Restriction enzyme

732:, with recognition sites that are usually undivided and palindromic and 4–8 nucleotides in length. They recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity—they usually require only Mg as a cofactor. These enzymes cleave the phosphodiester bond of double helix DNA. It can either cleave at the center of both strands to yield a blunt end, or at a staggered position leaving overhangs called sticky ends. These are the most commonly available and used restriction enzymes. In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily 600:. These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site. Cleavage at these random sites follows a process of DNA translocation, which shows that these enzymes are also molecular motors. The recognition site is asymmetrical and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a non-specific spacer of about 6–8 nucleotides. These enzymes are multifunctional and are capable of both restriction digestion and modification activities, depending upon the methylation status of the target DNA. The cofactors 460: 478: 1159:, with the ultimate goal of inducing target mutagenesis and aberrations of human-infecting viruses. The human genome already contains remnants of retroviral genomes that have been inactivated and harnessed for self-gain. Indeed, the mechanisms for silencing active L1 genomic retroelements by the three prime repair exonuclease 1 (TREX1) and excision repair cross complementing 1(ERCC) appear to mimic the action of RM-systems in bacteria, and the non-homologous end-joining (NHEJ) that follows the use of ZFN without a repair template. 420: 1076:. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA, since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a 1820: 1143:

dimerization being mediated in-situ through the FokI domain. Each zinc finger array (ZFA) is capable of recognizing 9–12 base pairs, making for 18–24 for the pair. A 5–7 bp spacer between the cleavage sites further enhances the specificity of ZFN, making them a safe and more precise tool that can be applied in humans. A recent Phase I clinical trial of ZFN for the targeted abolition of the CCR5 co-receptor for HIV-1 has been undertaken.

1806: 35: 743:, containing more than one subunit. They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg cofactors. Type IIE restriction endonucleases (e.g., NaeI) cleave DNA following interaction with two copies of their recognition sequence. One recognition site acts as the target for cleavage, while the other acts as an 639: 774:

Type III restriction enzymes (e.g., EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20–30 base pairs after the recognition site. These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation

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that speeds up or improves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both sequences at the same time. Type IIG restriction endonucleases (e.g., RM.Eco57I) do have a

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K, is known as the restricting host and appears to have the ability to reduce the biological activity of the phage λ. If a phage becomes established in one strain, the ability of that phage to grow also becomes restricted in other strains. In the 1960s, it was shown in work done in the laboratories

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group that mimics ribonucleases for specific RNA sequence and cleaves at a non-base-paired region (RNA bulge) of the targeted RNA formed when the enzyme binds the RNA. This enzyme shows selectivity by cleaving only at one site that either does not have a mismatch or is kinetically preferred out of

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requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence. DNA sequence analysis of restriction enzymes however show great variations, indicating that there are more than four types. All types of enzymes recognize specific short

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Others have proposed using the bacteria R-M system as a model for devising human anti-viral gene or genomic vaccines and therapies since the RM system serves an innate defense-role in bacteria by restricting tropism by bacteriophages. There is research on REases and ZFN that can cleave the DNA of

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Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences can also be classified by the number of bases in its recognition site, usually between 4 and 8 bases, and the number of bases in the sequence will determine how

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More than 3,600 restriction endonucleases are known which represent over 250 different specificities. Over 3,000 of these have been studied in detail, and more than 800 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a vital

864:) utilize guide RNAs to target specific non-palindromic sequences found on invading organisms. They can cut DNA of variable length, provided that a suitable guide RNA is provided. The flexibility and ease of use of these enzymes make them promising for future genetic engineering applications. 1142:

Artificial restriction enzymes created by linking the FokI DNA cleavage domain with an array of DNA binding proteins or zinc finger arrays, denoted zinc finger nucleases (ZFN), are a powerful tool for host genome editing due to their enhanced sequence specificity. ZFN work in pairs, their

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Since their discovery in the 1970s, many restriction enzymes have been identified; for example, more than 3500 different Type II restriction enzymes have been characterized. Each enzyme is named after the bacterium from which it was isolated, using a naming system based on bacterial

752:, are able to recognize and cut methylated DNA. Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites; this characteristic is widely used to perform in-vitro cloning techniques such as 3481:

Gigorescu A, Morvath M, Wilkosz PA, Chandrasekhar K, Rosenberg JM (2004). "The integration of recognition and cleavage: X-ray structures of pre-transition state complex, post-reactive complex, and the DNA-free endonuclease". In Alfred M. Pingoud (ed.).

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DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements, as summarised below:

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by restriction digest can also be generated that can give the relative positions of the genes. The different lengths of DNA generated by restriction digest also produce a specific pattern of bands after gel electrophoresis, and can be used for

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only one strand of the DNA, at the N-6 position of adenine residues, so newly replicated DNA will have only one strand methylated, which is sufficient to protect against restriction digestion. Type III enzymes belong to the beta-subfamily of

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groups to host DNA (methyltransferase activity), and HsdS is important for specificity of the recognition (DNA-binding) site in addition to both restriction digestion (DNA cleavage) and modification (DNA methyltransferase) activity.

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Wayengera M, Kajumbula H, Byarugaba W (2007). "Frequency and site mapping of HIV-1/SIVcpz, HIV-2/SIVsmm and Other SIV gene sequence cleavage by various bacteria restriction enzymes: Precursors for a novel HIV inhibitory product".

569:) cleave at sites a short distance from a recognition site; require ATP (but do not hydrolyse it); S-adenosyl-L-methionine stimulates the reaction but is not required; exist as part of a complex with a modification methylase ( 428:

often the site will appear by chance in any given genome, e.g., a 4-base pair sequence would theoretically occur once every 4^4 or 256bp, 6 bases, 4^6 or 4,096bp, and 8 bases would be 4^8 or 65,536bp. Many of them are

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palindrome is also a sequence that reads the same forward and backward, but the forward and backward sequences are found in complementary DNA strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being

1103:. In general, alleles with correct restriction sites will generate two visible bands of DNA on the gel, and those with altered restriction sites will not be cut and will generate only a single band. A 351:. Restriction enzymes of this type are more useful for laboratory work as they cleave DNA at the site of their recognition sequence and are the most commonly used as a molecular biology tool. Later, 616:, are required for their full activity. Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction digestion; HsdM is necessary for adding 760:. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits. Some recognize palindromic sequences while others have asymmetric recognition sites. 903:

In 2013, a new technology CRISPR-Cas9, based on a prokaryotic viral defense system, was engineered for editing the genome, and it was quickly adopted in laboratories. For more detail, read

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to leave short, single-stranded 5' protrusions. They require the presence of two inversely oriented unmethylated recognition sites for restriction digestion to occur. These enzymes

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was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes. These subgroups are defined using a letter suffix.

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Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, et al. (May 2009). "Precise genome modification in the crop species Zea mays using zinc-finger nucleases".

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at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each

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Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. (March 2007). "CRISPR provides acquired resistance against viruses in prokaryotes".

1463: 803:; as such, it is functionally equivalent to the M and S subunits of type I restriction endonuclease. Res is required for restriction digestion, although it has no 2920:

Jeltsch A, Pingoud A (February 1996). "Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems".

363:, thus showing that restriction enzymes can also be used for mapping DNA. For their work in the discovery and characterization of restriction enzymes, the 1978 171: 436:

palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backward on a single strand of DNA, as in GTAATG. The

432:, meaning the base sequence reads the same backwards and forwards. In theory, there are two types of palindromic sequences that can be possible in DNA. The 3679:

Meisel A, Bickle TA, Krüger DH, Schroeder C (January 1992). "Type III restriction enzymes need two inversely oriented recognition sites for DNA cleavage".

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The restriction enzymes studied by Arber and Meselson were type I restriction enzymes, which cleave DNA randomly away from the recognition site. In 1970,

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Dussoix D, Arber W (July 1962). "Host specificity of DNA produced by Escherichia coli. II. Control over acceptance of DNA from infecting phage lambda".

1091:(SNPs). This is however only possible if a SNP alters the restriction site present in the allele. In this method, the restriction enzyme can be used to 3318: 748:

single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active. Type IIM restriction endonucleases, such as

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Smith HO, Nathans D (December 1973). "Letter: A suggested nomenclature for bacterial host modification and restriction systems and their enzymes".

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experiments. For optimal use, plasmids that are commonly used for gene cloning are modified to include a short polylinker sequence (called the

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A palindromic recognition site reads the same on the reverse strand as it does on the forward strand when both are read in the same orientation

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group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA

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that the restriction is caused by an enzymatic cleavage of the phage DNA, and the enzyme involved was therefore termed a restriction enzyme.

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Naturally occurring restriction endonucleases are categorized into five groups (Types I, II, III, IV, and V) based on their composition and

5387: 4608:"Combining allele-specific fluorescent probes and restriction assay in real-time PCR to achieve SNP scoring beyond allele ratios of 1:1000" 1933:

Kessler C, Manta V (August 1990). "Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)".

1099:. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by 663:) are shown as magenta spheres and are adjacent to the cleaved sites in the DNA made by the enzyme (depicted as gaps in the DNA backbone). 5826: 502:. These often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the same location are known as 884:). Such artificial restriction enzymes can target large DNA sites (up to 36 bp) and can be engineered to bind to desired DNA sequences. 483:

Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (

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Tovkach A, Zeevi V, Tzfira T (January 2011). "Expression, purification and characterization of cloning-grade zinc finger nuclease".

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Jeltsch A, Kröger M, Pingoud A (July 1995). "Evidence for an evolutionary relationship among type-II restriction endonucleases".

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Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of

5801: 1738: 1714: 1690: 1665: 1641: 1569: 1481: 1457: 1409: 466: 364: 293:, a virus that infects bacteria, and the phenomenon of host-controlled restriction and modification of such bacterial phage or 3939:

Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (September 2010). "Genome editing with engineered zinc finger nucleases".

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Type I restriction enzymes were the first to be identified and were first identified in two different strains (K-12 and B) of

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to CATATG). Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes.

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Smith HO, Wilcox KW (July 1970). "A restriction enzyme from Hemophilus influenzae. I. Purification and general properties".

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Shimotsu H, Takahashi H, Saito H (November 1980). "A new site-specific endonuclease StuI from Streptomyces tubercidicus".

5816: 5810: 5611: 5361: 764: 442: 3023: 5659: 2033:"Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts" 360: 305:

and Giuseppe Bertani in the early 1950s. It was found that, for a bacteriophage λ that can grow well in one strain of

3534:"Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI" 317:

K, its yields can drop significantly, by as much as three to five orders of magnitude. The host cell, in this example

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technology that has many applications, for example, allowing the large scale production of proteins such as human

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that protect the organism against invading foreign DNA. Type III enzymes are hetero-oligomeric, multifunctional

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Lederberg S, Meselson M (May 1964). "Degradation of non-replicating bacteriophage dna in non-accepting cells".

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activity on its own. Type III enzymes recognise short 5–6 bp-long asymmetric DNA sequences and cleave 25–27 bp

2082:"Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution" 6355: 6108: 6049: 1720: 808: 6478: 6153: 5720: 5710: 1866: 579:

Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA

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Mierzejewska K, Siwek W, Czapinska H, Kaus-Drobek M, Radlinska M, Skowronek K, et al. (July 2014).

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Naito T, Kusano K, Kobayashi I (February 1995). "Selfish behavior of restriction-modification systems".

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Used by restriction enzymes to locate specific sequences of DNA on which to bind and subsequently cleave

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Villa-Komaroff L, Efstratiadis A, Broome S, Lomedico P, Tizard R, Naber SP, et al. (August 1978).

1247: 223: 4508:"A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes" 4409:

Murtola M, Wenska M, Strömberg R (July 2010). "PNAzymes that are artificial RNA restriction enzymes".

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Bourniquel AA, Bickle TA (November 2002). "Complex restriction enzymes: NTP-driven molecular motors".

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Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They form

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Williams RJ (March 2003). "Restriction endonucleases: classification, properties, and applications".

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Laboratory DNA science: an introduction to recombinant DNA techniques and methods of genome analysis

6350: 6304: 6247: 5615: 5464: 5378: 5250: 5246: 1672: 570: 563: 549: 539: 524: 400: 379:. The discovery of restriction enzymes allows DNA to be manipulated, leading to the development of 6252: 5484: 5354: 3143: 829: 5132:

Krieger M, Scott MP, Matsudaira PT, Lodish HF, Darnell JE, Zipursky L, Kaiser C, Berk A (2004).

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Wayengera M (2003). "HIV and Gene Therapy: The proposed model for a gene therapy against HIV".

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Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea".

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for the discovery of restriction enzymes and their application to problems of molecular genetics

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Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, et al. (April 2003).

4282:"TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain" 3424:"Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle)" 2745:"Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae" 1343: 1271: 1132: 1053:

Isolated restriction enzymes are used to manipulate DNA for different scientific applications.

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Artificial ribonucleases that act as restriction enzymes for RNA have also been developed. A

817: 799:). The Mod subunit recognises the DNA sequence specific for the system and is a modification 744: 601: 535: 492: 470: 452: 92: 4579: 4445: 459: 6345: 6158: 5993: 5988: 5921: 5581: 5406: 4231:

Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, et al. (October 2010).

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Pingoud A, Alves J, Geiger R (1993). "Chapter 8: Restriction Enzymes". In Burrell M (ed.).

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applications. Other artificial restriction enzymes are based on the DNA binding domain of

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Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, et al. (July 2009).

4052: 3999: 3897: 3842: 3781: 3692: 3294: 3259: 2984: 2933: 2843: 2760: 2582: 2457: 2146: 2009: 359:(SV40) DNA by restriction enzymes yields specific fragments that can be separated using 6242: 6146: 5479: 5469: 5347: 5002: 4977: 4953: 4928: 4904: 4877: 4853: 4828: 4765: 4740: 4673: 4648: 4355: 4330: 4306: 4281: 4257: 4232: 4173: 4140: 4116: 4091: 4072: 4016: 3983: 3964: 3862: 3811: 3712: 3607: 3582: 3558: 3533: 3401: 3358: 3281:

Yuan R (1981). "Structure and mechanism of multifunctional restriction endonucleases".

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Kelly TJ, Smith HO (July 1970). "A restriction enzyme from Hemophilus influenzae. II".

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Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (May 2009).

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are the most commonly used artificial restriction enzymes and are generally used in

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to function; multifunctional protein with both restriction digestion and methylase (

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Restriction enzymes likely evolved from a common ancestor and became widespread via

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Sistla S, Rao DN (2004). "S-Adenosyl-L-methionine-dependent restriction enzymes".

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Artificial restriction enzymes can be generated by fusing a natural or engineered

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Schiffer JT, Aubert M, Weber ND, Mintzer E, Stone D, Jerome KR (September 2012).

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Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B (January 2011).

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and Kent Wilcox isolated and characterized the first type II restriction enzyme,

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A DNA fragment resulting from the cutting of a DNA strand by a restriction enzyme

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the prokaryotic DNA and blocks cleavage. Together, these two processes form the

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