the SV40 viral genome, a groundbreaking method for inferring gene sequence information. This method of cutting a DNA molecule into smaller pieces is called a restriction enzyme digest, and it quickly became a powerful tool for generating physical maps of a multitude of genomes, which at the time was a precious revelation in the early stages of genome sequencing.
For this groundbreaking set of discoveries, Arber, Smith, and Nathans were jointly awarded the Nobel Prize in Physiology or Medicine in Given the vast genetic diversity among bacteria, it follows that different bacterial strains express different restriction enzymes, allowing them to balance their own genes against those of invading bacteriophages.
The known variety of restriction enzymes is staggering: To date, more than 4, different restriction enzymes that collectively recognize more than different recognition sequences have been isolated from a wide variety of bacterial strains.
Based on DNA sequence analysis, scientists know that there are many more restriction enzymes out there waiting to be characterized. The recognition sequences of these enzymes are typically four to six base pairs in length, and they are usually palindromic, which means that their recognition sequence reads the same in the 5' to 3' direction on both DNA strands. There are four different categories of restriction enzymes.
Type I restriction enzymes cut DNA at random locations far from their recognition sequence, type II cut within or close to their recognition sequence, type III cut outside of their recognition sequence, and type IV typically recognize a modified recognition sequence. Type II restriction enzymes, which cut within their recognition sequence, are the most useful for laboratory experiments.
When they act on a DNA molecule, restriction enzymes produce "blunt" ends when they cut in the middle of the recognition sequence, and they yield "sticky" ends when they cut at the recognition sequence in a staggered manner, leaving a 5' or 3' single-stranded DNA overhang. Any two blunt ends can be joined together, but only sticky ends with complementary overhangs can be connected to each other.
Restriction enzyme digestion continues to be one of the most common techniques used by researchers who carry out DNA cloning experiments.
0コメント