Nā mea hoʻopilikia i nā hopena PCR

I ka wā o ka hana PCR, loaʻa pinepine kekahi mau mea keakea.
Ma muli o ke kiʻekiʻe loa o ka PCR, ua manaʻo ʻia ka haumia kekahi o nā mea nui e pili ana i nā hopena PCR a hiki ke hoʻopuka i nā hopena maikaʻi ʻole.
He mea koʻikoʻi like nā kumu like ʻole e alakaʻi ai i nā hopena wahaheʻe-maikaʻi ʻole. Inā pāpā ʻia a hoʻopilikia ʻia paha kekahi a ʻoi aku paha o nā ʻāpana koʻikoʻi o ka hui PCR a i ʻole ka hopena hoʻonui ponoʻī, hiki ke keakea ʻia ka hoʻāʻo diagnostic. Hiki i kēia ke alakaʻi i ka emi ʻana o ka pono a me nā hopena wahaheʻe maikaʻi ʻole.
Ma waho aʻe o ke kāohi ʻana, hiki ke nalowale ka pono o ka waikawa nucleic target ma muli o nā kūlana hoʻouna a/a i ʻole ka mālama ʻana ma mua o ka hoʻomākaukau ʻana o ka laʻana. Ma ke ʻano kūikawā, ʻo nā mahana kiʻekiʻe a i ʻole ka mālama kūpono ʻole e alakaʻi i ka hōʻino ʻana o nā cell a me nā waikawa nucleic. ʻO ka hoʻopaʻa ʻana o nā cell a me nā ʻiʻo a me ka hoʻokomo ʻana o ka paraffin he mau kumu i ʻike nui ʻia o ka ʻāpana DNA a me kahi pilikia mau (e nānā i nā Kiʻi 1 a me 2). I kēia mau hihia, ʻaʻole e kōkua ka hoʻokaʻawale maikaʻi loa a me ka hoʻomaʻemaʻe ʻana.

Kiʻi 1 | Ka hopena o ka hoʻopaʻa ʻana i ka pono o ka DNA
Ua hōʻike ʻia ka electrophoresis gel Agarose ua ʻokoʻa loa ke ʻano o ka DNA i hoʻokaʻawale ʻia mai nā ʻāpana paraffin o nā autopsies. Aia ka DNA o nā lōʻihi ʻāpana awelika like ʻole i loko o nā extracts ma muli o ke ʻano hoʻopaʻa. Ua mālama ʻia ka DNA i ka wā i hoʻopaʻa ʻia ai i loko o nā laʻana hau maoli a i loko o ka formalin neutral buffered. ʻO ka hoʻohana ʻana i kahi fixative Bouin acidic ikaika a i ʻole unbuffered, formic acid-containing formalin i hopena i ka nalowale nui o ka DNA. ʻO ka hapa i koe ua ʻoki nui ʻia.
Ma ka ʻaoʻao hema, ua hōʻike ʻia ka lōʻihi o nā ʻāpana ma nā kilobase pairs (kbp)

Kiʻi 2 | Ka nalowale ʻana o ka pono o nā pahuhopu waikawa nucleic
(a) ʻO kahi hakahaka 3′-5′ ma nā kaula ʻelua e hopena i ka haki ʻana o ka DNA i manaʻo ʻia. E hana mau ʻia ka synthesis o ka DNA ma ka ʻāpana liʻiliʻi. Eia nō naʻe, inā nele kahi kahua annealing primer ma ka ʻāpana DNA, hana wale ʻia ka hoʻonui linear. I ka hihia kūpono loa, hiki i nā ʻāpana ke hoʻopiha hou kekahi i kekahi, akā e liʻiliʻi nā hua a ma lalo o nā pae ʻike.
(b) ʻO ka nalowale ʻana o nā kumu, ma muli o ka depurination a me ka hoʻokumu ʻana o ka thymidine dimer, e alakaʻi ana i ka emi ʻana o ka helu o nā H-bond a me ka emi ʻana o Tm. I ka wā hoʻomehana elongated, e heheʻe nā primers mai ka matrix DNA a ʻaʻole e anneal ʻoiai ma lalo o nā kūlana ʻoi aku ka paʻakikī.
(c) Hoʻokumu nā kumu thymine e pili ana i kahi dimer TT.
ʻO kekahi pilikia maʻamau e kū pinepine ana i nā diagnostics molekala ʻo ia ka hoʻokuʻu ʻana o nā waikawa nucleic target i hoʻohālikelike ʻia me ka unuhi ʻana o ka phenol-chloroform. I nā hihia koʻikoʻi, hiki ke pili kēia me nā hopena wahaheʻe. Hiki ke mālama ʻia ka manawa nui ma o ka hoʻolapalapa ʻana i ka lysis a i ʻole ka hoʻoheheʻe ʻana o ka enzymatic o nā ʻōpala cell, akā ʻo kēia ʻano hana pinepine ka hopena i ka haʻahaʻa o ka PCR ma muli o ka lawa ʻole o ka hoʻokuʻu ʻana o ka waikawa nucleic.

Ka pale ʻana i ka hana polymerase i ka wā o ka hoʻonui ʻana

Ma keʻano laulā, hoʻohana ʻia ka inhibition ma ke ʻano he manaʻo ipu e wehewehe i nā mea āpau e alakaʻi ai i nā hopena PCR suboptimal. Ma ke ʻano biochemical paʻa, ua kaupalena ʻia ka inhibition i ka hana o ka enzyme, ʻo ia hoʻi, hōʻemi a pale paha i ka hoʻololi ʻana o ka substrate-product ma o ka launa pū ʻana me ka pūnaewele hana o ka DNA polymerase a i ʻole kona cofactor (e laʻa, Mg2+ no Taq DNA polymerase).
Hiki i nā ʻāpana i loko o ka laʻana a i ʻole nā ​​​​buffers like ʻole a me nā extracts i loaʻa nā reagents ke kāohi pololei i ka enzyme a hoʻopaʻa paha i kāna mau cofactors (e like me EDTA), no laila e hoʻopau ai i ka polymerase a ma ka huli ʻana e alakaʻi ai i ka emi ʻana a i ʻole nā ​​​​hopena PCR maikaʻi ʻole.
Eia nō naʻe, ua kapa ʻia hoʻi nā pilina he nui ma waena o nā ʻāpana hopena a me nā waikawa nucleic i loaʻa i ka pahuhopu he 'PCR inhibitors'. Ke hoʻopilikia ʻia ka pono o ke kelepona e ka hoʻokaʻawale ʻana a hoʻokuʻu ʻia ka waikawa nucleic, hiki ke hana ʻia nā pilina ma waena o ka laʻana a me kona hopena a puni a me ka pae paʻa. No ka laʻana, hiki i nā 'scavengers' ke hoʻopaʻa i ka DNA kaula hoʻokahi a pālua paha ma o nā pilina non-covalent a hoʻopilikia i ka hoʻokaʻawale ʻana a me ka hoʻomaʻemaʻe ʻana ma ka hōʻemi ʻana i ka helu o nā pahuhopu e hiki hope ana i ka ipu hopena PCR.
Ma keʻano laulā, aia nā mea hoʻopaʻa PCR i ka hapa nui o nā wai o ke kino a me nā reagents i hoʻohana ʻia no nā hoʻokolohua diagnostic lapaʻau (urea i loko o ka mimi, hemoglobin a me heparin i loko o ke koko), nā mea hoʻohui meaʻai (nā ʻāpana organik, glycogen, momona, nā ion Ca2+) a me nā ʻāpana i ke kaiapuni (phenols, nā metala kaumaha).

Hiki ke loaʻa nā mea hoʻopaʻa PCR i ʻoi aku ka nui ma nā bacteria a me nā cell eukaryotic, ka DNA non-target, nā macromolecules DNA-binding o nā matrices tissue a me nā lako hana hoʻokolohua e like me nā mīkina lima a me nā plastics. ʻO ka hoʻomaʻemaʻe ʻana i nā waikawa nucleic i ka wā a ma hope paha o ka unuhi ʻana ke ʻano makemake ʻia no ka wehe ʻana i nā mea hoʻopaʻa PCR.
I kēia lā, hiki i nā lako hana hoʻokaʻawale automated like ʻole ke pani i nā protocol manual he nui, akā ʻaʻole i hoʻokō ʻia ka hoʻōla 100% a/a i ʻole ka hoʻomaʻemaʻe ʻana o nā pahuhopu. Hiki ke loaʻa nā mea hoʻopaʻa hiki i loko o nā waikawa nucleic i hoʻomaʻemaʻe ʻia a i ʻole ua hoʻomaka mua ʻia. Aia nā hoʻolālā like ʻole e hōʻemi i ka hopena o nā mea hoʻopaʻa. ʻO ke koho ʻana o ka polymerase kūpono hiki ke loaʻa ka hopena koʻikoʻi i ka hana inhibitor. ʻO nā ʻano hana ʻē aʻe i hōʻoia ʻia e hōʻemi i ka pale ʻana o ka PCR ʻo ka hoʻonui ʻana i ka nui o ka polymerase a i ʻole ka hoʻopili ʻana i nā mea hoʻohui e like me BSA.
Hiki ke hōʻike ʻia ka pale ʻana o nā hopena PCR ma o ka hoʻohana ʻana i ka mana hoʻomalu maikaʻi o ke kaʻina hana kūloko (IPC).
Pono e akahele e wehe i nā reagents a me nā hopena ʻē aʻe i loko o ka pahu unuhi, e like me ka ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol a me ka phenol, mai ka isolate nucleic acid ma o kahi ʻanuʻu holoi pono. Ma muli o ko lākou nui, hiki iā lākou ke hoʻāla a kāohi paha i ka PCR.