Molecular biological tshawb fawb txoj hauv kev ua lub luag haujlwm tseem ceeb hauv cov tshuaj niaj hnub, forensics thiab biology. Ua tsaug rau kev nce qib hauv kev tshawb fawb DNA thiab RNA, tus neeg muaj peev xwm kawm txog genome ntawm cov kab mob, txiav txim siab tus neeg sawv cev ntawm tus kab mob, paub txog qhov xav tau nucleic acid hauv kev sib xyaw ntawm cov kua qaub, thiab lwm yam.
Molecular biological tshawb fawb txoj kev. Dab tsi yog?
Rov qab rau xyoo 70 thiab 80s, cov kws tshawb fawb thawj zaug tau ua tiav hauv kev txiav txim siab tib neeg genome. Qhov kev tshwm sim no tau muab kev txhawb zog rau kev tsim kho ntawm noob caj noob ces thiab molecular biology. Kev tshawb fawb txog cov khoom ntawm DNA thiab RNA tau ua rau muaj qhov tseeb tias tam sim no muaj peev xwm siv cov nucleic acids txhawm rau kuaj mob, kawm cov noob.
Tau txais DNA thiab RNA
Molecular biological diagnostic txoj kev xav kom muaj cov khoom pib: ntau zaus nws yog nucleic acids. Muaj ntau txoj hauv kev los cais cov tshuaj no los ntawm cov hlwb ntawm cov kab mob nyob. Txhua tus ntawm lawv muaj nws tus kheej zoo thiab qhov tsis zoo, thiab nws yog qhov tsim nyogcoj mus rau hauv tus account thaum xaiv ib txoj kev rau cais cov ntshiab nucleic acids.
1. Tau txais DNA raws li Marmur. Cov txheej txheem muaj nyob rau hauv kev kho ib tug sib tov ntawm tshuaj nrog cawv, raws li ib tug tshwm sim ntawm cov ntshiab DNA precipitates. Qhov tsis zoo ntawm txoj kev no yog kev siv tshuaj tiv thaiv: phenol thiab chloroform.
2. Kev rho tawm DNA raws li Boom. Cov khoom siv tseem ceeb ntawm no yog guanidine thiocyanate (GuSCN). Nws pab txhawb rau nag lossis daus ntawm deoxyribonucleic acid ntawm cov substrates tshwj xeeb, uas tom qab ntawd nws tuaj yeem sau tau siv cov khoom tshwj xeeb. Txawm li cas los xij, GuSCN yog ib qho inhibitor ntawm PTC, thiab txawm tias ib feem me me ntawm nws uas nkag mus rau hauv cov nag lossis daus DNA tuaj yeem cuam tshuam rau cov txheej txheem ntawm polymerase saw cov tshuaj tiv thaiv, uas ua lub luag haujlwm tseem ceeb hauv kev ua haujlwm nrog nucleic acids.
3. Sedimentation ntawm impurities. Txoj kev sib txawv ntawm cov yav dhau los uas nws tsis yog cov molecules ntawm deoxyribonucleic acid uas yog precipitated, tab sis impurities. Ua li no, ion exchangers yog siv. Qhov tsis zoo yog tias tsis yog txhua yam khoom tuaj yeem ua rau nag.
4. Kev tshuaj ntsuam loj. Txoj kev no yog siv nyob rau hauv rooj plaub uas tsis muaj cov ntaub ntawv tseeb txog qhov muaj pes tsawg leeg ntawm DNA molecule, tab sis nws yog ib qho tsim nyog kom tau txais qee cov ntaub ntawv txheeb cais. Qhov no tau piav qhia los ntawm qhov tseeb tias cov qauv ntawm cov nucleic acid tuaj yeem raug puas tsuaj thaum kho nrog cov tshuaj ntxuav tes, tshwj xeeb yog alkalis.
Kev faib cov txheej txheem tshawb fawb
Txhua txoj kev tshawb fawb molecular biological tau muab faib ua peb pawg loj:
1. Amplification (siv ntau enzymes). Ntawm nohais txog PCR - polymerase saw cov tshuaj tiv thaiv, uas ua lub luag haujlwm loj hauv ntau txoj kev kuaj mob.
2. Tsis-amplifying. Cov txheej txheem no yog ncaj qha ntsig txog kev ua haujlwm ntawm kev sib xyaw ntawm nucleic acids. Piv txwv yog 3 blots, hauv situ hybridization, thiab lwm yam.
3. Cov txheej txheem raws li kev lees paub ntawm lub teeb liab los ntawm kev sojntsuam molecule uas khi rau ib qho kev sojntsuam DNA lossis RNA. Ib qho piv txwv yog Hybride Capture System (hc2).
Enzymes uas tuaj yeem siv rau hauv kev tshawb fawb molecular biology
Ntau txoj kev kuaj mob molecular suav nrog kev siv ntau yam enzymes. Hauv qab no yog cov feem ntau siv:
1. Kev txwv enzyme - "txiav" DNA molecule rau hauv qhov tsim nyog.
2. DNA polymerase - synthesizes ob-stranded molecule ntawm deoxyribonucleic acid.
3. Rov qab transcriptase (revertase) - siv los ua ke DNA ntawm RNA template.
4. DNA ligase - lub luag haujlwm rau kev tsim cov phosphodiester bonds ntawm nucleotides.
5. Exonuclease - tshem tawm cov nucleotides los ntawm cov ntu ntu ntawm deoxyribonucleic acid molecule.
PCR yog txoj hauv kev tseem ceeb ntawm DNA amplification
Polymerase saw cov tshuaj tiv thaiv (PCR) yog nquag siv nyob rau hauv niaj hnub molecular biology. Qhov no yog ib txoj hauv kev uas muaj ntau cov ntawv luam tau los ntawm ib qho DNA molecule (molecules yog amplified).
Lub luag haujlwm tseem ceeb ntawm PCR:
- kuaj mobkab mob;
- cloning ntawm DNA ntu, noob.
Cov ntsiab lus hauv qab no yuav tsum tau ua kom muaj cov tshuaj tiv thaiv polymerase: thawj DNA molecule, thermostable DNA polymerase (Taq los yog Pfu), deoxyribonucleotide phosphates (qhov chaw ntawm nitrogenous bases), primers (2 primers ib 1 DNA molecule) thiab lub kaw lus tsis yog nws tus kheej, nyob rau hauv uas txhua qhov kev tshwm sim tuaj yeem ua tau.
PCR muaj peb kauj ruam: denaturation, primer annealing thiab elongation.
1. Denaturation. Ntawm qhov kub ntawm 94-95 degrees Celsius, hydrogen bonds ntawm ob strands ntawm DNA tau tawg, thiab vim li ntawd peb tau txais ob qho tib si stranded molecules.
2. Primer annealing. Ntawm qhov kub ntawm 50-60 degrees Celsius, primers yog txuas mus rau qhov kawg ntawm ib leeg-stranded nucleic acid molecules los ntawm hom complementarity.
3. Elongation. Ntawm qhov kub ntawm 72 degrees, kev sib txuas ntawm tus ntxhais ob-stranded molecules ntawm deoxyribonucleic acid tshwm sim.
YDNA sequencing
Molecular biological tshawb fawb txoj kev feem ntau xav tau kev paub txog nucleotide ib ntus hauv deoxyribonucleic acid molecule. Sequencing yog nqa tawm los txiav txim cov kab mob caj ces. Molecular diagnostics ntawm lub neej yav tom ntej yuav yog raws li kev paub tau los ntawm tib neeg sequencing.
Cov hom kev sib txuas hauv qab no yog qhov txawv:
- Maxam-Gilbert sequencing;
- Sanger sequencing;
- pyrosequencing;
- noobsequencing.
