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Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

ABSTRACT: A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.

SUBMITTER: Ren X

PROVIDER: S-EPMC4856977 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA. Keywords: Comparison of fluorescent dyes Each dye pairs were hybridized on two slides. The same two samples were compared on all the slides used in the present study. The liver sample from a healthy animal was labeled with the high wavelength dyes (Oyster 650/Alexa 647/Cy5) and the liver sample from the sick animal was labeled with the low wavelength dyes (Oyster 550/Alexa 546/Cy3). Each slide was scanned 3 times, immediately after hybridization (first round), after 1 month of storage in darkness and after 24 hours on the bench (12 hours of daylight and 12 hours of artificial light). Total RNA from the infected and control liver samples were extracted using RNeasy midi kit (Qiagen, Denmark) and DNase treated using RNase-Free DNase Set (Qiagen). 3DNATM Array 900 expression array detection kits (Genisphere Inc.) were used for the labeling and cDNA synthesis reaction of the RNA in the present study. Three different pairs of fluorescent dyes Oyster 550/650 (Genisphere Inc.), Alexa 546/647 (Genisphere Inc.), and Cy3/Cy5 (Genisphere Inc.) were used in separate labeling reactions. Labeling was done according to the manufacturers protocol for large-scale cDNA synthesis. For the cDNA synthesis 9.2Âµg total RNA from each liver sample was used and mixed with 20U AmpliQ RTenzyme and 10x first strand buffer (Bie og Berntsen, RÃ¸dovre, Denmark). Hybridization and washing were performed according to the manufacturers instructions (Genisphere) using Corning hybridization chambers. For one cDNA hybridization reaction; 6.5Âµl cDNA from each synthesis, 0.5Âµl Salmon sperm (10Âµg/Âµl) and 13.5Âµl 2 x Formamide-Based Hybridization Buffer (3DNA Array 900, Genisphere) was used. A total hybridization mix of 29Âµl was applied under a 22I x 25 mm LifterSlip (Erie Scientific, Portsmouth, NH, USA) carefully avoiding air bubbles. Slides were incubated at 44oC in a water bath over night. Wash buffer 1 (3DNA Array 900, Genisphere) was preheated to 44oC for the post cDNA hybridization wash. For two 3DNA hybridization reaction mixes; 2.5Âµl of each Capture reagent and 26Âµl SDS-Based hybridization buffer was mixed to a final volume of 52Âµl. 26Âµl 3DNA hybridization mix was applied to each slide, and incubated in darkness in a water bath at 50oC for 4 hours. Wash buffer 1 was preheated to 60oC for the post 3DNA hybridization wash. Slides were scanned on a CCD ArrayWoRxe auto (Applied Precision, Issaquah, WA, USA) using different exposure times (0.3 for 595 nm and 1.2 for 685 nm).

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Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

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