$un 2) Recombinant DNA technology (RDT) Recombinant DNA. Regulatory sequence & shuctural que Sounes I RDT source. I Desired DNA Vector. Cautonomously replicating w DNA Prolachiang DNA Chimeric DNA INA (genoals Hon) cloning screening RDT enkymes:- Restriction endonuclease Many no. of pragnients well be produced be produced used for gene cloning separatory tlectrophorosis. Restriction Endonuclease (18):- RDT requires certain purified biological products 1 agents for achieving ito. calleg as genetic engineering fools. objectiver There are many tools & techniques useg in RDI. - certain purified enzymes are the basics tools to initiate RDT. (antoy diff. -These enzyme bels are grouped into y types such as ty Nuclease. üy Ligaves. ty polymerase. DJA modified enxynces. - dmong different purified enzyme tools, mestriction enqonlictease a of great importance. Defination property & RE: I the nuclease enzymes fut at unique requence. that produce internal os ite in the DNA molecules are called as RE. The site weith specific base bes cences are called as recognition sequences which consist 4-8 The f - base pair's d recognition sites are for different. different f specific sore restriction enzyme recognise palidro- noric sequence while other recognise non-palindromnule sequence. Types of RE:- Bareg an their recognition sites and cleavage pattern mestriction endonuclease are of hypes Type 1 "y Type stil Type III. •Type 14 are not used in RDT. - Type 1 the recognise specific sequence in lex DNA but wut the DNA awar duplex from the recognition site. away месе Type :- There are coneplex endonuclease which are recognition requence og about 15 bp These cleaved the DNA pom 1000bpaway about 1000bpa its recognition ste the blend git (TCA). 13 ez-E-LOK, Edo B. Type I enzymes are made up of 3 non identical mubunits. There molecular weight 44 lakh dalton ymes have both endonuclease f These enzymes miethylase activity. Type:- J I These are the stable, symmetrical endonucleares which ent either withen or immediately outside thet's recognition sequence These recognise specific sequence ang cut the specific sequence. Loncints Ridentical mubunits was th a molecular weight of 20000 to 1 ik dalton. 297 requires only my las cofactor for 114 activity ex. EcoRI, Hing To ete. Eg-Hind, EcoRI, Bam HI st present there are 350 types & RE ucith over sequence are known. different recognition reque 106 d ↓ A T T 111 A C T 3 51 E-coli &eco Bal 2 pellin dromi e sequence Brevi bacterium 4 ANG Recognition site same grochizomer Nomenclatur 1"one letter-Genus and f 51 letter-species. No-prom-strains discovered. Type: Intermediate between Type III, Th The veleave the DNA near the mecognihon site. ric target sites f recognition asymetric target, 20x on one wut the DNA nition requ requence upid 20bp away.eg .copt fil jst letter of an enzyme to Nomenclature the lot letter of an las per the genus from. wendtten in capital as per P15 com cchiel Et is derived. -The 279f 69 setter is written on talla as per the species hanie after that the strain no, its written comen followed by not the nestricted enkynie discovered from that organisms. Eco RI front RE from genus species trady type Recognition sequence of cleavage pattern: Type I RE have recognition sequences molly of pallindromes with rotational symmetric @they have rance recognition site and cleavage ste @echen restricated enzynees. are is alated from 2 different organisms havhey have recognition earage site are ang cleavage 9- Acp 718, Asp Kim I ara evochicomerl. After the cleavage the pallindromic. generete staggered ends. pellindromic sequences sticky'r with stick eq-Ecoli RI. @Sony & RE produces ends. even cuts with blunk y-Alu I. The eveymes uskich produces t produces staggered euls reful for producing molecule recoribinant DNA Sometimes the ende produced by Restricted enzymes required to modify as per the requirements. eg-Removal g by 5'p group og VECHOY DNA es dome be in order to alkaline phosphatele prevent vector circulixation during DNA ensertfentegration. Daddition of 'p'proups to 51 ost ham van be done by Ty polynucleotide kinase. Linker 4 adapter. molecules sonichinees are Jornog to the uut hanga nchich veontains restriction sites. Linkers are agged to blunt endeq DNA fragments. Diddaptans are used to link the ends of 2 DNA. molecules that have dif aifferent sequences at thetr different kinds of adapter There are dif purposes. for different Vector- vector és a DNA molecule that has ability replicate appropriate hast cell and as is integrated for to buh uwhich the DNA Invert nan cloning. A vector Omost have certain properties to act as a good vector. 94 followsing properties should have the ex Replicate autonomously. if kary and purspy. to isolate and 1) Easy to introduce into the host well. (0) Easy for detection & selection. host well. from the transfered Ability to integrate esther itself or the DNA Ansert it carries into the genome host well. genome q the vriy contain urique recognition altes for restriction enzymes. Contain atleast suttable elenients. regulatory Avector replicate in a host It has been Esclated. from see natura naturally occuring. vectors donot have required propekties. all the Hea 6 To convert a vector into vector into good vector certain quences are agged from different gends cources to meet the the require ther such good vectors are good ud vector is ured for for woning or for called modulus. & purposes either expression. wchen a wchen a vector is wed for cloning purpose It is called as cloning vestered use for expresion purpose velled as expressiont sector. expression vector when allows of fusion proteins compared encoded by sequence in synthesis in the veltor and als encoded by DNA Insert called as translational fusion vecter. @ hehen a vector perrcit the synthesis of pure proteins encoded by only DNA Inserts i called as transuriptional fusion vector. The vectors are obtained from different sources, plasmid vectors are wolated pom bacterdapmages for the purposes. DAJA cloning. cats & the bacteria en uchich most plasmid vectors have been obtained. the 12 RP plenud (prendomonas) natural | les milf sechd LOL-EL (ECON) Ecoli also supports the several types of rectors Inside it to replicate of express vectors like plasmids, cosmids, phasemid, shuttle vector, artificial chromosomes all can be replicated inside cocoli. [A] plasmid vector- This are small, circular, double stranded, extrachromosomal DNA pre bacterial cell. int in the present 2 They replicate indipendently due to its suwn origin rite oq replication. 2 plasmids are 1kbp-200kbpsixe and has limited now gencinta Most bacteria contain more than one copy of each plasm plasmiqusually 2-300. 02-300 Natural plasmidi 8 6. coli have been modified shortened reconstructed f Reconebineg uility reate plasmilde of to create ual th specific function. enhanced 6 Achary & N. cohen ferst fert praved the use of plaining for gene cloning vector in 1973. The eloning. ning vectors created from plesning vectors are PBR 322, R1010, PSF 1010, PUE 8, pecios, PC18. But good plasmid vector must have mini- ancounter DNA, relaxed replication mal of control, atleast I selectable marker 4 one mecognition site for RA feary Splasmid vector are selection vestor are a hyper- natural for derived based 1 type: af pol 322 :- the main por denote p- plaanid, Rodrigodz the name B- Boliver f & for Rodrigodz the scientists ucho developed thin. It is a kircular dooNA (Plasmid) containing 4345 bobp. It has 528 nestrichien site for 66 mestriction enzymes. Out of 66, 20 mestriction enzymes unique restriction site. unique me cut at It has & selectable gene markers Like- Lin Meschtant tetracyclin gene and amphijeliin mestictant gene The TRT' gene has 6 restrichon site. - ECO-RL T 3 pres (13114) SPH1 Grushichow Alie origin repl Bar 2 49- Pestrichiam mapf structural features of per BR322 of gene is inserted into ane gene of this mestriction any of sites, the tetracycline resistant gene become frachive. -Simcilarly of a gene in inserted en amphyeylon resistance Inactive. gene Into resbichon It also become Advantages:- 91 & a small peesmild. consisting of 4363 bp. The vcopy no. of portoss in 15 which wan be increased upto 2000 by adding chloram- -phenical to the bacterial culture Bacterial wells wan uptake DNA pom culture. so. more and large DNA segments it can carry into host. poboss has & selectable marker genes (leta f २ Anep) for the selection of reconcbinent by insertional inachvation method. ene into this • Regulation I expression of the gene plasmig is very good. used "base plasenid for as a base Pavitro construction of derived plasmid vectors such as plovic pluca, pucio. fricainide. by al velonning veces constructed from PAR 322 It consist of 2076 bp. - It has origen g lac Z resistant sequence & amphieytis no melpercation from pasta f jene from Excali gene fram The lac z gene ha nuuttiple clorning segments sequente (MCS).