Ngā āhuatanga whakararuraru i roto i ngā tauhohenga PCR

I te wā o te tauhohenga PCR, ka tūtaki pinepine ki ētahi āhuatanga whakararuraru.
Nā te tino tairongo o te PCR, e kiia ana ko te poke tētahi o ngā mea tino nui e pā ana ki ngā hua PCR, ā, ka taea te whakaputa i ngā hua teka-pai.
He rite tonu te hiranga o ngā pūtake maha e hua ai he hua kino-hē. Mēnā ka āraia, ka whakararuraruhia rānei tētahi, ētahi rānei o ngā wāhanga nui o te ranunga PCR, o te tauhohenga whakanui rānei, ka āraia pea te whakamātautau tātaritanga. Ka taea e tēnei te arahi ki te hekenga o te whai huatanga, tae atu ki ngā hua kino-hē.
Haunga te aukati, ka ngaro pea te pumau o te waikawa karihi ūnga nā ngā āhuatanga tuku me/ranei te rokiroki i mua i te whakarite tauira. Ina koa, ko te pāmahana teitei, ko te rokiroki koretake rānei ka arahi pea ki te kino o ngā pūtau me ngā waikawa karihi. Ko te whakapiri pūtau me te kiko me te whakauru paraffin he take rongonui o te pakaru o te DNA me te raruraru pumau (tirohia ngā Pikitia 1 me te 2). I roto i ēnei take, ahakoa te wehenga me te purenga tino pai, kāore e āwhina.

Pikitia 1 | Te pānga o te aukati i te tinana ki te pumau o te DNA
I whakaatuhia e te hikohiko agarose gel he rerekē te kounga o te DNA i wehea mai i ngā wāhanga paraffin o ngā tūpāpaku. I kitea ngā DNA o ngā roa maramara toharite rerekē i roto i ngā tangohanga i runga i te tikanga whakapiri. I tiakina te DNA i te wā i whakapirihia ai ki roto i ngā tauira tio taketake me te formalin kūpapa kua whakapūmauhia. Ko te whakamahinga o te whakapūmau Bouin waikawa kaha, te formalin rānei kei roto he waikawa formic kāore i whakapūmauhia, i hua ake ai he ngaronga nui o te DNA. He tino pakarukaru te toenga o te maramara.
I te taha maui, ka whakaatuhia te roa o ngā kongakonga i roto i ngā takirua kilobase (kbp)

Pikitia 2 | Te ngaronga o te pumau o ngā whāinga waikawa karihi
(a) Mā te āputa 3′-5′ i ngā miro e rua ka pakaru te DNA ūnga. Ka puta tonu te hanganga o te DNA i runga i te kongakonga iti. Heoi, ki te ngaro he pae whakamahana tuatahi i runga i te kongakonga DNA, ka puta noa te whakanui raina. I te wā tino pai, ka taea e ngā kongakonga te whakakī anō tetahi ki tetahi, engari he iti ngā hua, ā, kei raro iho i ngā taumata kitea.
(b) Ko te ngaronga o ngā turanga, ko te nuinga nā te tango i te purine me te hanganga o te thymidine dimer, ka heke te maha o ngā here-H me te hekenga o te Tm. I te wā whakamahana roa, ka rewa ngā primers mai i te DNA matrix, ā, e kore e whakamahana ahakoa i raro i ngā āhuatanga iti ake te uaua.
(c) Ka hangaia he dimer TT e ngā turanga thymine e tata ana.
Ko tētahi atu raruraru noa e puta pinepine ana i roto i ngā tātaritanga ngota ko te tukunga koretake o ngā waikawa karihi whāinga ki te whakataurite ki te tangohanga phenol-chloroform. I ngā wā tino kino, ka hono pea tēnei ki ngā hua kino teka. Ka taea te penapena i te nui o te wā mā te kohua i te wai, te nakunaku whākōkī rānei i ngā para pūtau, engari he maha ngā wā ka hua ake i tēnei tikanga he iti te aro o te PCR nā te korenga o te tukunga waikawa karihi.

Te aukati i te mahi polymerase i te wā whakanui

I te nuinga o te wā, ka whakamahia te aukati hei ariā ipu hei whakaahua i ngā āhuatanga katoa e hua ai ngā hua PCR kāore i te tino pai. I roto i te tikanga matū koiora tino tika, ka herea te aukati ki te mahi a te whākōkī, arā, ka whakaitihia, ka āraihia rānei te huringa hua-papa mā te taunekeneke ki te pae hohe o te DNA polymerase, ki tōna taupū rānei (hei tauira, Mg2+ mō te Taq DNA polymerase).
Ka taea e ngā wāhanga o te tauira, e ngā momo ārai me ngā tangohanga kei roto ngā tauhohenga te aukati tika i te whākōkī, te hopu rānei i ōna hoa mahi (hei tauira, te EDTA), ā, ka whakakore i te polymerase, ā, ka heke iho, ka puta rānei he hua PCR hē, he hua kino rānei.
Heoi, he maha ngā taunekeneke i waenga i ngā wāhanga tauhohenga me ngā waikawa karihi kei roto i te ūnga ka tohua hoki ko ngā 'aukati PCR'. Kia pakaru te hanganga o te pūtau i te wehenga, ā, kia tukuna te waikawa karihi, ka puta pea ngā taunekeneke i waenga i te tauira me tōna otinga me te wāhanga totoka e karapoti ana. Hei tauira, ka taea e ngā 'kaitātari' te here i te DNA kotahi-aho, takirua-aho rānei mā roto i ngā taunekeneke kore-tauhohenga, ā, ka whakararuraru i te wehenga me te purenga mā te whakaiti i te maha o ngā ūnga ka tae atu ki te oko tauhohenga PCR.
I te nuinga o te wā, kei roto i te nuinga o ngā wai tinana me ngā matū e whakamahia ana mō ngā whakamātautau tātaritanga haumanu (urea i roto i te mimi, te hemoglobin me te heparin i roto i te toto), ngā tāpiringa kai (ngā wāhanga rauropi, glycogen, ngako, ngā iona Ca2+) me ngā wāhanga i roto i te taiao (ngā phenol, ngā konganuku taumaha).

Ka kitea ngā aukati PCR e kitea whānuitia ana i roto i ngā huakita me ngā pūtau eukaryotic, te DNA kore-whainga, ngā macromolecules here-DNA o ngā matrices kiko me ngā taputapu taiwhanga pēnei i ngā karapu me ngā kirihou. Ko te purenga o ngā waikawa karihi i te wā e tangohia ana, i muri rānei i te tangohanga te tikanga pai mō te tango i ngā aukati PCR.
I ēnei rā, ka taea e ngā taputapu tango aunoa te whakakapi i ngā tikanga ā-ringa maha, engari kāore anō kia tutuki te whakaora me te/te purenga 100% o ngā ūnga. Tērā pea kei roto tonu i ngā waikawa karihi kua purea ngā aukati pea, kua whai hua rānei. He rerekē ngā rautaki hei whakaiti i te pānga o ngā aukati. Ko te whiriwhiri i te polymerase tika ka pā nui ki te mahi aukati. Ko ētahi atu tikanga kua whakamātauhia hei whakaiti i te aukati PCR ko te whakanui ake i te kukū polymerase, te whakamahi rānei i ngā tāpiringa pēnei i te BSA.
Ka taea te whakaatu i te aukati i ngā tauhohenga PCR mā te whakamahi i te mana whakahaere kounga tukanga ā-roto (IPC).
Me āta tango ngā matū katoa me ngā otinga kē atu i roto i te kete tango, pērā i te etanoru, te EDTA, te CETAB, te LiCl, te GuSCN, te SDS, te isopropanol me te phenol, mai i te waikawa karihi mā te horoi pai. I runga i te kukū, ka taea e rātou te whakahohe, te aukati rānei i te PCR.