PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9266082

Automated differential display using a fluorescently labeled universal primer.

N R Smith·M Aldersley·A Li·A S High·T P Moynihan·A F Markham·P A Robinson
View original source ↗Export metadata ↓

Abstract

We have modified the automated differential display reverse transcription polymerase chain reaction technique (DDRT-PCR) such that a single fluorescently labeled universal primer (d(F)CTCACG-GATCCGTCGATTTT) is used in all PCRs together with a selection of arbitrary primers. We term this fluorescent detection procedure FDDRT-PCR. Anchoring primers of general structure dTGGTCTCACGGATCCTCGA-(T)12 VN (where N can be any deoxynucleoside and V can be any deoxynucleoside other than thymidine) are used for the RT step, and the universal primer together with selected arbitrary primers are then used for the PCR amplification. Advantages of this approach are: (i) the fluorescently labeled universal primer is a constant feature in every PCR, so that changes in banding profile are highly likely to reflect the incorporation of different arbitrary 10-mer primers; (ii) artifacts that result from arbitrary 10-mer to arbitrary 10-mer primer amplifications are not observed by fluoresence detection on an automated gene scanner because such products are not fluorescently labeled; (iii) sample throughput and ease of data handling are increased when compared with the conventional radioactive/manual approach and (iv) using a single fluorescently labeled primer in all PCRs is highly cost-effective.

Explore related subjects

AutoradiographyBase SequenceDNA PrimersDNA, ComplementaryFluorescent DyesPolymerase Chain ReactionSequence Analysis, DNAComparative StudyResearch Support, Non-U.S. Gov'tTechnical ReportJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

N R Smith, M Aldersley, A Li, A S High, T P Moynihan, A F Markham, P A Robinson. 1997. Automated differential display using a fluorescently labeled universal primer.. https://doi.org/10.2144/97232st02

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Analysis of neurotransmitter receptor distribution patterns in the cerebral cortex.

The concentration of transmitter receptors varies between different locations in the human cerebral cortex, but also between the different cortical layers within the same area. Analyzing the regional differences in the laminar distribution patterns of various neurotransmitter receptor binding sites by means of quantitative receptor autoradiography may thus provide a functionally relevant insight into the organization of the cortex. Here we introduce an approach to the analysis of in vitro receptor autoradiographic data, providing a framework for the assessment of differences in both mean concentration and laminar distribution patterns across multiple subjects. Initially, laminar receptor distribution patterns for cortical areas are quantified by sampling density profiles in a series of regions of interest (ROIs) from digitalized autoradiographs and computing a mean profile per ROI. These ROI mean profiles are then corrected for distortions in the laminar pattern introduced by cortical folding and averaged to yield a mean profile per area, receptor and hemisphere. Differences in mean binding site concentration between areas are quantified by the asymmetry coefficient which is the difference of the mean concentrations divided by their sum. To quantify differences in laminar receptor distribution patterns between areas, the effects of absolute binding site concentration are first removed by dividing each profile by its mean value. Differences in the laminar pattern were then quantified by calculating the Euclidean distance between these mean corrected profiles. For single subject analysis, we propose a permutation test, comparing the differences between the mean profiles for two areas to differences between groups of profiles randomly assembled from all ROIs sampled in either area. Group inference can then be based on a between-subject conjunction analysis after correcting p-values to control for multiple comparisons. The feasibility of the presented approach is demonstrated by an exemplary analysis of the neurochemical differences between the ventral parts of the second and the third visual area.

Autoradiography↗
PubMed2006

Fate of cadmium in Elodea canadensis.

Elodea canadensis is a submersed macrophytes, widely distributed in stormwater treatment ponds and able to remove heavy metals from water. This study examines the Cd uptake, translocation, and efflux patterns in Elodea. Several experiments were set up in a climate chamber. To study the root and shoot Cd uptake, living and dead roots and shoots were treated with (109)Cd in one- and two-compartment systems. Furthermore, to examine Cd translocation and distribution, either roots or shoots were treated with (109)Cd. Finally, the efflux of Cd from roots and shoots, respectively, to the external solution was studied after loading whole plants with (109)Cd. Results from the two compartment studies show that Cd is accumulated via direct uptake by both roots and shoots of Elodea. The Cd accumulation proved not to be metabolically dependent in Elodea, and the apoplastic uptake in particular was decreased by Cd pretreatment. In one week, up to 23% of the root uptake was translocated to the shoots, while about 2% of the Cd accumulated by shoots was translocated to the roots. Thus, slight dispersion of Cd is possible, while metal immobilization will not be directly mediated via the Elodea plant. The efflux experiment proved that both shoots of dead plants and roots of living plants had a faster efflux than did shoots of living plants. This information is relevant for an understanding of the fate of Cd in stormwater treatment ponds with Elodea.

Autoradiography↗
PubMed2006

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

We have analysed the incorporation of [(3)H]sucrose and [(3)H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [(3)H]sucrose or [(3)H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2-3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [(3)H]Sucrose uptake was twice that of [(3)H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [(3)H]sucrose and [(3)H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [(3)H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [(3)H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [(3)H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Autoradiography↗