Restriction enzyme
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'AG↓CT 3'TC↑GA |
5'---AG CT---3' 3'---TC GA---5' |
Blunt end |
| BamHI[1] | Bacillus amyloliquefaciens |
5'GGATCC 3'CCTAGG |
5'G↓GATCC 3'CCTAG↑G |
5'---G GATCC---3' 3'---CCTAG G---5' |
Sticky end |
| EcoRI[1] | Escherichia coli |
5'GAATTC 3'CTTAAG |
5'G↓AATTC 3'CTTAA↑G |
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'GAT↓ATC 3'CTA↑TAG |
5'---GAT ATC---3' 3'---CTA TAG---5' |
Blunt end |
| EcoP15I | Escherichia coli |
5'CAGCAGN25NN 3'GTCGTCN25NN |
5'CAGCAGN25↓NN 3'GTCGTCN25NN↑ |
5'---CAGCAGN25 NN---3' 3'---GTCGTCN25NN ---5' |
Frayed end |
| HaeIII[1] | Haemophilus aegyptius |
5'GGCC 3'CCGG |
5'GG↓CC 3'CC↑GG |
5'---GG CC---3' 3'---CC GG---5' |
Blunt end |
| HhaI[1] | Haemophilus haemolyticus |
5'GCGC 3'CGCG |
5'GCG↓C 3'C↑GCG |
5'---GCG C---3' 3'---C GCG---5' |
Sticky end |
| HindII[8] | Haemophilus influenzae |
5'GTGGAC 3'CAGCTG |
5'GTG↓GAC 3'CAG↑CTG |
5'---GTG GAC---3' 3'---CAG CTG---5' |
Sticky end |
| HindIII | Haemophilus influenzae |
5'AAGCTT 3'TTCGAA |
5'A↓AGCTT 3'TTCGA↑A |
5'---A AGCTT---3' 3'---TTCGA A---5' |
Sticky end |
| HinfI | Haemophilus influenzae |
5'GANTC 3'CTNAG |
5'G↓ANTC 3'CTNA↑G |
5'---G ANTC---3' 3'---CTNA G---5' |
Frayed end |
| HpaI[2] | Haemophilus parainfluenzae |
5'GTTAAC 3'CAATTG |
5'GTT↓AAC 3'CAA↑TTG |
5'---GTT AAC---3' 3'---CAA TTG---5' |
Blunt end |
| HpaII | Haemophilus parainfluenzae |
5'CCGG 3'GGCC |
5'C↓CGG 3'GGC↑C |
5'---C CGG---3' 3'---GGC CG---5' |
Sticky end |
| KpnI[9] | Klebsiella pneumoniae |
5'GGTACC 3'CCATGG |
5'GGTAC↓C 3'C↑CATGG |
5'---GGTAC C---3' 3'---C CATGG---5' |
Sticky end |
| MspI | Moraxella |
5'CCGG 3'GGCC |
5'C↓CGG 3'GGC↑C |
5'---C CGG---3' 3'---GGC CG---5' |
Sticky end |
| NotI | Nocardia otitidis |
5'GCGGCCGC 3'CGCCGGCG |
5'GC↓GGCCGC 3'CGCCGG↑CG |
5'---GC GGCCGC---3' 3'---CGCCGG CG---5' |
Sticky end |
| PvuII | Proteus vulgaris |
5'CAGCTG 3'GTCGAC |
5'CAG↓CTG 3'GTC↑GAC |
5'---CAG CTG---3' 3'---GTC GAC---5' |
Blunt end |
| PstI[2] | Providencia stuartii |
5'CTGCAG 3'GACGTC |
5'CTGCA↓G 3'G↑ACGTC |
5'---CTGCA G---3' 3'---G ACGTC---5' |
Sticky end |
| SmaI | Serratia marcescens |
5'CCCGGG 3'GGGCCC |
5'CCC↓GGG 3'GGG↑CCC |
5'---CCC GGG---3' 3'---GGG CCC---5' |
Blunt end |
| SpeI | Sphaerotilus natans |
5'ACTAGT 3'TGATCA |
5'A↓CTAGT 3'TGATC↑A |
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'GAGCT↓C 3'C↑TCGAG |
5'---GAGCT C---3' 3'---C TCGAG---5' |
Sticky end |
| SalI[9] | Streptomyces albus |
5'GTCGAC 3'CAGCTG |
5'G↓TCGAC 3'CAGCT↑G |
5'---G TCGAC---3' 3'---CAGCT G---5' |
Sticky end |
| SphI[9] | Streptomyces phaeochromogenes |
5'GCATGC 3'CGTACG |
5'GCATG↓C 3'C↑GTACG |
5'---GCATG C---3' 3'---C GTACG---5' |
Sticky end |
| StuI[10] | Streptomyces tubercidicus |
5'AGGCCT 3'TCCGGA |
5'AGG↓CCT 3'TCC↑GGA |
5'---AGG CCT---3' 3'---TCC GGA---5' |
Blunt end |
| ScaI[9] | Streptomyces caespitosus |
5'AGTACT 3'TCATGA |
5'AGT↓ACT 3'TCA↑TGA |
5'---AGT ACT---3' 3'---TCA TGA---5' |
Blunt end |
| TaqI | Thermus aquaticus |
5'TCGA 3'AGCT |
5'T↓CGA 3'AGC↑T |
5'---T CGA---3' 3'---AGC T---5' |
Sticky end |
| XbaI[9] | Xanthomonas badrii |
5'TCTAGA 3'AGATCT |
5'T↓CTAGA 3'AGATC↑T |
5'---T CTAGA---3' 3'---AGATC T---5' |
Sticky end |
| XhoI[1] | Xanthomonas campestris |
5'CTCGAG 3'GAGCTC |
5'C↓TCGAG 3'GAGCT↑C |
5'---C TCGAG---3' 3'---GAGCT C---5' |
Sticky end |
References
- ↑ 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.
- ↑ 2.0 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.
- ↑ 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.
- ↑ Lesk, Arthur M (2008). Introduction to Bioinformatics (3rd ed.). Oxford: Oxford University Press. p. 75. ISBN 978-0-19-920804-3.
- ↑ 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.
- ↑ Rice P, Longden I, Bleasby A (2000). "EMBOSS: The European Molecular Biology Open Software Suite". Trends in Genetics 16 (6): 276–277. PMID 10827456.
- ↑ Markell, Scott; León, Darryl (2003). Sequence Analysis in a Nutshell. Sebastopol, CA: O'Reilly. p. 186. ISBN 0-596-00494-X.
- ↑ Panno, Joseph (2005). The Cell: Evolution of the First Organism. New York: Facts on File, Inc.. p. 134-135. ISBN 0-8160-4946-7.
- ↑ 9.0 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.
- ↑ 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
- Restriction Enzymes at TheLabRat.com
- REBASER The Restriction Enzyme Database
