Course Name:
Molecular Genetics
Topic:
Restriction & Modification System
Academic Year:
2026
Prepared by:
Bishu Sarkar
Lecturer
Department of Biochemistry and Molecular Biology,
Primeasia University
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Features and Functions of Restriction Endonuclease system
Typology: Lecture notes
2025/2026
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Download Restriction Enzymes: function and application and more Lecture notes Biology in PDF only on Docsity!
Course Name:
Molecular Genetics
Topic:
Restriction & Modification System
Academic Year:
Prepared by:
Bishu Sarkar
Lecturer
Department of Biochemistry and Molecular Biology,
Primeasia University
Prepared by Bishu Sarkar; Lecturer, Primeasia University
Course Contents:
Restriction Enzymes and their features
Cleavage of DNA by EcoRI and its protection from cleavage
The Production of Recombinant DNA Molecules
Classification of Restriction Endonucleases
Differences among Restriction Enzyme (RE) I, II, and III
DNA binding and cleavage by a type-II restriction endonuclease
Factors that affect restriction enzyme activity
Applications of Restriction Enzymes
Restriction Mapping with examples
Prepared by Bishu Sarkar; Lecturer, Primeasia University In addition, a useful feature of many restriction nucleases is that they make staggered cuts; that is, they cleave the two strands of a double helix at different points. Other restriction endonucleases cut both strands at the same place and produce blunt ended fragments. Because of the palindromic nature of the restriction sites, the staggered cuts produce segments of DNA with complementary single-stranded ends. Because all the resulting DNA fragments will have complementary single stranded termini, they will hydrogen bond with each other and can be rejoined under the appropriate renaturation conditions by using the enzyme DNA ligase to re-form the missing phosphodiester linkages in each strand.
Cleavage of DNA by EcoRI and its protection from cleavage
Figure
-^01
Prepared by Bishu Sarkar; Lecturer, Primeasia University All cleavage sites in the DNA of an organism must be protected from cleavage by the organism’s own restriction endonucleases; otherwise the organism would commit suicide by degrading its own DNA. In many cases, this protection of endogenous cleavage sites is accomplished by methylation of one or more nucleotides in each nucleotide sequence that is recognized by the organism’s own restriction endonuclease (Figure- 0 1). Methylation occurs rapidly after replication, catalyzed by site-specific methylases produced by the organism. Each restriction endonuclease will cleave a foreign DNA molecule into a fixed number of fragments, the number depending on the number of restriction sites in the particular DNA molecule.
The Production of Recombinant DNA Molecules
The construction of recombinant DNA molecules in vitro. DNA molecules isolated from two different species are cleaved with a restriction enzyme, mixed under annealing conditions, and are Figure
-^01
Prepared by Bishu Sarkar; Lecturer, Primeasia University Type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites. Example: EcoRI, HindIII, BamHI, NotI, PacI. Type III restriction enzymes: These enzymes recognize and methylate the same DNA sequence but cleave 24–26 bp away. They have two different subunits, in which one subunit (M) is responsible for recognition and modification of DNA sequence and other subunit (R) has nuclease action. Mg2+ ions, ATP are needed for DNA cleavage and process of cleavage is stimulated by SAM. Cleave only one strand. Example: EcoP15.
Differences among Restriction Enzyme (RE) I, II, and III
Property Type I RE Type II RE Type III RE Abundance Less common than Type II Most common Rare Recognition site Cut both strands at a non- specific location > 1000 bp away from recognition site Cut both strands at a specific, usually palindromic recognition site (4- 8 bp) Cleavage of one strand, only 24-26 bp downstream of the 3' recognition site Restriction and modification Single multifunctional enzyme Separate nuclease and methylase Separate enzymes sharing a common subunit Nuclease subunit structure Heterotrimer Homodimer Heterodimer Cofactors ATP, Mg2+, SAM Mg2+ Mg2+ (SAM) DNA cleavage requirements Two recognition sites in any orientation Single recognition site Two recognition sites in a head-to-head orientation Enzymatic turnover No Yes Yes DNA translocation Yes No No Site of methylation At recognition site At recognition site At recognition site
Prepared by Bishu Sarkar; Lecturer, Primeasia University
DNA binding and cleavage by a type-II restriction endonuclease
i. These enzymes have nonspecific contact with DNA and initially bind to DNA as dimmers. ii. The target site is then located by a combination of linear diffusion or “sliding” of the enzyme along the DNA over short distances, and hopping/jumping over longer distances. iii. Once the target restriction site is located, the recognition process (coupling) triggers large conformational changes of the enzyme and the DNA, which leads to activation of the catalytic center. iv. Catalysis results in hydrolysis of phosphodiester bond and product release.
Factors that affect restriction enzyme activity
The digestion activity of restriction enzymes depends on the following factors:
- Temperature: Most endonucleases digest the target DNA at 37 °C with few exceptions. Some work at lower temperatures (~25 °C, Sma 1) while Taq I works at 65 °C.
- Cofactors: Restriction endonucleases require certain cofactors or combination of cofactors to digest at the recognition site. All enzymes require Mg2+ as a cofactor for the endonuclease
Prepared by Bishu Sarkar; Lecturer, Primeasia University The other applications of restriction endonucleases include gene expression and mutation studies and examination of population polymorphisms.
- DNA fingerprint: Cleaving a single piece of DNA with multiple restriction enzymes creates a "DNA fingerprint." The pattern of fragments can be compared to similar DNA from another source treated with the same enzymes, to determine if the two are identical or different.
What is restriction map?
By simply, restriction map is the physical map of a DNA which shows the relative positions of restriction enzyme cleavage sites. Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites. After a DNA segment has been digested using a restriction enzyme, the resulting fragments can be examined using a laboratory method called gel electrophoresis, which is used to separate pieces of DNA according to their size.
Prepared by Bishu Sarkar; Lecturer, Primeasia University Worked problem 1 : A linear DNA of 13 kb is digested with Bam HI and Eco RI. Digestion with Bam HI resulted 9 kb and 4 kb fragments where digestion with Eco RI produced 7, 2,1 and 3 kb fragments. Draw the restriction map. Worked problem 2 : A linear piece of DNA that is 30 kb long is first cut with BamHI, then with HpaII, and, finally, with both BamHI and HpaII together. Fragments of the following sizes were obtained from this reaction: BamHI: 20-kb, 6-kb, and 4-kb fragments HpaII: 21-kb and 9-kb fragments BamHI and HpaII: 20-kb, 5-kb, 4 - kb, and 1-kb fragments Draw a restriction map of the 30-kb piece of DNA, indicating the locations of the BamHI and HpaII restriction sites.
Prepared by Bishu Sarkar; Lecturer, Primeasia University Let’s arrange the fragments alphabetically, What is Plasmid? A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria. What is Circular DNA? Circular DNA is DNA that forms a closed loop and has no ends. Examples include: ✓ Plasmids, mobile genetic elements ✓ Covenently closed circular DNA (cccDNA), formed by some viruses inside cell nuclei ✓ Circular bacterial chromosomes ✓ Mitochondrial DNA (mtDNA) ✓ Chloroplast DNA (cpDNA), ✓ Extrachromosomal circular DNA (eccDNA)