Wopambana mphoto ya Nobel Kary Mullis, yemwe anayambitsa DNA polymerase chain reaction, wamwalira

Wopambana mphoto ya Nobel waku America mu chemistry Kary Mullis adamwalira ku California ali ndi zaka 74. Malinga ndi mkazi wake, imfa inachitika pa August 7. Chifukwa ndi mtima ndi kupuma kulephera chifukwa cha chibayo.

James Watson mwiniwake, yemwe adatulukira molekyu ya DNA, atiuza za zomwe adathandizira pazachilengedwe komanso zomwe adalandira Mphotho ya Nobel.

Kuchokera m'buku la James Watson, Andrew Berry, Kevin Davis

DNA. Mbiri ya Genetic Revolution

Mutu 7. Matupi athu. Zochitika pamoyo

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Polymerase chain reaction (PCR) idapangidwa mu 1983 ndi biochemist Carey Mullis, yemwe amagwira ntchito ku Cetus. Kutulukira kwa zimene anachitazi kunali kochititsa chidwi kwambiri. Pambuyo pake Mullis anakumbukira kuti: “Lachisanu madzulo mu April 1983, ndinali ndi chisinthiko. Ndinali kuseri kwa gudumu, ndikutsika mumsewu wa m’mapiri wonyezimira, wokhotakhota ku Northern California, dziko la nkhalango za redwood.” N'zochititsa chidwi kuti zinali mumkhalidwe woterowo pamene kudzoza kunamukhudza. Ndipo sikuti kumpoto kwa California kuli ndi misewu yapadera yomwe imalimbikitsa kuzindikira; kungoti bwenzi lake linamuwonapo Mullis akuthamanga mosasamala m'njira yamitundu iwiri yamadzi oundana ndipo sizinamuvutitse ngakhale pang'ono. Mnzake anauza nyuzipepala ya New York Times kuti: “Mullis anali ndi masomphenya akuti adzafa mwa kugwera mumtengo wa redwood. Choncho, iye saopa chilichonse poyendetsa galimoto, pokhapokha ngati pali mitengo ya redwood yomwe imamera m'mphepete mwa msewu. Kukhalapo kwa redwoods mumsewu kunakakamiza Mullis kuti aganizire ndipo ... apa zinali, kuzindikira. Mullis adalandira Mphotho ya Nobel mu Chemistry pakupanga kwake mu 1993 ndipo kuyambira pamenepo wakhala wachilendo m'zochita zake. Mwachitsanzo, iye ndi wochirikiza chiphunzitso cha revisionist chakuti Edzi sichigwirizana ndi HIV, zomwe zinawononga kwambiri mbiri yake ndikusokoneza madokotala.

PCR ndi njira yosavuta. Kuti tichite izi, timafunikira zoyambira ziwiri zopangidwa ndi mankhwala zomwe zimayenderana ndi zingwe zosiyanasiyana zachidutswa cha DNA. Zoyambira ndi zigawo zazifupi za DNA ya chingwe chimodzi, chilichonse chili ndi ma pair 20 m'litali. Chodabwitsa cha oyambira ndikuti amafanana ndi zigawo za DNA zomwe ziyenera kukulitsidwa, ndiye kuti, template ya DNA.

(Chithunzi chojambulidwa) Kary Mullis, woyambitsa PCR

Kukhazikika kwa PCR kumachokera ku mapangidwe ophatikizana pakati pa template ndi ma primers, oligonucleotides afupikitsa. Chilichonse mwazoyambira chimakhala chothandizira chimodzi mwa zingwe za template yokhala ndi mizere iwiri ndikuletsa chiyambi ndi mapeto a dera lokulitsa. M'malo mwake, "matrix" yomwe imachokera ndi genome yonse, ndipo cholinga chathu ndikupatula zidutswa za chidwi kwa ife kuchokera ku izo. Kuti muchite izi, template ya DNA yokhala ndi zingwe ziwiri imatenthedwa mpaka 95 ° C kwa mphindi zingapo kuti ilekanitse zingwe za DNA. Siteji imeneyi imatchedwa denaturation chifukwa zomangira za haidrojeni zomwe zili pakati pa nsonga ziwiri za DNA zimasweka. Zingwezo zikangolekanitsidwa, kutentha kumatsitsidwa kuti zoyambira zigwirizane ndi template yokhala ndi chingwe chimodzi. DNA polymerase imayamba kubwerezabwereza kwa DNA pomangirira kumtunda kwa unyolo wa nucleotide. Enzayimu ya DNA polymerase imatengera chingwe cha template pogwiritsa ntchito choyambira ngati choyambira kapena chitsanzo pokopera. Chifukwa cha kuzungulira koyamba, timapeza kuwirikiza kotsatizana kwa gawo lina la DNA. Kenako timabwereza ndondomekoyi. Pambuyo pa mkombero uliwonse timapeza malo omwe tikuyembekezera mowirikiza kawiri. Pambuyo pa magawo makumi awiri ndi asanu a PCR (ndiko kuti, pasanathe maola awiri), tili ndi chigawo cha DNA chomwe chili ndi chidwi kwa ife mu kuchuluka kwa 225 kuposa koyambirira (ndiko kuti, takulitsa nthawi pafupifupi 34 miliyoni). M'malo mwake, pakulowetsamo tidalandira zosakaniza zoyambira, template ya DNA, enzyme ya DNA polymerase ndi maziko aulere A, C, G ndi T, kuchuluka kwa chinthu china (chochepa ndi zoyambira) chimakula mokulirapo, ndipo kuchuluka kwake Makope a DNA “aatali” amakhala amizere, motero zinthu zimayendera kwambiri.

Kukulitsa gawo lomwe mukufuna la DNA: polymerase chain reaction

M'masiku oyambilira a PCR, vuto lalikulu linali lotere: pambuyo pa kutenthetsa-kuzizira kulikonse, DNA polymerase iyenera kuwonjezeredwa kusakaniza komweko, chifukwa idatsekedwa pa kutentha kwa 95 ° C. Chifukwa chake, kunali kofunikira kuti muwonjezerenso zisanachitike 25 iliyonse. Njira zomwe zimachitikira zinali zosagwira ntchito bwino, zimafuna nthawi yambiri ndi enzyme ya polymerase, ndipo zinthuzo zinali zodula kwambiri. Mwamwayi, Mayi Nature anabwera kudzapulumutsa. Nyama zambiri zimamva bwino potentha kwambiri kuposa 37 ° C. N’chifukwa chiyani chiwerengero cha 37 °C chinakhala chofunika kwa ife? Izi zidachitika chifukwa kutenthaku ndi koyenera kwa E. coli, komwe enzyme ya polymerase ya PCR idachokera. M'chilengedwe pali tizilombo tating'onoting'ono tomwe mapuloteni awo, zaka mamiliyoni ambiri akusankhidwa kwachilengedwe, akhala akugonjetsedwa ndi kutentha kwakukulu. Zapangidwa kuti zigwiritse ntchito DNA polymerases kuchokera ku mabakiteriya a thermophilic. Ma enzymes awa adakhala otenthetsera ndipo amatha kupirira zinthu zambiri zomwe zimachitika. Kugwiritsa ntchito kwawo kunapangitsa kuti zitheke komanso kusinthira PCR. Imodzi mwa ma polymerases a DNA omwe amatha kutentha kwambiri anali atasiyanitsidwa ndi bakiteriya Thermus aquaticus, yomwe imakhala ku akasupe otentha a Yellowstone National Park, ndipo idatchedwa Taq polymerase.

PCR idakhala gawo lalikulu pantchito ya Human Genome Project. Kawirikawiri, ndondomekoyi si yosiyana ndi yomwe inapangidwa ndi Mullis, yangokhala yokha. Sitinadalirenso gulu la ophunzira omaliza maphunziro anzeru omwe anali kuthira madontho amadzimadzi m'machubu oyesera apulasitiki. M'ma laboratories amakono omwe amapanga kafukufuku wamtundu wa mamolekyulu, ntchitoyi imachitika pa makina otumizira ma robotiki. Maloboti a PCR omwe akuchita nawo projekiti yotsatizana yayikulu ngati Human Genome amagwira ntchito mosalekeza ndi ma polymerase ambiri osasunthika. Asayansi ena omwe akugwira ntchito ya Human Genome Project adakwiya kwambiri ndi ndalama zochulukirapo zomwe zidawonjezedwa ku mtengo wazinthu zogwiritsidwa ntchito ndi eni ake a PCR patent, chimphona chazamankhwala ku Europe Hoffmann-LaRoche.

“Mfundo ina yoyendetsera galimoto” inali njira yotsatirira DNA yokha. Maziko a mankhwala a njirayi sanalinso atsopano panthawiyo: Interstate Human Genome Project (HGP) inatengera njira yochenjera yomwe Fred Sanger adapanga kumbuyo kwa zaka za m'ma 1970. Zatsopanozi zinali mu sikelo ndi digiri ya automation yomwe kutsatizana kunatha kukwaniritsa.

Makina otsatizana adapangidwa koyambirira mu labotale ya Lee Hood ku California Institute of Technology. Anapita kusukulu yasekondale ku Montana ndikusewera mpira waku koleji ngati quarterback; Chifukwa cha Hood, timuyo inapambana mpikisano wadziko kangapo kamodzi. Maluso ake ogwirira ntchito pamodzi adathandizanso pa ntchito yake ya sayansi. Laboratory ya Hood inali ndi antchito a motley a akatswiri a zamankhwala, akatswiri a sayansi ya zamoyo, ndi mainjiniya, ndipo labotale yake posakhalitsa inakhala mtsogoleri pazatsopano zamakono.

M'malo mwake, njira yotsatirira yokha idapangidwa ndi Lloyd Smith ndi Mike Hunkapiller. Mike Hunkapiller, yemwe amagwira ntchito mu labotale ya Hood, adafikira Lloyd Smith ndi lingaliro la njira yotsatirira yomwe mtundu uliwonse wa maziko uzikhala wosiyana. Lingaliro loterolo likhoza kuchulukitsa kanayi magwiridwe antchito a Sanger. Ku Sanger, potsata machubu onse anayi (molingana ndi kuchuluka kwa maziko), ndi DNA polymerase, gulu lapadera la oligonucleotides lautali wosiyanasiyana limapangidwa, kuphatikiza ndondomeko yoyambira. Kenako, formamide anawonjezedwa kwa machubu olekanitsa unyolo ndipo polyacrylamide gel osakaniza electrophoresis anachitidwa pa njira zinayi. M'mabaibulo a Smith ndi Hunkapiller, didioxynucleotides amalembedwa ndi mitundu inayi yosiyana ndipo PCR imapangidwa mu chubu chimodzi. Kenako, pa polyacrylamide gel electrophoresis, mtengo wa laser pamalo enaake pa gel osakaniza umasangalatsa ntchito ya utoto, ndipo chojambulira chimazindikira kuti ndi nucleotide iti yomwe ikuyenda kudzera mu gel. Poyamba, Smith anali wopanda chiyembekezo - amawopa kuti kugwiritsa ntchito utoto wochepa kwambiri kungapangitse kuti madera a nucleotide asadziwike. Komabe, pokhala ndi chidziwitso chabwino cha umisiri wa laser, posakhalitsa anapeza njira yothetsera vutoli pogwiritsa ntchito utoto wapadera wa fluorochrome umene umapangidwa ndi fluoresce pamene ukukumana ndi cheza cha laser.

(Full Version podina - 4,08 MB) Kusindikiza bwino: Ma DNA amatsatiridwa pogwiritsa ntchito makina ojambulira, otengedwa m'makina otsatizanatsatizana. Mtundu uliwonse umagwirizana ndi chimodzi mwa maziko anayi

Mu mtundu wakale wa njira ya Sanger, imodzi mwa zingwe za DNA yowunikiridwa imakhala ngati template ya kaphatikizidwe ka chingwe chothandizira ndi enzyme DNA polymerase, ndiye kuti mndandanda wa zidutswa za DNA umasanjidwa mu gel molingana ndi kukula. Chidutswa chilichonse, chomwe chimaphatikizidwa mu DNA panthawi ya kaphatikizidwe ndipo chimalola kuwonetsetsa kotsatira kwa zinthu zomwe zimachitika, zimalembedwa ndi utoto wa fulorosenti wofanana ndi malo osungira (izi zinakambidwa pa p. 124); choncho, fulorosenti ya chidutswa ichi chidzakhala chizindikiritso cha maziko operekedwa. Ndiye chomwe chatsala ndikungozindikira ndikuwonera zomwe zikuchitika. Zotsatira zake zimawunikidwa ndi makompyuta ndikuwonetsedwa ngati nsonga zamitundu yambiri zomwe zimayenderana ndi ma nucleotide anayi. Chidziwitsocho chimasamutsidwa mwachindunji ku chidziwitso cha makompyuta, ndikuchotsa njira yowononga nthawi komanso nthawi zina zowawa zomwe zimapangitsa kuti kusanja kukhale kovuta kwambiri.

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