Dna manipulation enzymes

Powerpoint Presentation for B.Sc III Sem VI students as per syllabus of SGB amravati University

1. TOOLS OF rDNA TECHNOLOGY DNA Manipulation enzymes By Mr. Pranav Gadkar M.Sc Botany CSIR NET JRF, SET, GATE, UGC NET JRF

2. RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology is the set of techniques that enable the DNA from different sources to be identified, isolated and recombined so that new characteristics can be introduce into an organism.

3. Tools in rDNA technology Five kinds of ‘Biological tools’ used by scientists in the synthesis of recombinant DNA. Tools are grouped into five categories Enzymes Cloning vectors Foreign DNA DNA library Molecular Probes

4. Enzymes For DNA Manipulation Genetic Engineers needs to be able to cut and join DNA from different sources Certain modification may have to be carried out to the DNA during various steps required to produce, clone and identify rDNA Tools that enables these manipulation to be performed by enzymes

5. The following 5 different types of enzymes are used Nucleases Polymerases Ligases Kinases Phosphatase

6. NUCLEASES Enzymes that degrade nucleic acids by breaking phosphodiester bonds that link one nucleotide to next. Ribonucleases - attack RNA Deoxyribonucleases - attack DNA Nucleases are of two different kinds Exonucleases – remove nucleotides one at a time from the end of nucleic acid Endonucleases – able to break internal phosphodiester bonds within nucleic acid

7. Mung bean nuclease – an endonuclease which is specific for ssDNA and RNA.Purified from mung bean sprouts. Requires Zn2+ for catalytic activity. S1 Nucleases – endonuclease purified from Aspergillus oryzae. Degrades RNA or ssDNA RNaseA – endonuclease which digests ssRNA at 3’ end of pyrimidine residues RNaseH – endonuclease which digest strand of RNA-DNA hetero duplex does not digest ssDna or dsDna

8. Restriction Endonucleases A restriction endonuclease is a bacterial enzyme that cuts the dsDNA into fragments after recognizing specific sequences known as recognition or restriction site. Restriction term comes from the fact that these enzyme restrict the entry of foreign DNA into bacteria. Believed to be evolved by bacteria to resist viral attack.

9. Discovery Initial knowledge of RE came to light in 1950s. It was observed that some bacteria are immune to bacteriophage. The phenomenon was termed as host controlled restriction. It took several years then to understand the actual mechanism. The phage DNA would be degraded by an enzyme produced by the bacteria; while its own DNA would be protected by methylation.

10. 1960s – Warner Arber (with Matthew Messelson) – hypothesized and later proved the existence of REs and Methylases 1970 – Hamilton Smith identified and purified RE from H. influenzae Daniel Nathan used the RE purified by H. Smith. The three scientists got the Nobel.

11. Nomenclature Proposed by Smith and Nathans in 1973 The name of any restriction enzyme consist of 3 parts An abbreviation of genus and species of the organism to three letters, eg. Eco for Escherichia coli. A letter, number or combination of the two to indicate strain of relevant species. A roman numeral to indicate the order in which restriction modification system were found in the same organism or strain. For eg, the name of the EcoRI restriction was derived as: E Escherichia(genus) co coli(species) R RY13(strain) I First identified(order of identification)

12. Examples of restriction nomenclature

13. Characterstics of different kinds of restriction endonucleases

14. Type 1 and type111 are not useful for gene cloning because they cleave DNA at sites other than recognition sites cause random cleavage patterns. Type 11 endonucleases are widely used for cloning and mapping purposes because they recognize specific site and cleave at these sites

15. Recognition site Almost all REs recognize palindromic sequences. Palindromic sequences have two-fold rotational symmetry. e.g. GGATCC CCATGG GGAANNNTTCC CCTTNNNAAGG

16. Recognition sequences of some REs

17. Types of cuts made by type II REs Cohesive OR Staggered OR Sticky ends – The RE makes an uneven cut so that there is an overhang Blunt OR Flush ends – The RE cuts in such a way that the ends are uniform and there is no overhang

18. Isoschizomers – Different REs having the same recognition sites Msp I & Hpa II both recognize CCGG Neoschizomers – REs with same recognition sites but generating different ends: e.g. SmaI (CCC/GGG) and XmaI (C/CCGGG)

19. RE digestion in the laboratory In a microfuge tube: Add DNA 1/10 th volume by volume 10X buffer 1 unit per 21 μ g DNA enzyme (RE) Water to make up the volume Incubate at least one hour at 37°C Heat kill the enzyme at 70° for 10 minutes Analyze on agarose gel by after electrophoresis

20. Star Activity Some REs are sensitive to temperature and pH of the reaction system . At abnormal temperature (42°C) or elevated pH or low ionic strength the RE cleaves the DNA at random sites. For example, EcoRI * ( EcoRI star activity) cleaves the sequence N/AATTN, where N is any base, whereas EcoRI cleaves the sequence GAATTC.

21. Polymerases Polymerases may be of two types: DNA polymerase and RNA polymerase DNA polymerase helps in adding a dNTP to a growing DNA chain RNA polymerase helps in adding a NTP to a growing RNA chain Modified DNA polymerase klenow fragment is also used Reverse transcriptase synthesizes DNA strand complementary to RNA use in cDNA library

22. Ligases Ligases are enzymes that join two or more DNA fragments forming concatamers OR join a vector and insert fragment to form a recombinant DNA. DNA ligase is present in all cells. However, for rDNA experiments either T4 (phage) DNA ligase T4 DNA ligase seals single strand nicks between adjacent nucleotides.

23. The T4 enzyme requires ATP, while the E. coli enzyme requires NAD+. In each case the cofactor is split and forms an enzyme–AMP complex. The complex binds to the nick, which must expose a 5′ phosphate and 3′ OH group, and makes a covalent bond in the phosphodiester chain. The temperature used is 4 – 15°C so as to maintain the stability of hydrogen bonds.

24. Alkaline Phosphatase Alkaline phosphatase is an enzyme that removes the phosphate bond from the restriction digested DNA fragment. Alkaline phosphatase is obtained usually from calf serum. Once a DNA fragment is treated with alkaline phosphatase it cannot self ligate; especially in case of vector it may circularize and in case of genome DNA concatemers may form

25. Kinases T4 polynucleotide kinase obtained from E.coli cells infected with T4 phage Perform the reverse reaction to alkaline phosphate adding phosphate to 5 th end Mainly used for end labeling of DNA molecule

26. Linkers Linkers are additional DNA sequences containing restriction sites. Two such linkers are added to the foreign DNA. The Linker added foreign DNA is the restriction digested and ligated to the vector. Linkers are added when there is no suitable restriction endonuclease site in the foreign DNA.

27. Adaptors Synthetic molecules having a restriction site. One end has the cohesive end and the other end has a blunt end bearing a 5’phosphate group. Such two molecules are constructed and attached to the foreign DNA. The newly constructed foreign DNA with adaptors is then ligated to the vector. Adaptors are used when there is an internal restriction endonuclease site that might cut the foreign DNA into two parts.

28. Reference Principals of Gene manipulation-By Primrose, Twyman and Old Gene Cloning and DNA Analysis-By Terry A. Brown Cell and Molecular Biology –By Gerald Carp

29. Thank You!!