Iindlela Ezinto uphando begazi nokusetyenziswa kwazo

Ezinto eziphilayo iindlela zophando zidlala indima ebalulekileyo amayeza mihla, inzululwazi lwasenkundleni, kunye eziphilayo. Ngenxa phambili ekufundeni DNA kunye RNA, umntu uyakwazi ukuba bahlole genome yento ephilayo limisele arhente causative, ukuba baqaphele nucleic acid ayinqwenelayo umxube asidi, njl

iindlela Ezinto-eziphilayo. Yintoni na?

Buyela kwi 70-80s zobunzululwazi waba ngowokuqala ukuba izakhi zofuzo zabantu. Esi siganeko wabanika amandla okuqhubela kuphuhliso zobunjineli yemfuza ezinto eziphilayo. Study of iimpawu DNA kunye RNA kuye kwathetha ukuba ngoku kunokwenzeka ukuba ukusebenzisa le asidi acid ngenjongo loxilongo izifo, ukufunda sofuzo.

Ukulungiswa DNA kunye RNA

Ezinto eziphilayo iindlela zokuxilonga lifune ukuqala impahla: ngokuqhelekileyo oku nucleic acid. Kukho iindlela ezininzi ukuze ukhethe ezi zingangeni kwiiseli eziphilayo. Ngamnye alungileyo kunye neengxaki, yaye oko kufuneka ziqwalaselwe xa ukhetha indlela kusahlulwa nucleic ngendlela ecocekileyo.

1. Ukulungiswa DNA yi Marmur. Le ndlela iqulathe ekunyangeni umxube kwezinto utywala, ngokwemvula leyo DNA ecocekileyo. Icala elisezantsi le ndlela yeyona ukusetyenziswa kwezinto bako, phenol kunye chloroform.

2. Ukuba kude ye DNA kwi boom. Eyona nto ephambili esetyenziswa apha - nto guanidine Iamoniyam (GuSCN). Oku kukhuthaza yokulahlwa kwe deoxyribonucleic acid kwi substrates ezizodwa, ukusuka apho kunokuthi kamva kuqokelelwa ngokusebenzisa buffer okhethekileyo. Nangona kunjalo GuSCN - yinto inhibitor lwe TCP, nkqu inxalenye encinane yaloo kokuba ifakwe kwi-DNA anganefuthe ikhondo indlela polymerase chain, nto leyo ebalulekileyo xa usebenza asidi acid.

3. Umbilo of ukungcola. Le ndlela yohlukile engaphambili kuba molekyuli ngokwabo na ezifakwe dehoksiribonukleinovoy acid kunye ukungcola. Ukuze wenze oku, sebenzisa kubananisi ion. Esi sithintelo kukuba asingawo onke izinto ungayidlulisa.

4. zokuhlola Mass. Le ndlela iyasetyenziswa kwiimeko xa akuyomfuneko ukuba ube ulwazi oluchanekileyo malunga isakhiwo kwemolekyuli DNA, kunye nemfuneko ukufumana ezinye manani. Isizathu kukuba isakhiwo asidi acid zingamoshakala xa uphethe iisepha, ingakumbi alkalies.

Ucalulo yenze uphando

Zonke iindlela genome-bemvelo uphando bohlulwa babe ngamaqela ezinkulu ezintathu:

1. okhulisa (usebenzisa i nobuninzi enqanda). Oku kuquka PCR - polymerase chain reaction, nto leyo kudlala indima ebalulekileyo ezininzi iindlela diagnostic.

2. Neamplifikatsionnye. Eli qela iindlela wayanyaniswe ngqo umsebenzi kwimixube acid acid. Imizekelo iintlobo ezi-3 uyazicima, behleli-apho hybridization, njl

3. Iindlela ezisekelwe ukuqatshelwa umqondiso ovela kwemolekyuli uphenyo, lona lubopha izinto ukuya ethile DNA okanye RNA probe. Umzekelo - inkqubo hybridization Hybride isisombululo Capture System (hc2).

Enzayim enokusetyenziswa kwiindlela emolekyuli uphando eziphilayo

ubuchule lwemolekyuli ezininzi zibandakanya ukusetyenziswa kuluhlu olubanzi enqanda. Ngezantsi ezithi zisetyenziswe kakhulu:

1. ukukhawulelwa enzyme - "kunqunyulwa" i-DNA ukuya kwiindawo eziyimfuneko.

2. DNA polymerase - synthesizes a deoxyribonucleic acid molecule kabini-balahleke.

3. reverse transcriptase (reverse transcriptase) - esetyenziswayo nokulungelelanisa DNA ukusuka template RNA.

4. DNA ligase - linoxanduva ukuyilwa iibhondi phosphodiester phakathi nucleotides.

5. exonuclease - ususa nucleotides ukusuka ngokwezahlulo ekupheleni acid molecule ideoxyribonucleic.

PCR - indlela esisiseko DNA yokukhulisa

Polymerase Chain Reaction (PCR) sisetyenziswa ngokubanzi ezinto eziphilayo mihla. Le ndlela, apho zinemoletyhule DNA zingafumaneka inani elikhulu iikopi (molecule zwi).

Imisebenzi esemqoka PCR:

- ukubonwa kwezifo;

- ngezakhi ziqwempu DNA, Gene.

Ukwenza indlela polymerase chain kufuna ezi zinto zilandelayo: ekuqaleni DNA, i-Polymerase DNA thermostable (Taq okanye Pfu), phosphate deoxyribonucleotide (imithombo neziseko nitrogen), le ncwadi (2 primer 1 DNA) kwaye ngokwayo inkqubo buffer endaweni leyo aqhube bonke abasabela ngayo.

PCR iqulathe amanyathelo amathathu: nokuguqulwa, annealing primer kanye zobude.

1. nokuguqulwa. Kubushushu degrees 94-95 Celsius proshodit ndibutshitshise imixokelelwane hydrogen phakathi neentsontelo ezimbini DNA, yaye ngenxa yoko sifumana iimolekyuli ezimbini-balahleke single.

2. kwenziwa annealed. Kubushushu degrees 50-60 C kwenzeka kwenziwa Ukuvuma shinyi, iimolekyuli acid acid-ndixakene single ngokungqinelana uhlobo complementarity.

3. zobude. Kubushushu degrees 72 ke intombi kuhlelwe kabini ndixakene iimolekyuli acid ideoxyribonucleic.

DNA ukulandelelanisa

Ezinto iindlela zophando bemvelo badla kufuna ulwazi ngokulandelelana nucleotides kwi molecule deoxyribonucleic acid. Ukuze ubone ikhowudi yezofuzo ezilandelelaniswa. uxilongo Ezinto ikamva liya kusekwa kwi ulwazi olufunyenwe ekumisweni ngokulandelelana yabantu.

Ezi ndidi zilandelayo yokusombulula:

• Ukulandelelaniswa yi Maxam-Gilbert;
• ukulandelelanisa Sanger;
• pyrosequencing;
• nanoporovoe nolandelelwano.