sn technique - > FISH = Flunacnw Sn Attu hybrids zation os Thus metal mous a PNA of He gene Ceatled a probe) “that & complememtary w the gens of ment > thd proto is made radioactive oF is atachal te a Special molecule thet Fiusenss G4 undon ultavieles Cu) Ught = Tt ® added -» chromotomes from Kpecially treated celld (metaphase apraad de chnomeome peuponarien) Thai hawe bon Bpread ona microswpe blide => The probe birds ww the complementary DIA Sequence + the ENE ON “hw chnoMmesome ~The presence of mactioactiving of Puencreence from tt bound probe gwveals te lecation of the gene on a peutiuulen Chnormosome ~~ Tre we a flumpsconee in xitu hybridization has boon widly weed w identify The chromosomal Lecation ef human genes, Applications :— ) => Vad fr diagnosis of lymphoma | ex: B ce LyrPNOMA . (with Chromosoma! ‘rans location > detect gene amangement In cancer. SE Liteynee ex! JLukemia 7 ification nes in Breast cance) — dewd > amplifica 4 %: ’pa fingenprioning 2 the we of pen Acqunung aw Tdertify a pewen , % called Ona naenprinting i a pownrful 106! for criminal investigations ard other forensic application > The technique wed u pinpoint the culprit of the crime. ard ao in dispulme q ponenthood stone: = A sample of DNA from bleed, seman, hewi on oth body sisiuo collected from the crime Avene = TE tho domple & very Amat. PCR can be wrod tw amplify U 40 thet enough DNA is axnilble for tasting = Additional DNA Aomples ene tollectod from one or more SuApectn. => Fach DNA 4omple wt with ono or mose gwatriction enzymes. and The svsuiting DNA fragmens ow Aeponated by gel electro pheresis. = The frogmoms fn the gel one denatured and rrarsfevod w pinocellutose paper by poutrorn plotting. =3 One or mone madinactive probes & ~ hybridized gw He ninocelutose and a 10/ I cute radi ography = We gation of ponds produced by Dv prem to sample collected at te Crime Arone a ot on cpmpoved with the panerms proctuccd by pwa prem the Auopette.=) The ad used i in DNA Fregen preening dejeq war) viable negions a the genome , 4&0 ths chances Go pna from te0 pepople producing exactly Tue Samo paroliing fatten w Low. <- A match petween the aample fsorm thu scene and one ROM The pudspett Can crine provide oui dene hurt Yo Bubpect woe present at vAe scone cf he cr#me . = ee = —————— SSSDNA sequencing ey Sangen’s Fema ceennigque 4B cated Con nolled Aevmineine ah Sgmhesis We sea heen Aeammating agents £2 Suppnse “He Seqecence G Ww polynucleotide SAAT CH AMTAB' =) PNA Sample 4 taken 10 A differnt t0.T tubes =) IN all qubes, sho Klenow polymanade oithoul 6! enzyme ¢ DNA Ww BS exonuclease activity) and radio labotted TT ato prime ow addod- > Th av tubes gadicactive adNiP aw added => Sail amount of one 4 tne foun Aidoory yiborucleosids triphosphates Gad NTP ) is added & each esi thbe- = Tre produce of tt geaction. tren waist of a mixte of pra Grands 4 different length , each jeminating at a specific lone. => Tn firs) Yet tube, > date is added. We ddtTP will add to T, but cannot form the nox1 phospho cdiadior Vink, ont 50 funtius charn Nengthoning is Ateppod « n> ddtTP will Stop chains ay =) Tasted 4 dATP, the PYymeanone might add a norma AMP. ia which cose chan : P growth well continue yin tao next T.| > in The 3rd tot puke. ddcie % added | | uohich will ertain TTHAGC anda NAADCTC > Tn the Ath tube containing dd aTP will howe tru ard THAD => The wens « each tue one im foncnwaly lcxarnined on polyacrouslond cle ele cho p hora - ~ The position of “hase frogments wil wroapnd ow the choin length *, | ew on , =) chain with to nucleotide will move le. ~) Znuceottdo wil move maximum . otfen nulleotices wil be owanged in theins | order molecule Kite: | > h gw b thon auto- nodiograp hed. | => Ww oadiation pom %P labelled prime | will be qnaitable in ow pieces and wil) be sr os donk words in We x- may plate = From hw picts .,it ton bo infeed thot (Ath ard (pth poste ont T. 2 Siprilonly the position othr poses @P ba eee 8/il 5s Thus ihe pomeence of the newly br 4 Eimard OG enowr w» Tho complemen tog beyrenw vol) be present in the original unknown PVAi/i Blotting Biotirg B wel ww detect differnt tyre of macnomolecutes such 04 DNA. RNA ard proteln frm a mixtue . ‘Types | ) Bouthern Blottirg sechniqne | @) Northen Blowing technique | 3) hletern Bling technique D Southern Blowing technique :- | > his ued w detect a SpeU AC Segmem af DNA in ime whole qyenorme - | some bowed on DAA hybridization |prvb.ca | THD @ single grrantud piece “+4 PKA. labelled teithin with madiownotepe OF Lith nonnadioactive jabled , the nucleotide sequene o which |Coreplomantany we ho teuger DNA. | Ietopa | | => DNA ut wh neshiction enzymes % Electophonais on agar qel > A membrane jy positioned on ep af ex qet > Buffer drawn up Thte the y9o Layer of | Bottirg paper prysos Through Abe Gel | G@unying PNA on te ~hye membrane > @m DNA on the membrane Ub ffwe} > Plated ina hybridization bettie with A0LutiOn ‘That contecins a MAMA Vek ObeLd amin ord gpnity yorated n => The probe bins Cem naprired ire Complementary DNA tragmems on Tho membrene=> Aurora 2/i ~ prove weer igre hey oiert% feagment Application | Valle method in gene analyse => Forericatly applied w dotect minute | dmamities 4 DNA Cte TdeMrified pevonthood | théevar, napi srs), . | => Murant genes Buch os MoS , cystic filer’ | Bowie DMD, PkU Oo Well O4 prosenco «f | vinal DNA Cheparitia vinus Band Tdemfied . ©) tan be }i) Northoar ont ae Blotting te | => Tt i Wed w idenrerying Riva | => The total RNA i Yolated from tho cell, electrophoresed andthon botled on wa membrane => Then it i probed with mnadicactive ¢DNA > Thew wit be RNA - pyar ha bridization | 2 The is usod to detoct the na expression fn a tissue ¥ —_ (Sampe | INA exnacton ~~ | elecrrophoresis [-- - RNA Sepovarie] L oby size. \ = . \Auali zotor . f 4 “ labeled up ona fie nosThen boring Crransfen of RNA ay |, Membrane) —_ Pa as LORD Fixed to membrane a romans with hybridized with UV oF boat labeled probes3/n > By wing ths sechnique, we can iden... 4 eI > proteins oe Dolled from tne sigsun and electrophoresis & done > The epeuated proteins avo thon transfernod on a nfs collage mombran - > After fixottion, 1 w& probed with radioactive antihedly and quiterediogeaphed . lication > Confirmorory o& WIN teat > Tdontify specific protein Ina +s 7 thereby Sroufing the expression of a pari clan yeno erigen 4ampes Blotirg tank =_ 1S seperated | = i = peren ~~ tater - aL pel x Heelte g te) C <> cuawredtiogaph |) ims | ] oil ie aiiPCR = > PCR — Polgmenmve chein neaction 2 i & in vitro DNA amplification proceduve Step 4: Sepanation CDenermatisn) > the DNA Arends one Bepewoted by heating at ase for 15 scords to 9 minutes 4/i HEP S* Priming (Annealing) => Primers ome anneated by cooling tw s0°C for 05 & 9d mirucles => The prime aw annealed by cooling w ard then hybridize with thein complementad single - srrorted DNA producd in the fimt step 60c Step 3: Extension = New DNA Strands ane Bynthesi zed b. Tar polymerase. C derived from then mus cqyoti ass) => the polymmcse meaction iu take plaw at 19'c for 30 Seconds in presen 4 ANTP. =) eoth prance ow dupiatal DD The ade cs mepeated by automated insruren! — Temp cycler = Afler amplication procetuan, DNA hybridizction jechnique or South Wot cnakysin with o Auitable probe shows the prcrenes of te DNA in he karople tiAtwe-| [a] | 7. | tae 3?” a : Sf 5 cm : | > ° 44 primo Exttere\on - : Gevetuncin O00) te 50°C add denPs and 5 aaa Trett DNA ot by anal as 3st Camara 5: 4s*c) prime) '¢ polymonne | et {er { 3‘ mee 5‘ origina! DNA | Steps Step2 Sep3 ') Diagnosis =f bacterial and vino Aiscorsen | => PR can enuiti ply krau amour 5/it And fo blood samples, and othon becty frida cereaning only Very few Ubencle bacilli, Cestomegale virus and wiv i) In Medi wlegal cases: Ra NS =) DNA Fingerprints . ) Diagenaia of penstic diaorctore : = pce ® widely Used w ampity The gene Aegrems thot contato trewn mutations for agnosia oh inhentted dicases éuch od gickle cou anemia, bara shalawsemia . cystic Fibrosis | oye. 2 For preatet Aiagnesis of inherited Aaseares. => telps a idemiry individuals 94 hgh nok cana => IN foasil studiosCther typer Eck > Tr & wed & amplify Pra = Fasemially pormal vce ie precedéd by Nevenerse Hanscription Cts wnvent PNA te CDNA) ~> Prosone 4 Hrv RNA fm Weed fon be dotectou as eculy G4 hk weeks a fler infection. =) Tread of Taq polymonnte, + T th prlymerare from ‘thers cthenrmephilus may be wed. > Tha enzyme har both DNA polymerase and PUNeMs TranberproAs ectivities at high tompeties. =) TW atlows both CDNA “yrthos'A From mRNA followed by PCR amplicerion 6/n > Wanritation of ths number 4 virus preceny in tho sample can be calusteried ex: Vinal lead tin Hiv a eo) HBV DS So, the trectrem modalities can pe Planned and the mespons., tw Wien wuld bo or ced {2 = it & wed in proleuctes biolegy te ampli fy The ends of mersongen a RNA (MRNA) =? Otherwise catled one Aided Fe lor) anchored exe