Restriction Endonucleases PDF

Z Restriction Endonucleases by Dr.Ezat Mersal Introduction Discovery How Restriction Enzymes work Types of Restriction Enzymes Subunits of Restriction Enzymes Nomenclature of Restriction Enzymes Recognition Sequences of Restriction Enzymes Properties...

Z Restriction Endonucleases by Dr.Ezat Mersal Introduction Discovery How Restriction Enzymes work Types of Restriction Enzymes Subunits of Restriction Enzymes Nomenclature of Restriction Enzymes Recognition Sequences of Restriction Enzymes Properties of Restriction Enzymes Applications of Restriction Enzymes  Restriction Endonucleases are enzymes that produce internal cuts, called cleavage, In the DNA molecule.  Restriction Endonuclease (Restriction Enzyme) is a bacterial enzyme that cuts dsDNA into fragments after recognizing specific nucleotide sequence known as recognition or restriction site.  Restriction Enzymes are believed to be evolved by bacteria to resist viral attack.  Restriction Enzymes are also known as molecular scissor. © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved.  Restriction enzymes are categorized into three general groups. © 2015 John Wiley & Sons, Inc. All rights reserved.  Their composition.  Enzyme co-factor requirement.  The nature of their target sequence.  Position of their DNA cleavage site relative to the target sequence. © 2015 John Wiley & Sons, Inc. All rights reserved.  Restriction enzymes recognize a specific sequence of nucleotides, and produce a double-stranded cut in the DNA. these cuts are of two types: more specific ↳ unpairing pairing T © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved. © 2015 John Wiley & Sons, Inc. All rights reserved.  Type I restriction enzymes are complex endonucleases, and have recognition sequence of 15 bp; they cleave the DNA about 1000 bp away from the 5` end of the sequence “TCA” located within the recognition site. Eg:- EcoK-12,EcoB,etc.  The type I Restriction Enzyme work with the help of many co factor like as S- Adenosyl Methionine, Hydrolysed Adenosine Triphosphate (ATP) and Magnesium (Mg2+ ).  The type I Restriction Enzyme methylates the DNA sequence at the site of recognition. > seconus si - : " jjj  The type I Restriction Enzyme translocate the DNA sequence. © 2015 John Wiley & Sons, Inc. All rights reserved.  Type I restriction enzymes possess three subunits:  HsdR:is required for restriction.  HsdM:necessaryfor adding methyl groups to host DNA (methyltransferase activity).  HsdS: importantfor specificity of cut site recognition in addition to its methyltransferase activity. © 2015 John Wiley & Sons, Inc. All rights reserved.  Thetype II restriction enzymes are remarkably stable and induce cleavage either, in most cases, within or outside their recognition sequences, which are symmetrical.  The type II Restriction Enzyme work with the help of only single co factor like as Magnesium (Mg2+). © 2015 John Wiley & Sons, Inc. All rights reserved.  The 1st type II Enzyme to be isolated was > - Hind II in 1970. ↓  Onlytype II restriction endonucleases are used for restriction mapping and gene cloning in view of their cleavage only at specific site. © 2015 John Wiley & Sons, Inc. All rights reserved.  Type II restriction enzymes can generate two different types of cuts depending on whether they cut both strands at the centre of the recognition sequence:  Theformer cut will generate “blunt ends” with no nucleotide overhangs.  Thelatter, generates “sticky” or “cohesive” ends © 2015 John Wiley & Sons, Inc. All rights reserved.  These subgroups are defined using a letter suffix.  Type II B restriction enzymes.  Type II E restriction endonucleases.  Type II M restriction endonucleases.  Type II T restriction enzymes © 2015 John Wiley & Sons, Inc. All rights reserved.  Type III restriction enzymes are intermediate between type I and type II endonucleases, they cleave DNA in the immediate vicinity of their recognition sites, Eg;- EcoP1, EcoP15, Hinf III etc.  They cut DNA up to 20-30 base pairs away from the recognition site.  These enzymes contain more than one subunit.(ATP & Mg2+)  And require AdoMet and ATP cofactors for their roles in DNA methylation and restriction. © 2015 John Wiley & Sons, Inc. All rights reserved. & & © 2015 John Wiley & Sons, Inc. All rights reserved.  t  The recognition sequence of Type II endonucleases form palindrome with rotational symmetry.  Mostly Type II endonucleases have recognition sites of 4,5 or 6 bp. Which are predominantly GC rich. © 2015 John Wiley & Sons, Inc. All rights reserved. P 20-30 bp © 2015 John Wiley & Sons, Inc. All rights reserved.  Theyare used in gene cloning and protein expression experiments.  Restrictionenzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals (Restriction Fragment Length Polymorphism – RFLP).  Eachof these methods depends on the use of agarose gel electrophoresis for separation of the DNA fragments. -s © 2015 John Wiley & Sons, Inc. All rights reserved.  Provides different ways of manipulating DNA such as the creation of recombinant DNA, which has endless applications  Allows for the large scale production human insulin for diabetics using E. coli, as well as for the Hepatitis B and HPV vaccines  Cloning DNA Molecules ·  Studying nucleotide sequence © 2015 John Wiley & Sons, Inc. All rights reserved.  Basic genetics : a human approach / BSCS. Dubuque, IA, Kendall/Hunt Pub. Co., c1999. 147 p. QH431.B305 1999  Genes, ethnicity, and ageing. Edited by Lincoln H. Schmitt, Leonard Freedman, Rayma Pervan. Nedlands, Australia, Centre for Human Biology, University of Western Australia ; Singapore, River Edge, NJ, World Scientific, c1995. 100 p.QH455.G45 1995  Genetic polymorphisms and susceptibility to disease. Edited by M. S. Miller and M. T. Cronin. New York, Taylor & Francis, 2000. 266 p.  GENETIC ANALYSIS AN INTEGRATED APPROACH Mark F. Sanders , John L. Bowman Second edition 2015 ISBN 978 0-321-94890-8 (student edition) www.pearsonhighered.com