Molecular lom tshawb fawb txoj kev thiab lawv cov kev siv

Molecular lom tshawb fawb txoj kev ua si ib qho tseem ceeb luag hauj lwm nyob rau hauv niaj hnub tshuaj, forensic science, thiab biology. Tsaug rau kev kho nyob rau hauv txoj kev tshawb no ntawm DNA thiab RNA, tus neeg muaj peev xwm mus tshawb cov genome ntawm tus kab mob txiav txim seb lub causative tus neeg saib xyuas, kom paub txog cov kev kawm nucleic acid nyob rau hauv ib tug sib tov ntawm acids, thiab lwm yam

Molecular-lom txoj kev. Yuav ua li cas yog nws?

Rov qab nyob rau hauv lub 70-80s ntawm lub zaum yog tus thawj tau decipher tus tib neeg genome. Qhov kev tshwm sim muab impetus mus rau lub kev loj hlob ntawm kev tshuaj ntsuam genetic engineering thiab molecular biology. Txoj kev tshawb no ntawm thaj chaw ntawm DNA thiab RNA tau meant tias nws yog tam sim no tau siv cov nucleic acids rau lub hom phiaj ntawm tus kab mob mob, txoj kev tshawb noob.

Kev npaj ntawm DNA thiab RNA

Molecular lom diagnostic txoj kev yuav tsum pib cov ntaub ntawv uas: feem ntau qhov no nucleic acid. Muaj ob peb txoj kev yuav xaiv cov tshuaj los ntawm lub hlwb uas muaj sia nyob. Txhua yam nws muaj nws zoo thiab qhov tsis zoo, thiab nws yuav tsum tau raug xam tias thaum xaiv ib txoj kev cais nucleic acids nyob hauv cov ntshiab daim ntawv.

1. Kev npaj ntawm DNA los ntawm Marmur. Cov kev muaj nyob rau hauv kws kho lub ntoos sib xyaw cawv tshuaj, precipitates ua ntshiab DNA. Lub downside ntawm no txoj kev yog siv cov corrosive tshuaj, phenol thiab chloroform.

2. rho tawm ntawm DNA nyob rau boom. Lub ntsiab yam khoom uas yog siv no - yog guanidine thiocyanate (GuSCN). Nws yeej txhawb lub deposition ntawm deoxyribonucleic acid on tshwj xeeb substrates, los ntawm uas nws yuav tom qab sau los ntawm txhais tau tias ntawm ib tug tshwj xeeb tsis. Txawm li cas los GuSCN - yog ib tug inhibitor ntawm lub TCP, thiab txawm ib tug me me ib feem ntawm cov uas tau tso rau hauv DNA muaj feem xyuam rau cov chav kawm ntawm lub polymerase chain reaction, uas yog ib qho tseem ceeb thaum ua hauj lwm nrog nucleic acids.

3. Nag lossis daus ntawm impurities. Cov kev txawv los ntawm yav dhau los sawv daws yuav nyob rau hauv hais tias lub lwg me me lawv tus kheej yog tsis tso dehoksiribonukleinovoy acid thiab impurities. Ua li no, siv cov ion exchangers. Lub disadvantage yog tias tsis tag nrho cov tshuaj yuav kho.

4. pawg soj ntsuam. Qhov no yog siv nyob rau hauv tus neeg mob thaum koj tsis xav tau kom muaj tseeb cov lus qhia txog cov qauv ntawm cov DNA molecule, thiab tus yuav tau txais ib co txheeb cais. Yog vim li cas yog hais tias lub nucleic acid qauv yuav puas ntsoog thaum tuav detergents, tshwj xeeb yog alkalies.

Classification ntawm kev tshawb fawb txoj kev

Tag nrho molecular-lom txoj kev ntawm kev tshawb fawb yog muab faib ua peb loj pawg:

1. amplification (siv ib tug plurality ntawm enzymes). Qhov no muaj xws li PCR - polymerase chain reaction, uas plays ib qho tseem ceeb luag hauj lwm nyob rau hauv ntau yam ntawm lub diagnostic txoj kev.

2. Neamplifikatsionnye. Qhov no pab pawg neeg ntawm txoj kev yog txuam nrog lub chaw ua hauj lwm ntawm nucleic acid mixtures. Piv txwv yog 3 hom blots, nyob rau hauv situ hybridization, etc.

3. Txoj kev raws li nyob rau hauv lub Vocabulary ntawm lub teeb liab los ntawm cov kev sojntsuam molecule, uas uake rau ib tug DNA los yog RNA sojntsuam. Piv txwv li - hybridization system Hybride ntes System tshuaj (hc2).

Enzymes uas yuav siv tau nyob rau hauv molecular lom tshawb fawb txoj kev

Muaj ntau molecular diagnostic hom kev kawm cia siv ntau yam ntawm enzymes. Hauv qab no yog siv feem ntau:

1. txwv enzyme - "txiav" lub DNA molecule rau qhov tsim nyog qhov chaw.

2. DNA polymerase - synthesizes ib tug muab ob npaug rau-tso tseg deoxyribonucleic acid molecule.

3. Rov qab transcriptase (rov qab transcriptase) - siv coj los ua ke DNA los ntawm ib tug RNA template.

4. DNA ligase - yog lub luag hauj lwm rau cov tsim ntawm phosphodiester bonds ntawm nucleotides.

5. exonuclease - tuskheej nucleotides los ntawm lub kawg feem ntawm lub deoxyribonucleic acid molecule.

PCR - qhov yooj yim txoj kev ntawm DNA amplification

Polymerase saw tshuaj tiv thaiv (PCR) yog dav siv nyob rau hauv niaj hnub molecular biology. Qhov no txoj kev, nyob rau hauv uas ib tug tib DNA molecule yuav tau ib tug zoo tus naj npawb ntawm cov ntawv luam (amplifying molecule).

Lub ntsiab functions ntawm PCR:

- mob ntawm cov kab mob;

- cloning DNA theem, Noob.

Rau nqa tawm ib polymerase chain reaction yuav tsum tau cov nram qab no hais: thawj zaug DNA molecule, ib tug thermostable DNA polymerase (ntawm Taq los yog ntawm Pfu), deoxyribonucleotide phosphates (qhov chaw ntawm nitrogen bases), primers (2 primer 1 DNA molecule) nws tus kheej tsis system, uas tej zaum yuav ua tag nrho cov tshuaj.

PCR muaj peb cov kauj ruam: denaturation, primer annealing thiab elongation.

1. denaturation. Thaum ib tug kub ntawm 94-95 degrees Celsius proshodit ua txhaum lub hydrogen bonds nruab nrab ntawm ob chains ntawm DNA, thiab raws li ib tug tshwm sim uas peb tau txais ob zaug xwb-tso tseg molecules.

2. annealed primers. Thaum ib tug kub ntawm 50-60 degrees centigrade tshwm sim accession primers rau hauv lub xaus ntawm ib leeg-tso tseg nucleic acid molecules raws li lub hom complementarity.

3. elongation. Thaum ib tug kub ntawm 72 degrees yog tsim tus ntxhais ob tso tseg deoxyribonucleic acid molecules.

DNA sequencing

Molecular lom tshawb fawb txoj kev feem ntau yuav tsum tau kev paub txog cov theem ntawm nucleotides nyob rau hauv ib tug qauv ntawm deoxyribonucleic acid. Yuav kom txiav txim seb qhov tshuaj ntsuam genetic code sequenced. Molecular diagnostics ntawm lub neej yav tom ntej yuav tsum yog raws li nyob rau hauv txoj kev paub tau nyob rau hauv qhov kev txiav txim ntawm tus tib neeg ib theem zuj zus.

Cov nram qab no hom ntawm sequencing:

• sequencing los ntawm cov Maxam-Gilbert;
• Sanger sequencing;
• pyrosequencing;
• nanoporovoe sequencing.