Ukadaulo wozindikira mamolekyulu amagwiritsa ntchito njira za biology kuti azindikire momwe ma genetic athupi la munthu amagwirira ntchito komanso ma virus osiyanasiyana, kuti akwaniritse cholinga cholosera ndikuzindikira matenda.

M'zaka zaposachedwa, ndi kukweza ndi kubwereza teknoloji yowunikira maselo, kugwiritsidwa ntchito kwachipatala kwa matenda a maselo kumakhala kokulirapo komanso mozama, ndipo msika wofufuza za maselo walowa m'nthawi ya chitukuko chofulumira.

Wolembayo akufotokozera mwachidule matekinoloje omwe amapezeka pamsika, ndipo agawidwa m'magawo atatu: gawo loyamba limayambitsa ukadaulo wa PCR, gawo lachiwiri likuwonetsa ukadaulo wa nucleic acid isothermal amplification, ndipo gawo lachiwiri likuwonetsa ukadaulo wotsatizana.

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Gawo I: PCR Technology

PCR luso

PCR (polymerase chain reaction) ndi imodzi mwa matekinoloje a in vitro DNA amplification, omwe ali ndi mbiri yazaka zopitilira 30.

Ukadaulo wa PCR udapangidwa upainiya mu 1983 ndi Kary Mullis waku Cetus, USA.Mullis adafunsira patent ya PCR mu 1985 ndipo adasindikiza pepala loyamba la maphunziro a PCR pa Sayansi mchaka chomwecho.Mullis adapambana Mphotho ya Nobel mu Chemistry mu 1993.

Mfundo Zoyambira za PCR

PCR imatha kukulitsa zidutswa za DNA nthawi zopitilira miliyoni imodzi.Mfundo yake ndi yakuti pansi pa catalysis ya DNA polymerase, DNA ya strand ya kholo imagwiritsidwa ntchito ngati template, ndipo choyambira china chimagwiritsidwa ntchito ngati poyambira kuwonjezera.Imabwerezedwanso mu vitro kudzera munjira monga denaturation, annealing, ndi kukulitsa.Njira ya mwana wamkazi wa strand DNA yogwirizana ndi DNA ya template ya kholo.

Njira yokhazikika ya PCR imagawidwa m'magawo atatu:

1. Denaturation: Gwiritsani ntchito kutentha kwambiri kuti mulekanitse zingwe ziwiri za DNA.Zomangira za haidrojeni pakati pa zingwe ziwiri za DNA zimasweka pakutentha kwambiri (93-98°C).

2. Annealing: Pambuyo pa kupatukana kwa DNA yamitundu iwiri, kutentha kumatsitsidwa kuti choyambira chimangirire ku DNA ya chingwe chimodzi.

3. Kuwonjeza: DNA polymerase imayamba kuphatikizira tingwe tating'ono ta DNA kuchokera ku zoyambira zomwe zimamangidwa kutentha kutsika.Kuwonjezako kukatsirizidwa, kuzungulira kumatsirizika, ndipo chiwerengero cha zidutswa za DNA chimawirikiza kawiri.

Kubwereza masitepe atatuwa nthawi 25-35, chiwerengero cha zidutswa za DNA chidzawonjezeka kwambiri.

Luso la PCR ndikuti ma primers osiyanasiyana amatha kupangidwira mitundu yosiyanasiyana ya chandamale, kuti zidutswa za jini zomwe mukufuna zitha kukulitsidwa pakanthawi kochepa.

Mpaka pano, PCR akhoza kugawidwa m'magulu atatu, omwe ndi PCR wamba, fulorosenti kachulukidwe PCR ndi digito PCR.

Mbadwo woyamba wa PCR wamba

Gwiritsani ntchito chida chokulirapo cha PCR kuti mukweze jini yomwe mukufuna, kenako gwiritsani ntchito agarose gel electrophoresis kuti muzindikire zomwe zili, kuwunika koyenera kokha kungachitike.

Zoyipa zazikulu za m'badwo woyamba wa PCR:

-Kukhazikika pakukulitsa kosadziwika bwino komanso zotsatira zabodza.

-Kuzindikira kumatenga nthawi yayitali ndipo ntchito yake ndi yovuta.

-Kuyesa kwabwino kokha kungatheke.

M'badwo wachiwiri wa fluorescence kuchuluka kwa PCR

Fluorescence quantitative PCR (Real-Time PCR), yomwe imadziwikanso kuti qPCR, imagwiritsidwa ntchito poyang'anira kuchuluka kwa zinthu zokulirakulira kudzera pakuwunjika kwa ma siginecha a fulorosenti powonjezera ma probes a fulorosenti omwe angasonyeze kupita patsogolo kwa kachitidwe, ndikuweruza zotsatira kudzera munjira yokhotakhota ya fluorescence, ndikuthandizira kupindika kwa quantq.

Chifukwa teknoloji ya qPCR imachitika mu dongosolo lotsekedwa, kuthekera kwa kuipitsidwa kumachepetsedwa, ndipo chizindikiro cha fluorescence chikhoza kuyang'aniridwa kuti chizindikire kuchuluka kwake, kotero ndicho chimagwiritsidwa ntchito kwambiri pazochitika zachipatala ndipo chakhala teknoloji yaikulu mu PCR.

Zinthu za fulorosenti zomwe zimagwiritsidwa ntchito mu nthawi yeniyeni ya fulorosenti yochuluka ya PCR ikhoza kugawidwa mu: TaqMan fluorescent probes, ma beacon a maselo ndi utoto wa fulorosenti.

1) TaqMan fulorosenti kafukufuku:

Pakukulitsa kwa PCR, kafukufuku wina wa fulorosenti amawonjezeredwa ndikuwonjezera zoyambira.Kafukufukuyu ndi oligonucleotide, ndipo mapeto awiriwa amalembedwa ndi gulu la mtolankhani fulorosenti ndi gulu la quencher fluorescent.

Pamene kafukufukuyo ali bwino, chizindikiro cha fulorosenti choperekedwa ndi gulu la mtolankhani chimatengedwa ndi gulu lozimitsa;Panthawi ya kukula kwa PCR, ntchito ya 5'-3 ′ exonuclease ya Taq enzyme imadula ndikuwononga kafukufuku, kupangitsa mtolankhani gulu la fulorosenti ndi kuzimitsa Gulu la fulorosenti limalekanitsidwa, kotero kuti dongosolo lowunikira fulorosenti likhoza kulandira chizindikiro cha fluorescence, ndiye kuti, nthawi iliyonse pamene chingwe cha DNA chimapangidwira, kukulirakulira, kukulirakulira. chizindikiro ndi synchronized kwathunthu ndi mapangidwe PCR mankhwala.

2) utoto wa fulorosenti wa SYBR:

Mu PCR reaction system, utoto wochulukirapo wa SYBR fulorosenti umawonjezeredwa.Utoto wa fulorosenti wa SYBR ukapanda kuphatikizidwa mu DNA iwiri-strand, umatulutsa chizindikiro cha fulorosenti.Molekyu ya utoto wa SYBR yomwe sinaphatikizidwe mu unyolo sidzatulutsa chizindikiro chilichonse cha fulorosenti, potero kuwonetsetsa chizindikiro cha fulorosenti Kuwonjezeka kwa zinthu za PCR kumagwirizana kwathunthu ndi kuwonjezeka kwa zinthu za PCR.SYBR imangomangiriza ku DNA yazingwe ziwiri, kotero kuti piritsi losungunuka lingagwiritsidwe ntchito kudziwa ngati PCR ikuchita mwachindunji.

3) Ma beacons a mamolekyulu

Ndilo kafukufuku wamtundu wa oligonucleotide wopangidwa ndi tsinde womwe umapanga mawonekedwe atsitsi pafupifupi 8 kumapeto kwa 5 ndi 3.Ma nucleic acid amatsatizana pa malekezero onse awiriwa amaphatikizana, zomwe zimapangitsa gulu la fulorosenti ndi gulu lozimitsa kukhala lolimba.Pafupi, sichidzatulutsa fulorosenti.

Pambuyo popanga mankhwala a PCR, panthawi ya annealing, gawo lapakati la beacon ya molekyulu limaphatikizidwa ndi ndondomeko yeniyeni ya DNA, ndipo jini ya fulorosenti imasiyanitsidwa ndi jini ya quencher kuti ipange fluorescence.

Kuipa kwakukulu kwa PCR ya m'badwo wachiwiri:

Kumverera kulibe, ndipo kuzindikira kwa zitsanzo zochepa sikulondola.

Pali mphamvu yakumbuyo, ndipo zotsatira zake zimatha kusokonezedwa.

M'badwo wachitatu wa digito PCR

Digital PCR (DigitalPCR, dPCR, Dig-PCR) imawerengera nambala yachitsanzo chandamale chandamale pozindikira pomaliza, ndipo imatha kuzindikira kuchuluka kwachulukidwe kolondola popanda kugwiritsa ntchito zowongolera zamkati ndi ma curve okhazikika.

Digital PCR imagwiritsa ntchito kuzindikira kwa mapeto ndipo sizidalira mtengo wa Ct (kuzungulira kozungulira), kotero kuti machitidwe a digito a PCR sakhudzidwa kwambiri ndi mphamvu ya amplification, ndipo kulolerana kwa PCR reaction inhibitors kumakhala bwino, ndi kulondola kwakukulu ndi kubereka.

Chifukwa cha mawonekedwe a kukhudzika kwakukulu komanso kulondola kwakukulu, sikusokonezedwa mosavuta ndi PCR reaction inhibitors, ndipo imatha kukwaniritsa kuchulukira kokwanira popanda zinthu zokhazikika, zomwe zakhala malo ofufuzira ndikugwiritsa ntchito.

Malinga ndi mitundu yosiyanasiyana ya unit reaction, imatha kugawidwa m'mitundu itatu: microfluidic, chip ndi droplet system.