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Restriction enzyme

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Solution structure of the restriction enzime EcoRI as published in the Protein Data Bank.

The restriction enzymes or restriction endonucleases are special enzymes that cut (cleave) the double-stranded DNA by recognizing specific short sequences of nucleotides called restriction sites of the enzyme.[1] The recognition sequences generally range between 4 and 8 nucleotides, and most of them are palindromic. The palindromic forms can be in mirror-like or inverted repeat palindromes .

Types

Restriction enzymes are generally classified into three types: I, II and III.

  • Type I: cleave at sites remote from recognition site;
  • Type II: cleave within the site or at short specific distances from recognition site.
  • Type III: cleave at sites a short distance from recognition site;

Form of use

Restriction enzymes are bacterial enzymes that act identifying sequences of specific base pair DNA molecules and cleaving them at these points. Different species of bacteria produce various restriction nuclease in order to protect them against the virus by the degradation of the viral DNA that may enter the bacteria.[2] They have high specificity: each enzyme recognizes and cleaves only a particular sequence of nucleotides, generally constituting of 4 to 8 pairs of nitrogenous bases. A restriction enzyme will cleave the DNA into a set of fragments, called restriction fragments, which are determined by the locations of restriction sites.[3]

Applications

Restriction endonucleases are employed to digest genomic DNA in order to be performed gene analysis by Southern blot. Another application of restriction enzymes is its use on the location of mutations in restriction maps. A mutation in certain cleavage sites will result in a change in the size of fragments produced by a specific enzyme enabling the identification of mutations in the map.[4]

Another use of restriction endonucleases is to be employed to probe the methylation state of DNA. About 98% of the CpG dinucleotides are methylated with the exceptions that typically mark the 5' ends of genes.[5] Both restriction enzymes HpaII and MspI recognize and cleave at CCGG sites (see table below). However, MspI will cleave both methylated sites (CMeCGG) and unmethylated sites while HpaII will only cleave unmethylated sites.[5] Comparing the action of the two enzymes in a sample makes it possible to distinguish restriction sites of interest.

Restriction enzymes databases

EMBOSS is a free open source software analysis package specially developed for use in molecular biology and bioinformatics.[6] One of the tools of EMBOSS is redata that search the REBASE database for information on a specified restriction enzyme.[7]

Examples of restriction enzymes

Enzyme Source recognized sequence Cleavage sites Cleavage Type of end
AluI[1] Arthrobacter luteus
5'AGCT 
3'TCGA 
5'AGCT 
3'TCGA 
5'---AG  CT---3'
3'---TC  GA---5'
Blunt end
BamHI[1] Bacillus amyloliquefaciens
5'GGATCC
3'CCTAGG
5'GGATCC
3'CCTAGG
5'---G     GATCC---3'
3'---CCTAG     G---5'
Sticky end
EcoRI[1] Escherichia coli
5'GAATTC
3'CTTAAG
5'GAATTC
3'CTTAAG
5'---G     AATTC---3'
3'---CTTAA     G---5'
Sticky end
EcoRII Escherichia coli
5'CCWGG
3'GGWCC
5'CCWGG
3'GGWCC
5'---     CCWGG---3'
3'---GGWCC     ---5'
Sticky end
EcoRV Escherichia coli
5'GATATC
3'CTATAG
5'GATATC
3'CTATAG
5'---GAT  ATC---3'
3'---CTA  TAG---5'
Blunt end
EcoP15I Escherichia coli
5'CAGCAGN25NN
3'GTCGTCN25NN
5'CAGCAGN25NN
3'GTCGTCN25NN
5'---CAGCAGN25   NN---3'
3'---GTCGTCN25NN   ---5'
Frayed end
HaeIII[1] Haemophilus aegyptius
5'GGCC
3'CCGG
5'GGCC
3'CCGG
5'---GG  CC---3'
3'---CC  GG---5'
Blunt end
HhaI[1] Haemophilus haemolyticus
5'GCGC
3'CGCG
5'GCGC
3'CGCG
5'---GCG  C---3'
3'---C  GCG---5'
Sticky end
HindII[8] Haemophilus influenzae
5'GTGGAC
3'CAGCTG
5'GTGGAC
3'CAGCTG
5'---GTG   GAC---3'
3'---CAG   CTG---5'
Sticky end
HindIII Haemophilus influenzae
5'AAGCTT
3'TTCGAA
5'AAGCTT
3'TTCGAA
5'---A     AGCTT---3'
3'---TTCGA     A---5'
Sticky end
HinfI Haemophilus influenzae
5'GANTC
3'CTNAG
5'GANTC
3'CTNAG
5'---G   ANTC---3'
3'---CTNA   G---5'
Frayed end
HpaI[2] Haemophilus parainfluenzae
5'GTTAAC
3'CAATTG
5'GTTAAC
3'CAATTG
5'---GTT  AAC---3'
3'---CAA  TTG---5'
Blunt end
HpaII Haemophilus parainfluenzae
5'CCGG
3'GGCC
5'CCGG
3'GGCC
5'---C    CGG---3'
3'---GGC   CG---5'
Sticky end
KpnI[9] Klebsiella pneumoniae
5'GGTACC
3'CCATGG
5'GGTACC
3'CCATGG
5'---GGTAC  C---3'
3'---C  CATGG---5'
Sticky end
MspI Moraxella
5'CCGG
3'GGCC
5'CCGG
3'GGCC
5'---C    CGG---3'
3'---GGC   CG---5'
Sticky end
NotI Nocardia otitidis
5'GCGGCCGC
3'CGCCGGCG
5'GCGGCCGC
3'CGCCGGCG
5'---GC   GGCCGC---3'
3'---CGCCGG   CG---5'
Sticky end
PvuII Proteus vulgaris
5'CAGCTG
3'GTCGAC
5'CAGCTG
3'GTCGAC
5'---CAG  CTG---3'
3'---GTC  GAC---5'
Blunt end
PstI[2] Providencia stuartii
5'CTGCAG
3'GACGTC
5'CTGCAG
3'GACGTC
5'---CTGCA  G---3'
3'---G  ACGTC---5'
Sticky end
SmaI Serratia marcescens
5'CCCGGG
3'GGGCCC
5'CCCGGG
3'GGGCCC
5'---CCC  GGG---3'
3'---GGG  CCC---5'
Blunt end
SpeI Sphaerotilus natans
5'ACTAGT
3'TGATCA
5'ACTAGT
3'TGATCA
5'---A  CTAGT---3'
3'---TGATC  A---5'
Sticky end
Sau3A Staphylococcus aureus
5'GATC
3'CTAG
5'GATC
3'CTAG
5'---     GATC---3'
3'---CTAG     ---5'
Sticky end
SacI[9] Streptomyces achromogenes
5'GAGCTC
3'CTCGAG
5'GAGCTC
3'CTCGAG
5'---GAGCT  C---3'
3'---C  TCGAG---5'
Sticky end
SalI[9] Streptomyces albus
5'GTCGAC
3'CAGCTG
5'GTCGAC
3'CAGCTG
5'---G  TCGAC---3'
3'---CAGCT  G---5'
Sticky end
SphI[9] Streptomyces phaeochromogenes
5'GCATGC
3'CGTACG
5'GCATGC
3'CGTACG
5'---GCATG  C---3'
3'---C  GTACG---5'
Sticky end
StuI[10] Streptomyces tubercidicus
5'AGGCCT
3'TCCGGA
5'AGGCCT
3'TCCGGA
5'---AGG  CCT---3'
3'---TCC  GGA---5'
Blunt end
ScaI[9] Streptomyces caespitosus
5'AGTACT
3'TCATGA
5'AGTACT
3'TCATGA
5'---AGT  ACT---3'
3'---TCA  TGA---5'
Blunt end
TaqI Thermus aquaticus
5'TCGA
3'AGCT
5'TCGA
3'AGCT
5'---T   CGA---3'
3'---AGC   T---5'
Sticky end
XbaI[9] Xanthomonas badrii
5'TCTAGA
3'AGATCT
5'TCTAGA
3'AGATCT
5'---T  CTAGA---3'
3'---AGATC  T---5'
Sticky end
XhoI[1] Xanthomonas campestris
5'CTCGAG
3'GAGCTC
5'CTCGAG
3'GAGCTC
5'---C  TCGAG---3'
3'---GAGCT  C---5'
Sticky end

References

  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Hartl, Daniel L (2008) (in Portuguese). Princípios de Genética de População [A Primer of Population Genetics] (3rd ed.). São Paulo: Funpec Editora. p. 7. ISBN 978-85-7747-022-8. 
  2. 2.0 2.1 2.2 Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walters, Peter (2008). Molecular Biology of the Cell (5th ed.). New York and London: Garland Science. p. 532-535. ISBN 0-8153-4105-9. 
  3. Griffiths, Anthony J. F.; Wessler, Susan R.; Lewontin, Richard C.; Carroll, Sean B (2008). Introduction to Genetic Analysis (9th ed.). New York: W. H. Freeman. p. 717-718. ISBN 978-0-7167-6887-6. 
  4. Lesk, Arthur M (2008). Introduction to Bioinformatics (3rd ed.). Oxford: Oxford University Press. p. 75. ISBN 978-0-19-920804-3. 
  5. 5.0 5.1 Alphey, Luke (1997). DNA Sequencing:From Experimental Methods to Bioinformatics. New York: Springer/Bios Scientific Publishers. p. 92. ISBN 0-387-91509-5. 
  6. Rice P, Longden I, Bleasby A (2000). "EMBOSS: The European Molecular Biology Open Software Suite". Trends in Genetics 16 (6): 276–277. PMID 10827456. 
  7. Markell, Scott; León, Darryl (2003). Sequence Analysis in a Nutshell. Sebastopol, CA: O'Reilly. p. 186. ISBN 0-596-00494-X. 
  8. Panno, Joseph (2005). The Cell: Evolution of the First Organism. New York: Facts on File, Inc.. p. 134-135. ISBN 0-8160-4946-7. 
  9. 9.0 9.1 9.2 9.3 9.4 9.5 Krieger, M.; Scott, M. P.; Matsudaira, P. T.; Lodish, H. F.; Darnell, J. E.; Zipursky, L; Kaiser, C.; Berk, A (2004). Molecular Cell Biology (5th ed.). New York: W.H. Freeman and Company. ISBN 0-7167-4366-3. 
  10. Shimotsu, H.; Takahashi, H.; Saito, H (1980). "A new site-specific endonuclease StuI from Streptomyces tubercidicus". Gene 11 (3–4): 219–25. PMID 6260571. 

External links