Tiknoolajiyada ogaanshaha molecular waxay isticmaashaa hababka bayoloji molecular si ay u ogaato muujinta iyo qaab-dhismeedka walxaha hidde-sideyaasha ee jidhka bini'aadamka iyo cudur-sidaha kala duwan, si loo gaaro ujeedada saadaalinta iyo ogaanshaha cudurrada.

Sanadihii la soo dhaafay, iyada oo la cusboonaysiiyay iyo dib u habeynta tignoolajiyada ogaanshaha molecular, adeegsiga kiliinikada ee ogaanshaha unugyada ayaa noqday mid aad iyo aad u ballaaran oo qoto dheer, suuqa baarista molecular wuxuu galay xilli horumar degdeg ah.

Qoraagu wuxuu soo koobayaa teknoolojiyadda ogaanshaha molecular ee caanka ah ee suuqa, wuxuuna u qaybsan yahay saddex qaybood: qaybta koowaad waxay soo bandhigaysaa tignoolajiyada PCR, qaybta labaadna waxay soo bandhigaysaa tignoolajiyada amplification isothermal nucleic acid, qaybta labaadna waxay soo bandhigaysaa tignoolajiyada isku xigxiga.

01

Qaybta I: Farsamada PCR

Tiknoolajiyada PCR

PCR ( falcelinta silsiladda polymerase) waa mid ka mid ah tignoolajiyada kordhinta DNA-da gudaha, oo leh taariikh in ka badan 30 sano.

Tignoolajiyada PCR waxaa hormood ka ahaa 1983 Kary Mullis oo Cetus, USA ah.Mullis wuxuu codsaday patent PCR 1985 wuxuuna daabacay waraaqdii tacliimeed ee PCR ee ugu horeysay isla sanadkaas.Mullis waxa uu ku guulaystay abaalmarinta Nobel Prize ee Kimistariga 1993kii.

Mabaadi'da aasaasiga ah ee PCR

PCR waxay ku kordhin kartaa jajabyada DNA-da bartilmaameedka ah in ka badan hal milyan jeer.Mabda'a ayaa ah in iyada oo la raacayo catalysis DNA polymerase, DNA strand strand ee waalidka loo isticmaalo qaab template ah, iyo asal gaar ah ayaa loo isticmaalaa barta bilawga kordhinta.Waxa lagu soo koobaa in vitro iyada oo loo marayo tillaabooyin ay ka mid yihiin denaturation, annealing, iyo fidinta.Habka DNA strand inanta oo kaabaya template strand template DNA.

Habka caadiga ah ee PCR waxa loo qaybiyaa saddex tallaabo:

1. Denaturation: Isticmaal heerkul sare si aad u kala saarto labada qaybood ee DNA.Xidhiidhada hydrogen ka dhexeeya labada xadhig ee DNA waxay ku jabaan heerkul sare (93-98°C).

2. Annealing: Ka dib marka la kala saaro DNA-da laba-geesoodka ah, heerkulka ayaa hoos loo dhigayaa si uu asalku ugu xidho DNA-da hal-xadhigleed.

3. Kordhinta: polymerase-ka DNA-ga waxa uu bilaabaa in uu isku xidho xargaha is-kaabaya ee ku teedsan xadhkaha DNA-da ee ka yimaadda furayaasha ku xidhan marka heerkulku hoos loo dhigo.Marka kordhinta la dhammeeyo, wareeg ayaa la dhammeeyaa, tirada jajabyada DNA-da ayaa labanlaabma.

Dib u celinta saddexdan tillaabo 25-35 jeer, tirada jajabyada DNA-da ayaa si aad ah u kordheysa.

Xariifnimada PCR waa in curiyeyaal kala duwan loo qaabayn karo hiddo-wadaha bartilmaameedka ah ee kala duwan, si jajabyada hidde-sidaha bartilmaameedka loo kordhiyo muddo gaaban gudaheed.

Ilaa hadda, PCR waxa loo qaybin karaa saddex qaybood, kuwaas oo kala ah PCR caadiga ah, PCR-ga tirada badan ee fluorescent iyo PCR dhijitaalka ah.

Jiilka koowaad ee PCR caadiga ah

Isticmaal aaladda caadiga ah ee PCR-ga si aad u xoojiso hidda-wadaha bartilmaameedka ah, ka dibna isticmaal agarose gel electrophoresis si aad u ogaato badeecada, kaliya falanqaynta tayada ayaa la samayn karaa.

Khasaaraha ugu weyn ee jiilka koowaad PCR:

-U nugul kor u qaadis aan gaar ahayn iyo natiijooyin been abuur ah.

-In la ogaado waxay qaadataa wakhti dheer, qalliinkuna waa dhib badan yahay.

-Kaliya imtixaan tayo leh ayaa la samayn karaa.

PCR-ga jiilka labaad ee fluorescence quantitative

Fluorescence quantitative PCR (Real-Time PCR), oo sidoo kale loo yaqaan qPCR, ayaa loo isticmaalaa in lagu kormeero ururinta alaabada la xoojiyay iyada oo loo marayo ururinta calaamadaha fluorescent iyadoo lagu darayo shaybaarro fluorescent ah oo tilmaamaya horumarka nidaamka falcelinta, iyo in lagu xukumo natiijooyinka iyada oo loo marayo qalooca fluorescence, waxaana lagu qiyaasi karaa qiimaha qalooca iyo caawinta.

Sababtoo ah tignoolajiyada qPCR waxaa lagu fuliyaa nidaam xiran, suurtogalnimada wasakheynta waa la dhimay, iyo calaamadda fluorescence waxaa lagu kormeeri karaa ogaanshaha tirada, sidaas darteed waa tan ugu ballaaran ee loo isticmaalo waxqabadka kiliinikada waxayna noqotay tignoolajiyada ugu sareysa PCR.

Walxaha fluorescent ee loo isticmaalo wakhtiga dhabta ah ee PCR-ga ee fluorescent waxa loo qaybin karaa: TaqMan fluorescent probes, beacons molecular iyo midabyo fluorescent.

1) Baadhista TaqMan fluorescent:

Inta lagu jiro kor u qaadida PCR, baaritaan gaar ah oo fluorescent ah ayaa lagu daraa iyadoo lagu darayo labo ka mid ah furayaasha.Baadhitaanku waa oligonucleotide, labada darafna waxaa siday u kala horreeyaan ugu calaamadsan yihiin koox fluorescent weriye ah iyo koox florescent ah.

Marka baaritaanka uu yahay mid sax ah, calaamada fluorescent ee ay sii daayaan kooxda weriyaha waxaa nuuga kooxda demisay;inta lagu guda jiro kor u qaadida PCR, 5'-3' firfircoonida exonuclease ee Taq enzyme ayaa jejebisa oo hoos u dhigta baaritaanka, samaynta weriyaha fluorescent group iyo quencher Kooxda fluorescent waa la kala soocaa, si nidaamka kormeerka fluorescence uu u heli karo signalka fluorescence, taas oo ah, mar kasta oo xarig DNA ah la kordhiyo, isugeyn buuxda ayaa la sameeyay molecule molecule. oo leh samaynta alaabta PCR.

2) Midabaynta fluorescent SYBR:

Nidaamka falcelinta PCR, xad dhaaf ah oo SYBR ah oo dheeha fluorescent ah ayaa lagu daray.Ka dib markii SYBR fluorescent dheega aan si gaar ah loogu dhex darin DNA-da laba-geesoodka ah, waxay soo saartaa calaamada fluorescent.Unugyada dheeha ee SYBR ee aan lagu darin silsiladda ma sii dayn doono wax calaamad ah oo fluorescent ah, taas oo hubinaysa calaamadda fluorescent Kor u kaca alaabta PCR ayaa si buuxda ula mid ah kororka alaabta PCR.SYBR kaliya waxay ku xidhan tahay DNA-da laba-geesoodka ah, markaa qalooca dhalaalka waxaa loo isticmaali karaa in lagu go'aamiyo in falcelinta PCR ay tahay mid gaar ah.

3) Calaamadaha molecular

Waa baadhista oligonucleotide oo laba-geesood leh oo samaysa qaab-dhismeedka timaha biinanka ah ee ku dhow 8 saldhig ee 5 iyo 3 darafyada.Taxanayaasha aashitada nuclei-ka ee labada daraf ayaa si isdhaxgal ah loo lammaaniyay, taasoo keenaysa in kooxda fluorescent iyo kooxda demisaa ay adkaadaan.Xir, ma soo saari doonto fluorescence.

Ka dib marka la soo saaro badeecada PCR, inta lagu jiro habka nuugista, qaybta dhexe ee iftiinka molecular waxaa lagu dhejiyaa taxane DNA oo gaar ah, iyo hidda-wadaha fluorescent ayaa laga soocaa hiddaha quencher si uu u soo saaro fluorescence.

Dhibaatooyinka ugu waaweyn ee jiilka labaad ee PCR:

Dareenka ayaa wali maqan, iyo ogaanshaha shaybaarada koobiga hooseeya sax maaha.

Waxaa jira saameyn qiyamka asalka ah, natiijaduna waxay u nugul tahay faragelinta.

Jiilka saddexaad ee PCR dhijitaalka ah

PCR dhijitaalka ah (DigitalPCR, dPCR, Dig-PCR) waxay xisaabisaa nambarka koobiga ee isku xigxiga bartilmaameedka iyada oo loo marayo ogaanshaha barta dhamaadka, oo waxay samayn kartaa ogaanshaha saxda ah ee saxda ah iyada oo aan la isticmaalin kontaroolada gudaha iyo qalooca caadiga ah.

PCR dhijitaalka ah waxay isticmaashaa ogaanshaha barta dhamaadka kumana xidhna qiimaha Ct (mareegta wareegga), markaa falcelinta PCR-ga dhijitaalka ah aad ayay u saamaynaysaa hufnaanta kordhinta, iyo dulqaadka horjoogayaasha falcelinta PCR waa la wanaajiyay, iyadoo saxsanaan sare leh iyo dib u soo saarid.

Sababo la xiriira sifooyinka dareenka sare iyo saxnaanta sare, si fudud uma faragalin ka hortagga falcelinta PCR, waxayna gaari kartaa qiyaas dhab ah oo run ah iyada oo aan la helin alaabada caadiga ah, taas oo noqotay goob cilmi baaris iyo codsi ah.

Marka loo eego noocyada kala duwan ee unugga falcelinta, waxa loo qaybin karaa saddex nooc: microfluidic, chip iyo droplet system.