i we a Hi atures Racer oe eae naman number! Oder ings Gugino = Use of living organisms or enzymes from tmzymes were isolated from thar! qopticatio organisms to produce products and processes of bacteria ; es are palindromes i.e. the useful to humans. = Restriction envy re ace eet + Biotechnology : the same forwards 28 wel backwards, wom cells, + _ GITAAC - ap Integration of natural science and organisms, cells, fx 51 = GTTARC ay parts thereof and molecular analogues for the 3) CAATIG - 5 products and services (EFR) when a restriction endonuclease acy, a ANY 7 3 7 ¢ it cleaves both the strands of 1, can Term coined by Karl Ereky (1919). Palindrom os nes cut the two ws * Toole of Recombinant DNA Technology : molecule, $e ing blunt ends others ext’ ss vetricé te ger Restriction Enzymes : Enzymes used for cutting of Se taesicaly leaving sticky ends. er DNA at specific locations e _ = Belong to larger group of enzymes called oily ends are useful in inant Th Nucleases, which are two types exonuclease aca m endonucleases ‘= Restriction enzymes belong to a large cas, lik = They are also known as molecular scissors. enzyme nucleases. pr * Some Important Facts Regarding Nucleases Tr Restriction Enzymes os Tr * ‘és These enzymes recognize short sequences of . Endonucl K double stranded DNA as target for cleavage. ae Nile 7 : + Remove = e = Different enzymes recognize different but specific hucleotides from specific site sequences. the ends of DNA. within DNA Typical restriction site is 4-6 nucleotide base pair res Jong, known as recognition sequence. He DNAs ¢ f | _ " u hat | These enzymes help in sealing the gaps in Dy = About $00 restriction enzymes are known that fragments, which are joined by complemen have been isolated from 230 strains of bacter base paiiiiys Restriction enzymes are named after the bacteria |, They act as molecular glue, join DNA fragmens's that produce them. forming phosphodiester bond. Example : ECO RI ‘When both vector and desired DNA cut by t& Eco + The bacteria E.coli same enzyme, the resultant DNA fragments bie Ho > Nass OF tiedtiain the same kind of sticky ends and these can joined together using DNA ligases. (B) Cloning Vectors : Cloning Vectors Bacteriophages + Itis an extra chromosomal genetic element of + Itcan rey i i DNA that is capable of replicating thecontelofchomecenetel® Independen independently of the host chromosome. + Have thei + Plasmids are generally circular molecules, minbeeetele tumber per cell, have very high copy found in both Prokaryotes (e.g. bacteria) and Senome within the bacterial cells. Eukaryotes (e.g. yeast) 50Riotechnology + Principles and Processes Features required Meilitate cloning int 10 vector ah @ Vous vectors for cloning genes iy plants and animals Origin of Replication : The origin of replication i a particular sequence in a genuine at which replication is intiated. sencine at which An fragment of DNA when inked tothis sequen Cane made forepitcae within thehostea Selectable Cloning Marker : Sites Selectable Marker : Selectable markers are the genes which lin ideising and timing pontransformants and selectively permitting rowth of the transformants. aed a They often possess antibiotic resistance ger they protect the organism from sletve agent like antibiotics, that would normally kill it or prevent its growth. ‘The genes encoding resistance to antibiotics such ‘as ampicillin, Chloramphenicol, tetralcycline or Kanamycin are considered useful selectable marker for E.Coli. ‘The normal E.Coli cells do not carry resistance against any of these antibiotics. Jreating and the DNA with high Ca? which allows DNA uptake by cells. Recombinant DNA can be forced into bacterial cells by incubating the cells with recombinant DNA nice followed by placing them briefly at 42°C and then putting them back onthe ice. injection. Electroporation _+ Collsare exposed torapid pulse of hhigh voltage current to render the plasma membrane permeable to recombinant DNA process of Recombinant DNA Technology + DNA recombinant technolo The term another organism when it reproduces Microinjection + Recombinant DNA is directly injected to the nucleus of animal cell through micro gy refers to the transfer of a segment of =m Cloning Sites : The vectar must also have on striction endonucteas recognition site ‘enable foreign DNA ta be inserted into the vector ing the generation of a recombinant DNA ‘The plasmid plRIZ2 carries within its sequence an origin of replication and twa antibiotic resistance genes : amp%ampicillin resistance) and tet" (tetracycline resistance) The amp" gene has Pst 1 cecagnition site while tet! gene has recognition sites for Hind [ll Barn Ht Vand Sal t Vectors for Cloning Genes in Plants and Animals ‘A cloning vector is a carrier DNA molecule in ‘which a desired DNA fragment can be integrated in such a way that the carrier molecule does not lose its capacity for self replication. It is used to introduce desired (donor) DNA into host cells. Competent Host : DNA usually can not get across cell membrane since it is a hydrophilic molecule. In order to force bacteria to take up the recombinant DNA, the bacterial cells must be made competent to take up. ‘Competent Host Particle Bombardment » Tiny high velocity particles of tungsten oF gold are coated with recombinant DNA and then shor into cells. This ‘method isalso known as biolistics orgene gun. Lipofecation + Recombinant DNA is coated with lipid which allows it to pass through the plasmamembrane. DNA from one organism to, Biology CRACK NCERT Kumar, 1 specific Location = kay. ng of DNA TN donor is identified 5 va fe S from the o jy Isolate the DNA Se quance -e : : at al etn aa NEY pNa it ‘oi cs | gent eC ONA 8 ly rented actin (int cele oH von a te thangs te attach with any DNA mmoecule that conan, = RNA ean be removed by te termplementary sticky ends in P enzym, protein can be Fer con can be separated 4, OHA i dition of chilled |=» The fragment of DIA " DNA is precipitated by the ad Te a thy! alcohol. Sperms or eggs are modified 3 by introduction of functional gene. = Change due to therapy would beheritable. > eliminatessymptomsof the disorder.ee Xt eed dae thes hain, are the, fide bon B\ ctional tive or ghose Lemar ant is Aptian: Implementing Gene Therapy Treatment vivo {Outside the body} Cells from the patient's blood removed! andgrown inlaba ea Theyare then exposedtavirnceny ‘The virus enters the cells anu} partofthe DNAofthecells The cells are allowed to grow i cl ' OW in the laboratory befo beingretumed othe patient byinjectioninesca bone marrow are carrying the desired gene. the desired gene become In vivo IInside the body} Nocells are removed from the patient'sbody. Instead vectors are used to deliver the desired gene to cellsin the patient’ sbody. 4 Transgenesis Animals : © Transgenic refer to the phenomenon of in and maintain a stable heritable character, troduction of exogenous DNA into the genome of an animal to create Importance of Transgenic Animals > Apowerful + Good model for > Several proteins produced > Transgenic tool for understanding the by transgenic animals are mice are being studying human diseases. Today important formedicaland developed for the gene ‘Transgenic modelsexist_ —_ pharmaceutical use in testing expression. _—_forhuman diseases, application. the safety of such as cancer, cystic In 1997, the first transgenic _—_-vaccines before fibrosis, rheumatoid cow, Rosie, produced they are used arthritis and Alzheimer’s human protein-enriched on humans. milk (2.4 gmi/licre) + Ethical Issues © Ethics includes ‘moral principles’ that control or influences a person's behaviour. = Itis mandatory to adopt some ethical standards to evaluate the morality of all human activities that might help or harm living organisms. * Biopatent : = A patent is the right granted by a govemment to Prevent others from commercial use of researcher's invention. ic researches are called © Patent for bioscient biopatents, A patent is granted for ‘ninvention An ‘The process Aconcept incudinga improvement of _——_or design Product inanearlier generating invention, a product In India as per Indian patent Act (1970) greater importance is given to the process af obtaining a product rather than to the material or the object itself, (> Strainsof microorganisms Variouskinds of tissue-cultured cells, jenetically modified varietiesof Biopatents plantsand animals. =e eyerdes Ly Specific DNA sequences. ‘Theproteins encoded by DNA sequence Biotechnological methods ‘5 Products, processand application Biopiracy : When big organization and multinational companies exploit patent biological resources or bioresources of other nations without proper authorization from the countries concern such exploitation is called biopiracy.Es Std.-12 : Biology CRACK NCERT | Turmeric, Neem and Basmati rice are our bioresources. Examples of biopiracy of economically important plants. - (1) America obtained patent for germplasm of our Basmati rice. ia (2) Biomolecules of many plants are patented in | other countries. BEE (3) Isolate useful genes and then obtain patent for them. (4) Pirate or steal the traditional knowledge and publish the same as new finding. A plant Pentadiplandra brazzeana of West Africa, | produces a protein called brazzein. . America obtained a patent for brazzeain and fused it in maize. Through this genetically modified variety they manufacture sugar.