Ua make ʻo Kary Mullis ma Kaleponi i ka makahiki 74 i ka makahiki 7. Wahi a kāna wahine, ua make ma ʻAukake XNUMX. ʻO ke kumu o ka naʻau a me ka hanu ʻana ma muli o ka maʻi pneumonia.
ʻO James Watson ponoʻī, ka mea ʻike i ka mole DNA, e haʻi iā mākou e pili ana i kāna hāʻawi ʻana i ka biochemistry a no laila ua loaʻa iā ia ka makana Nobel.
Mahele mai ka puke na James Watson, Andrew Berry, Kevin Davis
DNA. Moolelo o ka Genetic Revolution
Mokuna 7. Genome kanaka. ʻO ke kūlana ola
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Ua hana ʻia ka polymerase chain reaction (PCR) i ka makahiki 1983 e ka biochemist Carey Mullis, nāna i hana ma Cetus. He mea kupaianaha ka ʻike ʻana o kēia ʻano. Ua hoʻomanaʻo ʻo Mullis: "I kekahi ahiahi Pōʻalima i ʻApelila 1983, ua loaʻa iaʻu kahi epiphany. Aia wau ma hope o ka huila, e holo ana ma ke alanui mauna ʻāwiliwili ma ka ʻĀkau o Kaleponi, ka ʻāina o nā ululāʻau ʻulaʻula. He mea kupanaha i loko o ia ʻano kūlana i loaʻa ai ka hoʻoikaika ʻana iā ia. A ʻaʻole ia he mau alanui kūikawā ma ka ʻākau o Kaleponi e hoʻoikaika i ka ʻike; ʻO ia wale nō ka ʻike ʻana o kāna hoaaloha iā Mullis e holo wikiwiki ana ma ke ala kaʻa ʻelua hau a ʻaʻole ia i pilikia iki iā ia. Ua haʻi aku kekahi hoaaloha i ka New York Times: "Ua ʻike ʻo Mullis e make ʻo ia ma ka hāʻule ʻana i kahi lāʻau ʻulaʻula. No laila, ʻaʻole ʻo ia e makaʻu i kekahi mea i ke kalaiwa, ke ʻole nā kumu lāʻau ʻulaʻula e ulu ana ma ke alanui. ʻO ka loaʻa ʻana o nā lāʻau ʻulaʻula ma ke alanui ua koi ʻo Mullis e noʻonoʻo a ... eia ʻo ia, he ʻike. Ua loaʻa iā Mullis ka Nobel Prize in Chemistry no kāna hana i ka makahiki 1993 a ua lilo ʻo ia i malihini i kāna mau hana. No ka laʻana, he kākoʻo ʻo ia i ka manaʻo revisionist ʻaʻole pili ʻo AIDS i ka maʻi HIV, ka mea i hoʻohaʻahaʻa nui i kona inoa ponoʻī a hoʻopilikia i nā kauka.
He mea maʻalahi ka PCR. No ka hoʻokō ʻana, pono mākou i ʻelua mau mea hoʻohui kemika i hoʻohui ʻia i nā ʻaoʻao ʻē aʻe o nā kaula like ʻole o ka ʻāpana DNA i makemake ʻia. He mau ʻāpana pōkole o ka DNA kaula hoʻokahi, ʻo kēlā me kēia ma kahi o 20 mau papa kumu ka lōʻihi. ʻO ka hiʻohiʻona o nā primers e pili ana i nā ʻāpana DNA e pono e hoʻonui ʻia, ʻo ia hoʻi, ka DNA template.
(Pili ke kiʻi) ʻO Kary Mullis, ka mea nāna i hoʻokumu i ka PCR
Hoʻokumu ʻia ka kikoʻī o ka PCR ma ka hoʻokumu ʻana i nā paʻakikī hoʻohui ma waena o ka template a me nā primers, nā oligonucleotides synthetic pōkole. Hoʻohui ʻia kēlā me kēia kumu mua i kekahi o nā kaula o ka laʻa kaulua a kaupalena ʻia ka hoʻomaka a me ka hopena o ka ʻāpana i hoʻonui ʻia. ʻO ka ʻoiaʻiʻo, ʻo ka hopena "matrix" he genome holoʻokoʻa, a ʻo kā mākou pahuhopu ke hoʻokaʻawale i nā ʻāpana hoihoi iā mākou mai ia mea. No ka hana ʻana i kēia, ua hoʻomehana ʻia ke kāʻei DNA kaulua i 95 ° C no kekahi mau minuke e hoʻokaʻawale i nā kaula DNA. Kapa ʻia kēia kaʻina denaturation no ka mea ua haki nā paʻa hydrogen ma waena o nā kaula DNA ʻelua. Ke hoʻokaʻawale ʻia nā kaula, e hoʻohaʻahaʻa ʻia ka mahana e hiki ai i nā primers ke hoʻopaʻa i ka template stranded hoʻokahi. Hoʻomaka ka DNA polymerase i ka hana hou ʻana o DNA ma o ka hoʻopaʻa ʻana i kahi kaulahao nucleotide. Hoʻopili hou ka enzyme DNA polymerase i ke kaula maʻamau me ka hoʻohana ʻana i ka primer ma ke ʻano he kumu mua a i ʻole he laʻana no ke kope ʻana. Ma muli o ka pōʻaiapuni mua, loaʻa iā mākou ka papalua kaʻina o kekahi ʻāpana DNA. A laila hana hou mākou i kēia kaʻina hana. Ma hope o kēlā me kēia pōʻai, loaʻa iā mākou kahi wahi i ʻike ʻia ma ka nui pālua. Ma hope o iwakāluakūmālima mau pōʻai PCR (ʻo ia hoʻi, ma lalo o ʻelua mau hola), loaʻa iā mākou ka ʻāpana DNA o ka hoihoi iā mākou ma kahi nui he 225 mau manawa kiʻekiʻe ma mua o ka mea mua (ʻo ia hoʻi, ua hoʻonui mākou iā ia ma kahi o 34 miliona mau manawa). ʻO kaʻoiaʻiʻo, ma ka hoʻokomo i loaʻa iā mākou kahi hui o nā primers, DNA template, ka DNA polymerase enzyme a me nā kumu manuahi A, C, G a me T, ʻo ka nui o kahi huahana hopena kikoʻī (i kaupalena ʻia e nā primers) e ulu nui, a me ka helu. ʻO nā kope DNA "lōʻihi" he laina laina, no laila e lanakila nā huahana hopena.
Ka hoʻonui ʻana i ka ʻāpana DNA i makemake ʻia: polymerase chain reaction
I nā lā mua o ka PCR, ʻo ka pilikia nui kēia: ma hope o kēlā me kēia pōʻai hoʻomaha hoʻomehana, pono e hoʻohui ʻia ka DNA polymerase i ka hui ʻana, no ka mea, ua hoʻopau ʻia i ka mahana o 95 ° C. No laila, pono e hoʻohui hou i mua o kēlā me kēia o nā pōʻai 25. ʻAʻole maikaʻi ke kaʻina hana, pono ka manawa nui a me ka enzyme polymerase, a he pipiʻi loa ka mea. ʻO ka mea pōmaikaʻi, ua hele mai ʻo Mother Nature e hoʻopakele. Ua ʻoluʻolu nā holoholona he nui i ka mahana i ʻoi aku ka kiʻekiʻe ma mua o 37 °C. No ke aha i lilo ai ka helu 37 °C i mea nui no mākou? Ua hana kēia no ka mea ʻoi aku ka maikaʻi o kēia wela no E. coli, kahi i loaʻa mua ai ka enzyme polymerase no PCR. I loko o ke ʻano, aia nā microorganism nona nā protein, ma luna o nā miliona mau makahiki o ke koho kūlohelohe, ua lilo i mea kūʻē i nā wela kiʻekiʻe. Ua manaʻo ʻia e hoʻohana i nā polymerases DNA mai ka bacteria thermophilic. Ua lilo kēia mau enzymes i thermostable a ua hiki ke pale aku i nā pōʻai hopena. ʻO kā lākou hoʻohana ʻana i hiki ke maʻalahi a hoʻomaʻamaʻa i ka PCR. Ua hoʻokaʻawale ʻia kekahi o nā polymerases DNA thermostable mua mai ka bacteria Thermus aquaticus, e noho ana ma nā pūnāwai wela o Yellowstone National Park, a ua kapa ʻia ʻo Taq polymerase.
Ua lilo koke ʻo PCR i mea hana o ka Human Genome Project. Ma keʻano laulā, ʻaʻole ʻokoʻa ke kaʻina hana mai ka mea i hoʻomohala ʻia e Mullis, ua hana ʻia. ʻAʻole mākou i hilinaʻi hou i ka lehulehu o nā haumāna puka ʻeleʻele e hoʻokahe ikaika ana i nā kulu wai i loko o nā paipu hoʻāʻo plastik. I loko o nā laboratories hou e hana ana i ka noiʻi molecular genetic, hana ʻia kēia hana ma nā conveyors robotic. Hoʻohana mau ʻia nā robots PCR i kahi papahana sequencing e like me ka Human Genome me ka nui o ka polymerase paʻa wela. Ua huhū kekahi poʻe ʻepekema e hana ana i ka Human Genome Project i ka uku ʻana i ke kumukūʻai kiʻekiʻe i hoʻohui ʻia i ke kumukūʻai o nā mea hoʻohana ʻia e ka mea nona ka PCR patent, ʻo ka hui ʻoihana lāʻau lapaʻau ʻEulopa ʻo Hoffmann-LaRoche.
ʻO kekahi "kumu hoʻokele hoʻokele" ʻo ia ke ʻano hoʻonohonoho DNA ponoʻī. ʻAʻole hou ke kumu kemika o kēia ʻano hana i kēlā manawa: ua hoʻohana ka Interstate Human Genome Project (HGP) i ke ʻano hana akamai like a Fred Sanger i hoʻomohala ai i ka waena waena o 1970s. Aia ka mea hou i ka unahi a me ka pae o ka automation i hiki ke hoʻokō.
Ua hoʻokumu mua ʻia ka hoʻonohonoho ʻokoʻa ma ka hale hana ʻo Lee Hood ma California Institute of Technology. Ua hele ʻo ia i ke kula kiʻekiʻe ma Montana a pāʻani i ka pôpeku kolepa ma ke ʻano he quarterback; Mahalo iā Hood, ua lanakila ka hui i ka hoʻokūkū mokuʻāina ma mua o hoʻokahi manawa. Maikaʻi nō hoʻi kona mākaukau hana hui i kāna ʻoihana ʻepekema. Ua mālama ʻia ka hale hana ʻo Hood e kekahi poʻe chemist, nā mea olaola, a me nā ʻenekinia, a ua lilo koke kāna hale hana i alakaʻi i ka ʻenehana hou.
ʻO ka ʻoiaʻiʻo, ua hoʻokumu ʻia ke ʻano hana hoʻonohonoho automated e Lloyd Smith lāua ʻo Mike Hunapilller. Ua hoʻokokoke ʻo Mike Hunkapiller, a laila e hana ana ma ke keʻena hana ʻo Hood, iā Lloyd Smith me kahi noi no kahi ala hoʻonohonoho hoʻomaikaʻi ʻana e ʻokoʻa ai kēlā me kēia ʻano kumu. Hiki i ia manaʻo ke hoʻohālua i ka pono o ke kaʻina hana Sanger. Ma Sanger, i ka wā e hoʻopili ai i kēlā me kēia o nā paipu ʻehā (e like me ka helu o nā kumu), me ke komo ʻana o DNA polymerase, ua hoʻokumu ʻia kahi ʻano oligonucleotides o nā lōʻihi like ʻole, me kahi kaʻina kumu. A laila, ua hoʻohui ʻia ka formamide i nā paipu no ka hoʻokaʻawale kaulahao a ua hana ʻia ka polyacrylamide gel electrophoresis ma nā ala ʻehā. Ma ka mana o Smith a me Hunkapiller, ua kapa 'ia ka dideoxynucleotides me eha mau kala like 'ole a ua hana 'ia ka PCR ma ka paipu hookahi. A laila, i ka wā polyacrylamide gel electrophoresis, kahi kukuna laser ma kahi kikoʻī o ka gel e hoʻohauʻoli i ka hana o nā kala, a hoʻoholo ka mea ʻike i ka nucleotide e neʻe nei ma o ka gel. I ka wā mua, he pessimistic ʻo Smith - ua makaʻu ʻo ia ʻo ka hoʻohana ʻana i nā ʻano haʻahaʻa haʻahaʻa loa o ke kala e alakaʻi i nā ʻāpana nucleotide ʻaʻole hiki ke ʻike ʻia. Eia naʻe, i ka loaʻa ʻana o ka ʻike maikaʻi loa i ka ʻenehana laser, ua loaʻa koke iā ia kahi ala i waho o ke kūlana me ka hoʻohana ʻana i nā mea pena fluorochrome kūikawā e fluoresce ke ʻike ʻia i ka radiation laser.
(Ka mana piha - 4,08 MB) Paʻi maikaʻi: Hoʻopili ʻia ke kaʻina DNA me ka hoʻohana ʻana i kahi sequencer maʻalahi, loaʻa mai kahi mīkini hoʻonohonoho maʻalahi. Pili kēlā me kēia kala i kekahi o nā kumu ʻehā
Ma ke ʻano maʻamau o ke ʻano Sanger, hana kekahi o nā kaula o ka DNA i kālai ʻia ma ke ʻano he kumu hoʻohālike no ka synthesis o kahi kaula hoʻohui e ka enzyme DNA polymerase, a laila hoʻokaʻawale ʻia ke kaʻina o nā ʻāpana DNA i kahi gel ma ka nui. ʻO kēlā me kēia ʻāpana i hoʻokomo ʻia i loko o ka DNA i ka wā synthesis a ʻae i ka ʻike ʻana o nā huahana hopena i hōʻailona ʻia me ka wai kala fluorescent e pili ana i ke kumu hope (ua kūkākūkā ʻia kēia ma ka ʻaoʻao 124); No laila, ʻo ka fluorescence o kēia ʻāpana he mea hōʻike no kahi kumu i hāʻawi ʻia. A laila, ʻo ka hana ʻana a me ka nānā ʻana i nā huahana hopena. Hoʻopili ʻia nā hopena e ka lolouila a hōʻike ʻia ma ke ʻano he kaʻina o nā piko kala like ʻole me nā nucleotides ʻehā. A laila hoʻoili pololei ʻia ka ʻike i ka ʻōnaehana ʻike kamepiula, e hoʻopau ana i ke kaʻina hoʻokomo ʻikepili ʻeha a i kekahi manawa i paʻakikī loa ka hoʻonohonoho ʻana.
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No Khabrozhiteley 25% ho'ēmi me ka hoʻohana coupon - PCR
Source: www.habr.com