The gene of interest is amplified in a regular polymerase chain reaction PCR , which produces a primer pair that, once annealed to the vector of interest, is extended in a linear amplification reaction. Thus, this method relies on amplified genes functioning as primers. However, this approach also has limitations. Second, this method relies on digestion with Dpn I, which cleaves methylated DNA, to remove parental plasmids. In this paper, we describe a simple and fast method for performing gene reconstitution by modified restriction-free MRF cloning.
This new method is independent of the existence of restrictions sites and Dpn I treatment. The efficiency was not significantly affected by the insert length up to 20 kb. Figure 1 shows the scheme for MRF cloning. To replace a gene Fig. Schematic representation of MRF cloning.
Compatible cohesive ends of insert gene or vector were created by two single-primer linear PCRs performed in parallel, followed by annealing of the two PCR products. Inserts and acceptors with compatible cohesive ends were then assembled by ligation. The resultant PCR products were gel purified. We first tested this protocol to reconstitute the E.
Based on the initial success of the protocol, we continued to employ it to generate the constructs needed for our studies. As shown in Fig. As the efficiency of cohesive ligation is higher than that of blunt-end ligation, the parental DNA of the products of second-round PCR did not need to be removed. In each transformation, we routinely checked eight colonies at random from each transformation by colony PCR with a forward primer annealing to vector and a reverse primer annealing to the inserted gene.
The agarose gel in Fig. Plasmid alone, prior to PCR, shows two major bands Fig. The ligation of insert into plasmid vector is performed by T4 DNA ligase using a molar ratio of vector to insert.
The inserted genes were amplified by colony PCR. The presence of forward and reverse cloning sites were confirmed by DNA sequencing Fig. Gel electrophoresis separation of double-primer and single-primer PCR products. DNA sequencing reveals that genes were correctly placed in the plasmid. In routine application of our cloning method, we created 46 constructs from E. The E. Under our test conditions, we achieved an average cloning efficiency of DNA sequencing revealed that all genes were correctly placed in the plasmid.
In this study, we describe a new cloning method. The technique uses two rounds of PCR to obtain inserts and acceptors with compatible cohesive ends, which are then ligated. Using this method, we made 46 constructs with inserts of variable size. The average cloning efficiency was For convenience, we only used the vector pET22b for E. Our results showed that cloning efficiency was not significantly affected by the different inserts, thus providing a glimpse of the wide choice in inserts that can be used as a template, which then can be used as an alternative method for multiple fragment assembly and library construction.
We noticed that cloning efficiency was not altered dramatically by fragment length. As shown in Table 2 and Additional file 7 : Figure S3, this method was suitable for the cloning of large DNA sequences up to 20 kb in size. In contrast to traditional restriction enzyme cloning, the method described here provides a much more flexible approach to gene cloning.
Therefore, it represents a cost-effective and simple solution for high-throughput cloning applications. Fortunately, the high-fidelity polymerases recently developed for cloning, e. Therefore, it is no longer challenging to amplify large DNA fragments for use in our method. We developed a novel cloning method that provides an alternative approach to DNA assembly.
This method is independent of restriction sites and Dpn I treatment, and does not introduce undesired operational sequences at the junctions of functional modules.
This new method simplifies complex cloning procedures in which long stretches of DNA can be inserted into circular plasmids in an unrestricted way, and the efficiency does not decrease for long inserts up to 20 kb. The simplicity of both primer design and the procedure itself makes the method suitable for high-throughput studies. The protein of interest is expressed without the addition of extra residues originating from the cloning procedure, making it an attractive alternative method for structural genomics.
Human cDNAs were purchased from Clontech. Oligonucleotide primers were purchased from Invitrogen. PCR purification and gel extraction kits were purchased from Qiagen. All other chemicals used in the study were of molecular biology grade.
In brief, the longer and shorter DNA fragments were mixed at a molar ratio of — The reaction was incubated at room temperature for 2 h. For each transformation, eight colonies were selected randomly for colony PCR to verify insertion. Insert-positive constructs were confirmed by DNA sequencing. Mid-log phase E. To pellet DNA, the sample was centrifuged at rpm for 10 min. Plasmids were isolated using the Spin Miniprep kit Qiagen.
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Thieme F, Marillonnet S. Quick and clean cloning. Simple and effective gap-repair cloning using short tracts of flanking homology in fission yeast. Swiss microbiologist Werner Arber was one of the recipients of the Nobel Prize in Physiology or Medicine, an award he earned for his discovery with Stuart Linn of restriction enzymes, otherwise known by his daughter Sylvia as "servants with scissors.
Bacteriophages are viral particles that invade bacteria and replicate their own DNA independently of the bacterial chromosomal DNA. Those phages that grew poorly were said to be "restricted" by their host.
Arber wanted to know why. Arber proposed that bacterial cells in this case, E. Specifically, he theorized that only those bacteriophages that had previously been in contact with the same bacterial strain could successfully infect new host cells, and that the previous exposure somehow modified the phage DNA in a way that protected it from restriction.
Phages with unmodified DNA, on the other hand, were immediately broken down by enzymes. This occurred because the host cell enzymes recognized these phages as foreign, cleaving their DNA and restricting their growth. Arber further proposed that there were specific sites in the genome at which restriction activities occurred. Arber and Linn referred to the enzyme responsible for this "endonucleolytic scission" as endonuclease R, a name later changed to EcoB.
It didn't take long for other scientists to identify a second restriction enzyme in E. Soon after the discovery of EcoB and EcoK, microbiologists Hamilton Smith and Kent Wilcox isolated and characterized the first restriction enzyme from a second bacterial species , Haemophilus influenzae.
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.
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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. Journal of Molecular Biology. Purification and general properties. Journal of Molecular Biology 51 , — Southern, E. Detection of specific sequences among DNA fragments separated by gel electrophoresis.
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