PCR (polymerase chain reaction) ndi imodzi mwamatekinoloje a in-vitro DNA amplification, omwe ali ndi mbiri yopitilira zaka 30.

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

Mfundo Zoyambira za PCR

PCR imatha kukulitsa zidutswa za DNA nthawi zopitilira miliyoni imodzi.Mfundoyi ili pansi pa catalysis ya DNA polymerase, pogwiritsa ntchito chingwe cha makolo DNA ngati template ndi choyambirira ngati poyambira kukulitsa.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.Chomangira cha haidrojeni pakati pa zingwe ziwiri za DNA chimasweka pakutentha kwambiri (93-98 ℃).

2.Annealing: Pambuyo pa DNA yamitundu iwiri yolekanitsidwa, tsitsani kutentha kotero kuti primer ikhoza kumangirira ku DNA ya chingwe chimodzi.

3. Kuwonjeza: DNA polymerase imayamba kupanga zingwe zowonjezera m'mizere ya 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 zoyambira zosiyanasiyana zimatha kupangidwira mitundu yosiyanasiyana ya chandamale, kuti tizidutswa ta jini titha 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:

1.Kutalikirana ndi kukulitsa kosaneneka komanso zotsatira zabodza.

2.Kuzindikira kumatenga nthawi yayitali ndipo ntchitoyo ndi yovuta.

3.Only Mkhalidwe mayeso angathe kuchitidwa

M'badwo Wachiwiri Real-Time PCR

Real-Time PCR, yomwe imadziwikanso kuti qPCR, imagwiritsa ntchito ma probes a fulorosenti omwe amatha kuwonetsa momwe kachitidwe kachitidwe kachitidwe kakuyendera, ndikuyang'anira kuchuluka kwa zinthu zokulirakulira kudzera pakusonkhanitsa ma siginecha a fulorosenti, ndikuweruza zotsatira zake kudzera pamapindikira a fulorosenti.Itha kuwerengedwa mothandizidwa ndi mtengo wa Cq ndi curve yokhazikika.

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 fulorosenti probe, ma beacon a maselo ndi utoto wa fulorosenti.

1) TaqMan fulorosenti kafukufuku:

Pakukulitsa kwa PCR, kafukufuku wina wa fulorosenti amawonjezedwa ndikuwonjezera zoyambira.Kafufuzidwe ndi oligonucleotide, ndipo mapeto onse amalembedwa ndi mtolankhani fulorosenti gulu ndi quencher fulorosenti gulu.

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) Chiwonetsero cha 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, palibe fulorosisi yopangidwa.

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 chikoka cha mtengo wakumbuyo, ndipo zotsatira zake zimatha kusokonezedwa.

Pakakhala PCR inhibitors mu reaction system, zotsatira zodziwikiratu 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 kukulitsa bwino, ndipo kulolerana kwa PCR reaction inhibitors kumapangidwa 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 yayikulu: microfluidic, chip ndi droplet system.

1) Microfluidic digito PCR, mdPCR:

Kutengera ukadaulo wa microfluidic, template ya DNA imasiyanitsidwa.Tekinoloje ya microfluidic imatha kuzindikira kukweza kwa nano-kukweza kapena kutulutsa madontho ang'onoang'ono, koma madontho amafunikira njira yapadera yotsatsira ndikuphatikizidwa ndi dongosolo la PCR.mdPCR pang'onopang'ono yatengedwa ndi njira zina m'malo.

2) Droplet-based digital PCR, ddPCR:

Gwiritsani ntchito matekinoloje opangira madontho amadzi mumafuta kuti muzitha kuyika madontho, ndikugawaniza zomwe zili ndi ma nucleic acid kukhala madontho masauzande a nanoscale, iliyonse yomwe ilibe molekyulu ya chandamale ya nucleic acid yomwe imayenera kuzindikirika, kapena Muli mamolekyu amodzi kapena angapo a nucleic acid kuti ayesedwe.

3) Chip-based digital PCR, cdPCR:

Ntchito Integrated madzimadzi njira luso kulemba microtubes ambiri ndi microcavities pa zowotcha pakachitsulo kapena galasi khwatsi, ndi kulamulira otaya njira kudzera mavavu osiyana ulamuliro, ndi kugawa madzi chitsanzo mu nanometers wa kukula chomwecho mu zitsime anachita kwa digito PCR Reaction kukwaniritsa quantification mtheradi.

Zoyipa zazikulu za m'badwo wachitatu wa PCR:

Zida ndi reagents ndi okwera mtengo.

Zofunikira zamtundu wa template ndizokwera.Ngati chiwerengero cha template chikuposa kuchuluka kwa microsystem, sikungatheke kuwerengera, ndipo ngati kuli kochepa kwambiri, kulondola kwa quantification kudzachepetsedwa.

Zonama zabodza zitha kupangidwanso ngati palibe kukulitsa kwina.