Restriction Endonuclease (Cutting of DNA)

in gene cloning technique the cutting of DNA is essential. With the help of restriction endonuclease, it has been done. It also describes the restriction digest of a DNA molecule.

1. Restriction Endonuclease (Cutting of DNA) Dr.R.Bhuvaneswari [email protected]

2. Why the cutting of DNA is essential ? Gene cloning requires DNA molecules, to cut in a very accurate and reproducible manner . Vector is a DNA molecule that is used as a vehicle to artificially carry the genetic material into another cell, where it can be replicated or expressed. Each vector molecule, cleaved at a single position to open the circle , only then the gene of interest can be inserted.

3. Cutting of DNA molecule DNA molecules are macromolecules that stores the genetic information of living organisms . DNA is has double helical Structure, joining adjacent nucleotides with the help of covalent bond called Phosphodiester Bond. The phosphodiester bonds present in nucleotides in DNA molecules are very stable unless they are physically stretched or exposed to enzymes called nucleases .

4. DNA-double helical structure Nucleotide Nucleotide

5. Nucleases Nucleases are enzymes capable of hydrolyzing (i.e breaking) phosphodiester bonds in DNA molecules . It is found in both plants and animals. Nuclease is c lassified into two major groups: Exonucleases: If the enzyme digest nucleotides from the exterior (outside) of the DNA molecules . Endonucleases: If the enzyme digest nucleotides in the interior (inside)of a DNA molecule . Note : Ribonuclease acts only in RNA Deoxyribonuclease acts only in DNA

6. Nuclease

7. Restriction Enzyme Restriction enzyme or Molecular scissors: It is a Nuclease enzyme . It consists of protein that recognizes a specific sequence of DNA bases and cuts in its particular subunit. This enzyme is produced in bacteria. B acterium  is made up of single cell ,some of which are pathogenic, which infect mammals and cause diseases(Bacteria –Plural).   Bacteriophage  are viruses that specifically infect bacteria  and it kills the bacterium during the process of producing more bacteriophage

8. Restriction Endonuclease Bacteria  use restriction enzymes to kill viruses, these enzymes attack the viral DNA and break it into useless fragments (Makes inactive and digested ). Some strains of bacteria are immune to bacteriophage infection, it is called as host controlled restriction. Restriction occurs in the bacterium, produces an restriction endonuclease that degrades the phage DNA, before it replicates.

9. Restriction Enzymes-Action

10. Recognition site / Palindromic sequence Restriction enzyme comes into contact with a (Phage) DNA Specific sequence with a shape that matches a part of the enzyme, called the  recognition site , it wraps around the DNA and causes a break in both strands (Nucleotides)of the DNA  molecule. Recognition sites are usually only short 4 to 8  nucleotides . This recognition site will have the palindromic sequence,  is a  sequence  made up of nucleic acids (DNA/RNA) that is a mirror image of the same when read from 5' to 3‘.

11. blunt end sticky end or cohesive end Blunt ends and Sticky ends Restriction enzymes recognize a specific sequence of nucleotides , and produce a double-stranded cut in the DNA . These cuts are of two types:

12. Blunt ends and Sticky ends Many r estriction endonuclease do a simple double stranded cut in the middle of the recognition sequence, and produce the blunt or flush end. These blunt ended fragments can be joined to any other DNA fragment with blunt ends . Blunt end is of considerable importance in the design of a gene cloning experiments.

13. Blunt ends and Sticky ends A large number of Restriction endonuclease cut DNA in a slightly different way (i.e ) not cut at exactly the same position and produce the sticky end or cohesive end. DNA fragments with complimentary sticky ends can be combined to create new molecules which allows the creation and manipulation of DNA sequences from different sources .

14. Blunt ends and Sticky ends One important feature of sticky end is that, restriction endonuclease with different recognition sequence may produce the same sticky ends . Example: BamHI- Recognition sequence - G GATC C BgtII Recognition sequence- -A GATC T Both produce GATC sticky ends

15. Blunt ends and Sticky ends Fragments of DNA produced by cleavage with either of these enzymes can be joined to each other if it carry a complementary sticky end. The cleaved sticky end may join and produce a new fragment .

16. Recognition sequence calculation Recognition sequence for a particular restriction endonuclease in a DNA molecule of known length can be calculated mathematically. (E.g.) Tetra nucleotide sequence GATC may present the sequence again after 256 nucleotide bases. 4 4 = 4X4X4X4=256. 4 6 =4096 Nucleotide bases. GC= 50% content present in nucleotide.

17. Type I Restriction Enzymes Type I- They are complex and have multi-subunit , it may have both endonuclease and methylase activities. They require ATP , Mg +2 and S- A denosyl L-methionine , which is required for cleavage. They are single, multi-functional enzyme which are capable of both restriction and modification activities. It recognizes 15bp in length and cleavage site is 1000bp . They show specificity for recognition site but not for cleavage. They produce heterogeneous type of fragments . So mostly it is not prepared in gene cloning techniques. Eg . EcoB, EcoK

18. Example of Restriction Enzymes Restriction Enzyme

19. Type II Restriction Enzymes Type II- restriction enzymes are made up of single subunit, cleave within or at short distance from DNA recognition sequence. They are simple enzymes having single polypeptide. They have separate methylase and endonuclease activity. It requires mostly Mg +2 as cofactor. This enzyme performs only the restriction function. It is the most common restriction enzyme used in gene cloning The recognition and cutting site is the same one. They generally recognize 6 nucleotides, some recognize 4 , 5,or 8bp also. Eg. PvuI, PvuII, EcoRI Cleave DNA to generate different “ends”

20. Type III Restriction Enzymes Type III restriction enzymes are intermediate between type I and type II endonucleases , they cleave DNA in the immediate vicinity (nearby) of their recognition sites. (E.g.) EcoP1, Hind III, EcoP15 etc. They cut DNA upto 20-30 base pairs away from the recognition site . These enzymes contain more than one subunit and requires S- Adenosyl L-Methionine and ATP & Mg 2+ cofactors for their roles in DNA methylation and restriction reactions.

21. Restriction Enzyme-Digest The required amount of DNA, depends on the nature of the experiment. In this study 2µg of λ DNA was present in the 16µl of the sample. Use micropipette for accurate measurement. Take restriction enzyme in a pure form with known concentration . B efore adding the enzyme the solution containing the DNA must be adjusted to provide the correct condition to ensure maximal activity of the enzyme. Most of the restriction endonuclease function effectively work at pH 7.4 . Different enzymes vary in their requirements for ionic strength provided usually by NaCl and Mg 2 + concentration ( all type 2 restriction endonuclease required Mg 2+ in order to function).

22. Restriction Enzyme-Digest Reducing agent dithiothreitol will stabilize the enzyme and prevent its inactivation . The suitable buffer for B gl II( Bacillus globigii II) is given in the table below: 2µl of buffer is taken and added in the reaction mixture. A suitable final volume for the restriction mixture would be fixed as 20µl. Component Concentration (mM) Tris HCl pH7.4 500 Dithiotheritol 10 MgCl 2 100 NaCl 500

23. Restriction Enzyme-Digest One unit of enzyme is defined as the quantity needed to cut 1 microgram of DNA in 1 hour. Bgl II is frequently obtained concentration of 4 units per µl, so 0.5 µl it will be sufficient to cleave the DNA (2µg of λ DNA4). The final ingredients in the reaction mixture are : 2µg of λ DNA in 16 µl +2 µl of buffer+ 0.5µl Bgl II + 1.5 water giving a final volume of 20µl . The last factor to consider is incubation temperature most restriction endonucleases work best at 37 o C but a few have different requirements.

24. Restriction Enzyme-Digest Taq I Thermas aquaticus lives at high temperature in habitats such as hot springs. Restriction enzyme digests Taq I, it must be incubated at 65 o C to obtain maximum enzyme activity. After one hour restriction should be completed. DNA fragments produced by restriction are to be used in cloning experiments, then the enzyme must somehow be destroyed. So that it does not accidently digest other DNA molecules that may be added at a later stage.

25. Restriction Enzyme-Digest There are several ways of killing the enzyme. For (E.g) short incubation at 70 C in sufficient to kill the enzyme. For other method is phenol extraction or the addition of EDTA which binds Mg 2+ ions preventing restriction endonuclease action . Analyzing the result of restriction endonuclease cleavage. A restriction digest will produce the number of DNA fragments, sizes of which depends on the exact positions of the recognition sequences for the endonuclease in the original molecule.

26. Restriction Enzyme-Digest Determining the number of sizes of the fragments is needed, depending on that, the restriction endonucleases are to be used in gene cloning. Initially DNA molecules are determined by its viscous nature . Large DNA molecule has high viscous solution but it is more difficult to identify the size of the individual cleavage . The Electrophoresis technique is now used to identify the DNA molecule.

27. Restriction Enzyme- Application Restriction endonucleases are synthesized by bacteria and used for its defense mechanism . Restriction enzymes recognize the source of incoming (foreign) DNA and destroy,   if it recognized it as foreign DNA . Restriction endonucleases spot (recognize) specific sequences in the incoming DNA and digest the DNA into fragments. They  cut the DNA either at specific sites or more randomly. They are used to digest DNA for gene analysis .  

28. Restriction Enzyme- Application Restriction enzymes are used in genetic engineering to cut the gene of the interest from source DNA (specific gene) and to introduce ( insert) them into the P lasmid vectors . It is used for construction of recombinant DNA and gene cloning experiments .

29. R eferences Gene cloning by T.A Brown https :// commons.wikimedia.org/wiki/File:RNA%27s_Degradation_Process.png . https:// commons.wikimedia.org/wiki/File:229_Nucleotides-01.jpg https://commons.wikimedia.org/wiki/File:Restriction_enzyme_Eco_RI.JPG https:// freesvg.org/open-scissors-icon-vector-drawing https:// commons.wikimedia.org/wiki/File:EcoRV_Restriction_Site.rsh.svg https:// commons.wikimedia.org/wiki/File:EcoRI_restriction_enzyme_recognition_site.svg

30. Thank You