Tools of genetic engineering
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This document summarizes several enzymes used in recombinant DNA techniques, including nucleases, ligases, polymerases, and modifying enzymes. It describes what each enzyme type does, such as nucleases breaking phosphodiester bonds in DNA, ligases joining DNA fragments, polymerases synthesizing new DNA strands, and modifying enzymes altering DNA structures through actions like methylation or phosphate addition/removal. Examples are given of several specific enzymes from each category, along with their sources, recognition sequences if applicable, and general functions in genetic engineering applications.
Tools of genetic engineering
- 1. Enzymes used in Recombinant DNA Prepared by: Dr. E. Gayathiri Department of Plant Biology and Plant Biotechnology Guru Nanak College
- 2. – 1. Nucleases – 2. Ligases – 3. Polymerases – 4. DNA modifying enzymes – Alkaline phosphatase (AP ) – Methylase – Ribonuclease (RNase) – Terminal transferase – Deoxyribonuclease (Dnase)
- 3. Nucleases – Nucleases are enzymes that degrade DNA molecules by breaking the phosphodiester – categorized into – (i) exonucleases and (ii) endonucleases. Source: The structure behind the simplicity of ... thescinder.com, Restriction Endonucleases [HD Animation ... youtube.comGene-editing nucleases ... youtube.comGene-Editing Therapy Using CRISPR ...news.utexas.edu
- 4. Why don’t bacteria destroy their own DNA with their restriction enzymes? Source: Microbiology and Molecular Biology Reviews - ASM
- 5. Restriction Enzymes – They were discovered in the late 1960’s. – They are proteins produced in a bacteria cell that cut DNA at a specific site. Also known as restriction endonucleases Haemophilus Influenzae and Type II ... mmg-233-2014-genetics-genomics.fandom.com, EndonucleaseWikipedia en.wikipedia.org,DNA digested by Restriction Enzyme ... quora.com
- 6. Restriction Endonuclease – Named for bacterial genus, species, strain, and type: – Example: Eco R 1 – Genus: Escherichia – Species: coli – Strain: R – Order discovered: 1 Source: Restriction Enzyme EcoR1 – YouTube , youtube.com
- 7. EcoR1- Restriction enzyme 5'-...GAATTC...-3' 3'-...CTTAAG...-5' EcoRI site 5'-...G|AATTC...-3' 3'-...CTTAA|G...-5' Sticky end
- 8. SmaI Restriction enzyme Source: Restriction enzymes & DNA ligase ...khanacademy.org
- 9. A palindromic sequence – Recognition sites have symmetry (palindromic) – Eg: MALAYALAM (English word) – Bam H1 site: 5’-GGATCC-3’ 3’-CCTAGG-5’
- 10. Restriction Enzymes are used in the following areas DNA fingerprinting DNA typing/profiling DNA sequencing Gene splicing/recombinant DNA Transformation Human Genome Project
- 11. DNA ligase – DNA ligase is a DNA-joining enzyme. – If two pieces of DNA have matching ends, ligase can link them to form a single, unbroken molecule of DNA.
- 12. Polymerases – DNA polymerases are enzymes that catalyse the synthesis of a new DNA strand from a pre-existing strand. – The enzyme adds deoxyribonucleotides to the free 3’-OH of the chain undergoing elongation. Source: Anatomy of a Polymerase - How Function ... neb.com, RNA polymerase II - Wikipediaen.wikipedia.org, Polymerase GIFs - Get the best GIF on GIPHYgiphy.com
- 13. Alkaline phosphatase (AP) – This group of enzymes removes the phosphate – group from 5' terminus of the DNA molecule. It is active at alkaline pH – hence the name ‘alkaline phosphatase’. Commercially, it is obtained from three major sources, viz., E. coli (bacteria), calf intestine and arctic shrimp. Source: Meprin β induces activities of A fasebj.org, Alkaline Phosphatase and Poly ... youtube.com
- 14. Polynucleotide kinase (PNK) – This group of enzymes perform a role completely opposite to the one performed by AP. – PNK catalyses the transfer of a phosphate group from ATP to the 5' terminus of the DNA molecule. polynucleotide 5'-hydroxyl-kinase catalyzes the chemical reaction ATP + 5'-dephospho-DNA ADP + 5'-phospho-DNA
- 15. Terminal transferase This group of enzymes catalyses the addition of one or more deoxyribonucleotides to the 3' terminus of the DNA molecule.
- 16. Deoxyribonuclease (DNase) – This group of enzymes catalyses the addition of one or more deoxyribonucleotides to the 3' terminus of the DNA molecule. – The enzyme can work on both double stranded as well as single stranded DNA molecules without the need of any primers. Source: DNase I activity ... link.springer.com
- 17. Methylase – Methyltransferase or methylase catalyzes the transfer of methyl group (-CH3) to its substrate. – The process of transfer of methyl group to its substrate is called methylation. – Methylation is a common phenomenon in DNA and protein structure. Source:IntechOpen
- 18. Ribonuclease (RNase) – Nuclease that can catalyze hydrolysis of ribonucleotides from either single – stranded or double stranded RNA sequence are called ribonucleotides (RNase). – RNase are classified into two types depending on position of cleavage, i.e. – Endoribonuclease – Exoribonuclease Source: https://www.sigmaaldrich.com/catalog/product/sigma/r4875?lang=en®ion=IN
- 19. THANK YOU Source: 7 Important Molecular Tools in Genetic explorebiotech.com