ASSIGNMENT

RESTRICTION ENZYME DIGESTION

DEPARTMENT OF MICROBIOLOGY AND MOLECULAR GENETICS

Submitted to: Ms.Zarwa Submitted by: Aqsa Imtiaz Roll no. : MMG-11-05 Kushbakhth Rizwan MMG-11-06

RESTRICTION ENZYME DIGESTION

Introduction: Restriction enzymes are enzymes isolated from bacteria are Nucleases which can cleave the sugar-phosphate backbone of DNA, found in bacteria. As they cut within the molecule, they are commonly called restriction endonucleases. They specifically cleave the nucleic acids at specific nucleotide sequence called Restriction sites to generate a set of smaller fragments called restriction fregments.[1] Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA.[2] For the first isolation of a restriction enzyme, HindII in 1970, and the subsequent discovery and characterization of numerous restriction endonucleases, the 1978 Nobel Prize for Physiology was awarded to Daniel Nathans, Werner Arber, and Hamilton O. Smith. Their discovery led to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. While recognition sequences vary between 4 and 8 nucleotides, many of them are palindromic, that read the same backwards and forwards. There are two types of palindromic sequences that can be possible in DNA. The mirror-like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on a single strand of DNA strand, as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands.[1]

Types of restriction and modication (R-M) system Type I enzymes :- Type I restriction enzymes exhibit both restriction and DNA modification activities.They require the cofactors such as Mg2+ ions, Sadenosylmethionine (SAM) and ATP for their activity. Type I restriction endo nucleases cleaves DNA at nonspecific sites and that can be 1000 base pair or more from recognition sequence. Type II enzymes :- Type II enzymes and their corresponding modification methyltransferases act as separate proteins. Class II restriction endonucleases are generally used as the key material in molecular biology and recombinant DNA techniques, including genome mapping, RFLP analysis, DNA sequencing, and cloning. Type III enzymes :- Like Class I enzymes, Type III enzymes possess both restriction and modification activities.They recognize specific sequences and cleave 25 - 27 base pairs outside of the recognition sequence, in a 3 direction. They require Mg2+ ions for their activity.[1] Applications: Restriction enzymes are powerful tools of molecular genetics used to: Map DNA molecules Analyze population polymorphisms Rearrange DNA molecules Prepare molecular probes Create mutants[4]

Protocol:Requirments: 1l 10x Buffer 6.5l H2O 2l DNA 0.5l Enzyme

Procedure:

Setting up the restriction digestion reaction 1. Place the vials containing restriction enzyme (EcoR I and Hind III) on ice. 2. Thaw the vials containing substrate (lambda DNA) and assay buffer. 3. Prepare two different reaction mixtures using the following constituents. Reaction 1 (EcoR I digestion) L DNA 20ul 2x assay buffer 25ul EcoR I 3ul

Reaction 2 (Hind III digestion) L DNA 20ul 2x assay buffer 25ul Hind III 3ul

2. Incubate the vial at 37oc for 1 hour. 3. Meanwhile, prepare a 1% agarose gel for electrophoresis. 4. After an hour add 5ul of gel loading buffer to vials 5. Load the digested samples, 10ul of control DNA and 10ul of marker, note down the order of loading. 6. Electrophorase the samples at 50-100 V for 1-2 hrs. 7. Stain the agarose gel with 1X staining dye. 8. Destain to visualize the DNA bands.[5]

References:1. Roberts RJ; Murray, Kenneth (November 1976). "Restriction endonucleases". CRC Crit. Rev. Biochem. 2. Arber W, Linn S (1969). "DNA modification and restriction". Annu. Rev. Biochem. 3. Pingoud A, Jeltsch A (September 2001). "Structure and function of type II restriction endonucleases". 4. Sistla S, Rao DN (2004). "S-Adenosyl-L-methionine-dependent restriction enzymes". Crit. Rev. Biochem. 5. Bourniquel AA, Bickle TA (November 2002). "Complex restriction enzymes: NTP-driven molecular motors".