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qPCR

qPCR. Līga Saulīte LU BF maģ . 1. kurss Stud apl.nr . ls10115. Kvantitatīvā polimerāzes ķēdes reakcija = reālā laika polimerāzes ķēdes reakcija qPCR = qRT-PCR = RT- qPCR. qPCR attīstība. Kary Mullis atklāj PCR (1983). 95 o C. NP(1993). 60 o C. 72 o C.

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qPCR

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  1. qPCR Līga Saulīte LU BF maģ. 1. kurss Studapl.nr. ls10115

  2. Kvantitatīvā polimerāzes ķēdes reakcija = reālā laika polimerāzes ķēdes reakcija qPCR = qRT-PCR = RT-qPCR

  3. qPCR attīstība KaryMullis atklāj PCR (1983) 95 oC NP(1993) 60 oC 72 oC http://www2.warwick.ac.uk/fac/sci/chemistry/research/arodger/arodgergroup/people/msrhab/research/pcr/ http://unlmbsc.wordpress.com/educator-and-student-materials-2/

  4. qPCR http://www.gene-quantification.de/chemistry.html

  5. Nepieciešamie materiāli • SYBR Green • Nuclease-free ūdens • Praimeris (tiešais + reversais) • cDNS • qPCR iekārta http://www.innovateus.net/innopedia/what-does-sybr-green-mean http://www.usascientific.com/non-skirted-96-well-pcr-plate.aspx http://thelabworldgroup.blogspot.com/2012_12_01_archive.html

  6. Metodes princips http://www.gene-quantification.de/chemistry.html

  7. Ct = Cq https://www.abbottmolecular.com/technologies/real-time-pcr/maxratio-data-analysis.html http://www.sigmaaldrich.com/life-science/molecular-biology/whole-genome-amplification/single-cell-wga/qpcr-data.html http://en.wikipedia.org/wiki/File:Qpcr-cycling.png

  8. Rezultātu apstrāde http://www.gene-quantification.de/habilitation.html

  9. Rezultātu apstrāde Livaketal. 2001

  10. +/- • Nav nepieciešama papildus vizualizēšana pēc reakcijas • Vienlaicīga daudzu paraugu apstrāde • Augsta jutība • Nepieciešams mazs sākuma materiāla daudzums • Informatīvi dati • Augstas izmaksas • SYBR Green nespecifiska saistīšanās (sekundārās struktūras) • Augsta jutība pret kļūdām • Sarežģīta un laikietilpīga datu analīze

  11. Pielietojums • Klīniskie pētījumi • Mikrobioloģiskie pētījumi • Gēnu ekspresijas pētījumi • Patogēnu detekcija • ĢMO detekcija • Klīniska vīrusu kvantificēšana

  12. Saulīte u.c.(nepublicēti dati)

  13. Izmantotie literatūras avoti • Derveaux S., Vandesompele J., Hellemans J. 2010. How to do successfulgeneexpressionanalysisusingreal-time PCR. Methods 50, 227-230pp. • Heid C.A., Stevens J., Livak K.J., Williams P.M. 1996. Realtimequantitative PCR. GenomeRes. 6, 986-994 pp. • Livak K.J., Schmitigen T.D. 2001. AnalysisofrelativegeneexpressiondatausingReal-timequantitative PCR andthe 2-ΔΔCtmethod. Methods 25, 402-408 pp. • Pfaffl M.W. 2010. TheongoingevolutionofqPCR. Methods 50, 215-216 pp. • http://www.biosistemika.com/workshops/qpcr-basics • http://www.gene-quantification.de/habilitation.html

  14. Jautājums auditorijai Kurā cikla solī un pēc kāda parametra qPCR tiek kvantificēti dati?

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