Fasahar gano kwayoyin halitta tana amfani da hanyoyin nazarin kwayoyin halitta don gano furci da tsarin kwayoyin halittar jikin dan Adam da kwayoyin cuta daban-daban, ta yadda za a cimma manufar hasashen da gano cututtuka.

A cikin 'yan shekarun nan, tare da haɓakawa da haɓaka fasahar gano ƙwayoyin ƙwayoyin cuta, aikace-aikacen asibiti na ƙwayoyin cuta ya zama mafi girma kuma mai zurfi, kuma kasuwar gwajin ƙwayoyin cuta ta shiga cikin saurin ci gaba.

Marubucin ya taƙaita fasahohin gano ƙwayoyin ƙwayoyin cuta na yau da kullun a kasuwa, kuma ya kasu kashi uku: kashi na farko ya gabatar da fasahar PCR, kashi na biyu ya gabatar da fasahar haɓaka haɓakar haɓakar acid nucleic acid, kashi na biyu kuma ya gabatar da fasaha ta sequencing.

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Sashe na I: Fasahar PCR

PCR fasaha

PCR (polymerase chain reaction) yana ɗaya daga cikin fasahar haɓaka DNA a cikin vitro, tare da tarihin sama da shekaru 30.

Kary Mullis na Cetus, Amurka ne ya fara aikin fasahar PCR a 1983.Mullis ya nemi takardar shaidar PCR a cikin 1985 kuma ya buga takarda ta farko ta PCR akan Kimiyya a cikin wannan shekarar.Mullis ya lashe kyautar Nobel a fannin ilmin sinadarai a shekarar 1993.

Ka'idodin asali na PCR

PCR na iya haɓaka gutsuttsuran DNA da aka yi niyya da fiye da sau miliyan ɗaya.Ka'idar ita ce, a ƙarƙashin catalysis na DNA polymerase, ana amfani da DNA strand na iyaye azaman samfuri, kuma ana amfani da takamaiman madaidaicin azaman wurin farawa don haɓakawa.Ana maimaita shi a cikin vitro ta matakai kamar denaturation, annealing, da tsawo.Tsarin DNA na 'ya mace wanda ya dace da samfurin mahaifa na DNA.

Daidaitaccen tsari na PCR ya kasu kashi uku:

1. Denaturation: Yi amfani da babban zafin jiki don raba DNA biyu igiyoyi.Haɗin hydrogen tsakanin igiyoyin DNA biyu sun karye a yanayin zafi mai girma (93-98°C).

2. Annealing: Bayan an raba DNA mai madauri biyu, ana saukar da zafin jiki ta yadda na'urar zata iya ɗaure ga DNA mai ɗaci ɗaya.

3. Tsawaitawa: DNA polymerase yana farawa don haɗa madaidaitan igiyoyi tare da igiyoyin DNA daga abubuwan da aka ɗaure lokacin da aka saukar da zafin jiki.Lokacin da aka gama tsawaita, ana kammala zagayowar, kuma adadin gutsuttsuran DNA ya ninka sau biyu.

Maimaita waɗannan matakai guda uku sau 25-35, adadin guntuwar DNA zai ƙaru sosai.

Hazakar PCR ita ce, ana iya ƙera maɓalli daban-daban don nau'ikan kwayoyin halitta daban-daban, ta yadda za a iya haɓaka gutsutsutsun halittar da aka yi niyya cikin ɗan gajeren lokaci.

Ya zuwa yanzu, ana iya raba PCR zuwa kashi uku, wato PCR na yau da kullun, PCR mai kyalli da kuma PCR na dijital.

Farkon ƙarni na talakawa PCR

Yi amfani da kayan haɓakawa na PCR na yau da kullun don haɓaka asalin manufa, sannan amfani da agarose gel electrophoresis don gano samfurin, ƙididdiga masu inganci kawai za a iya yin.

Babban rashin amfani na ƙarni na farko na PCR:

-Maganin haɓakawa mara iyaka da sakamako mai kyau na ƙarya.

- Gano yana ɗaukar lokaci mai tsawo kuma aikin yana da wahala.

- Gwajin inganci kawai za a iya yi.

PCR na ƙarni na biyu mai kyalli

Fluorescence quantitative PCR (Real-Time PCR), wanda kuma aka sani da qPCR, ana amfani da shi don saka idanu da tarin samfuran da aka haɓaka ta hanyar tarawar siginar kyalli ta hanyar ƙara ƙirar haske wanda zai iya nuna ci gaban tsarin amsawa, kuma don yin hukunci da sakamakon ta hanyar ƙirar haske, kuma ana iya ƙididdige ƙididdige ƙididdige ƙididdige ƙididdige ƙididdige ƙididdige ƙididdige ƙididdige ƙididdigewa da taimako.

Saboda ana aiwatar da fasahar qPCR a cikin rufaffiyar tsarin, yuwuwar kamuwa da cuta ta ragu, kuma ana iya lura da siginar kyalli don gano ƙima, don haka ita ce mafi yawan amfani da ita a cikin aikin asibiti kuma ta zama babbar fasaha a cikin PCR.

Abubuwan da aka yi amfani da su a cikin PCR na gaske ana iya raba su zuwa: TaqMan masu kyalli, tayoyin kwayoyin halitta da rini mai kyalli.

1) TaqMan bincike mai kyalli:

A lokacin haɓakawa na PCR, ana ƙara takamaiman bincike mai kyalli yayin ƙara nau'i-nau'i biyu.Binciken shine oligonucleotide, kuma ƙarshen biyu ana lakafta su da ƙungiyar masu kyalli mai ba da rahoto da ƙungiyar masu kyalli.

Lokacin da binciken ya kasance cikakke, siginar mai kyalli da ƙungiyar masu ba da rahoto ke fitarwa tana ɗaukar ƙungiyar masu kashewa;a lokacin haɓakawa na PCR, aikin 5'-3' exonuclease na Taq enzyme yana rarrafe kuma ya ƙasƙantar da binciken, yana sa ƙungiyar mai ba da rahoto ta ƙungiyar fluorescent da quencher Ƙungiyar fluorescent ta rabu, don haka tsarin kula da hasken wuta zai iya karɓar siginar hasken wuta, wato, duk lokacin da aka haɓaka DNA strand, an haɓaka siginar da aka yi da siginar da ke haifar da haɓakawa da haɓakawa gaba ɗaya. tare da samuwar samfurin PCR.

2) SYBR rini mai kyalli:

A cikin tsarin amsawar PCR, an ƙara yawan rini na SYBR mai kyalli.Bayan SYBR mai kyalli ba a haɗa shi ta musamman a cikin madauri biyu na DNA ba, yana fitar da siginar kyalli.Kwayoyin rini na SYBR wanda ba a haɗa shi cikin sarkar ba ba zai fitar da kowane sigina mai kyalli ba, don haka tabbatar da siginar kyalli Haɓaka samfuran PCR gaba ɗaya yana aiki tare tare da haɓaka samfuran PCR.SYBR kawai yana ɗaure ga DNA mai ɗaure biyu, don haka za a iya amfani da yanayin narkewa don sanin ko aikin PCR ya keɓanta.

3) Tambarin kwayoyin halitta

Wani bincike ne mai suna oligonucleotide mai tushe mai tushe wanda ya samar da tsarin gashin gashi na kusan sansanoni 8 a ƙarshen 5 da 3.Matsalolin acid nucleic a ƙarshen duka an haɗa su gaba ɗaya, yana haifar da ƙungiyar masu kyalli da ƙungiyar masu kashewa.Kusa, ba zai haifar da haske ba.

Bayan da aka samar da samfurin PCR, yayin aikin cirewa, ana haɗe tsakiyar ɓangaren fitilar kwayar halitta tare da takamaiman jerin DNA, kuma an raba kwayar halitta mai walƙiya da kwayar halitta don samar da haske.

Babban rashin amfanin PCR na ƙarni na biyu:

Har yanzu ba a sami hankali ba, kuma gano ƙananan kwafi ba daidai ba ne.

Akwai tasirin ƙimar baya, kuma sakamakon yana da sauƙin shiga tsakani.

PCR dijital na ƙarni na uku

PCR na dijital (DigitalPCR, dPCR, Dig-PCR) yana ƙididdige adadin kwafin jerin abubuwan da aka yi niyya ta hanyar gano ƙarshen ƙarshen, kuma yana iya yin daidaitaccen gano ƙididdige ƙididdigewa ba tare da amfani da sarrafawa na ciki da madaidaitan lanƙwasa ba.

PCR na dijital yana amfani da gano ƙarshen ƙarshen kuma baya dogara da ƙimar Ct (kofin sake zagayowar), don haka ƙimar PCR na dijital ba ta da tasiri ta haɓakar haɓakawa, kuma ana haɓaka haƙuri ga masu hana halayen PCR, tare da babban daidaito da haɓakawa.

Saboda halaye na babban hankali da daidaito mai girma, masu hana masu hanawa na PCR ba su da sauƙin shiga tsakani, kuma yana iya cimma cikakkiyar ƙididdigewa ba tare da daidaitattun samfuran ba, wanda ya zama wurin bincike da aikace-aikacen.

Dangane da nau'ikan nau'ikan nau'ikan amsawa, ana iya raba shi zuwa nau'ikan nau'ikan guda uku: microfluidic, guntu da tsarin droplet.