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PCR primers comparisons for a successful Tuber spp. DNA region amplification in routine identifications
Tina Unuk, Hojka Kraigher, Tine Grebenc, 2020, original scientific article

Abstract: Since late 20th century DNA sequencing became the method of choice method in precision species identification. The ITS region is one of the official fungal barcoding DNA markers, although in some cases sequencing of the ITS re-gion may, due to misidentification, mislabeling or nomen-clature errors in public databases, lead to incorrect or insuf-ficient identification, as is currently a case in the genus Tu b e r. The aim of this study was to test, which ITS primer pairs are most appropriate and optimal for Tu b e r species DNA region amplification. Thereby we (1) compared ampli-fication success for different Tu b e r species using fungal spe-cific primer pair ITS1f and ITS4 and (2) compared amplifi-cation success using different ITS primer pair combinations in amplifying DNA region an example species Tuber aesti-vum. Based on results, Tuber aestivum was one of the most reluctant Tu b e r species in this study and in most cases failed to amplify with the above primer pair. After comparing dif-ferent ITS primer pairs, we conclude that the primer pair ITS5 and ITS7 is the most appropriate primer pair for ampli-fication DNA region of T. ae stiv um as it resulted in high am-plification success from ectomycorrhizal root tips. Based on sequences, gained from public databases, we found that ITS1f and ITS6 primers have a mismatch in one base pair compared to the target sequence of Tuber aestivum, thus re-sulting in poor or no amplification success. Although prim-er pair ITS5 and ITS7 in our study was proven to be the most appropriate primer pair in amplifying DNA region Tu b e r aestivum species, further analysis about appropriateness of it for a general barcoding and identification of ectomycorrhiza in complex community samples is needed.
Keywords: Tuber spp., ITS region, PCR amplification, ITS primers
Published in DiRROS: 30.07.2020; Views: 1072; Downloads: 791
.pdf Full text (6,81 MB)
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Vpliv glivnih in rastlinskih sekundarnih metabolitov na verižno reakcijo s polimerazo (PCR)
Nejc Thaler, Marko Bajc, 2013, review article

Abstract: Sekundarni metaboliti so organske spojine, ki jih najdemo pri glivah in rastlinah, kjer imajo vlogo obrambnih in signalnih molekul ali zagotavljajo druge selekcijske prednosti, niso pa neposredno vpleteni v rast, razvoj in razmnoževanje organizma. Pri delu s tehnikami DNA so pogosto ravno sekundarni metaboliti tisti, ki posredno ali neposredno vplivajo na uspešnost verižne reakcije s polimerazo (PCR) ali reverezno transkriptazo, in sicer tako, da otežujejo celično lizo, povzročajo razpad nukleinskih kislin ali neposredno ovirajo delovanje encima polimeraze pri pomnoževanju tarčne DNK. Glavna ovira pri aplikaciji tehnike PCR v rutinski diagnostiki je priprava visoko kvalitetne DNA brez inhibitorjev. Še posebej to velja pri izolaciji DNA iz lesnatih rastlin (Minafra in sod., 1992) in vzorcev tal (Tsai in Olson, 1991). Večina standardnih postopkov izolacije nukleinskih kislin ne odstrani rastlinskih polisaharidov in polifenolnih komponent, ki imajo lahko neposreden vpliv na pomnoževanje s PCR (Demeke in Adams, 1992). Poskusi za premostitev tovrstnih ovir vključujejo bolj dovršene metode za izolacijo nukleinskih kislin in PCR, ki vključujejo uporabo pospeševalcev PCR za odstranitev ali zmanjšanje vpliva inhibitorjev PCR. Ta pregled je osredotočen na pristope za odstranitev ali zmanjšanje vplivov rastlinskih in glivnih sekundarnih metabolitov iz vzorcev tal, različnih rastlinskih tkiv in razkrojenega lesa zaradi pomena tovrstnih raziskav za gozdarstvo in lesarstvo.
Keywords: sekundarni metaboliti, izolacija DNA, pomnoževanje DNA, verižna reakcija, polimeraza, PCR
Published in DiRROS: 12.07.2017; Views: 3352; Downloads: 1469
.pdf Full text (685,41 KB)

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