aunp-modified sers substrates in a microscope slide format (ram-sers-au-5)  (Ocean Insight)

Journal: Upsala Journal of Medical Sciences

Article Title: From early methods for DNA diagnostics to genomes and epigenomes at high resolution during four decades – a personal perspective

doi: 10.48101/ujms.v129.11134

Figure Lengend Snippet: In-house manufactured modified robot for printing microarrays and silicon rubber reaction chamber for minisequencing reactions: (a) To automate printing of the minisequencing primers on a microscope, a low-cost industrial robot for etching a gluing (Isel Automation, Eiterfeld) was modified with an in-house manufactured tweezer-like tip for printing oligonucleotide primers in circular arrays on microscope slides in a format compatible with multichannel pipets. The robot had a capacity to print minisequencing detection primers for 300 samples per microscope slide. (b) Reusable miniaturized silicon rubber reaction chambers were prepared in-house molded on an inverted 384-well microtiter plate with V-shaped wells as a mold. Liquid silicon rubber (Elastosil RT 601 A/B, Wacker-Chemie GmbH) was poured into the mold, leaving about 1–2 mm of the tip of the wells uncovered. After allowing the rubber to harden over night, the grids containing 384 cone-shaped reaction chambers were cut to match the size of microscope slides. A rubber grid was placed over the primer arrays to form 80 separate reaction chambers. The reaction chambers had a glass surface with the primer array as bottom and the molded cone-shaped silicon rubber as wells, with open tops for pipet tips to fit into the chambers. Prior to adding the reaction mixtures, the rubber grid is firmly pressed against the glass surface in a custom-made aluminum rack with a Plexiglas cover containing drill holes for the pipet tips, through which the reaction mixtures are added. An aluminum rack, which can be heated, holds three microarrays with 80 reaction chambers.

Article Snippet: We used a microarray format on microscope slides for SNP genotyping with fluorescence detection using four dideoxy nucleotides (ddNTPs) labeled with Tamra (tetramethyl-6-carboxyrhodamine) in four separate reaction mixtures (NEN Life Science Products).

Techniques: Modification, Microscopy

Journal: Upsala Journal of Medical Sciences

Article Title: From early methods for DNA diagnostics to genomes and epigenomes at high resolution during four decades – a personal perspective

doi: 10.48101/ujms.v129.11134

Figure Lengend Snippet: Principle of allele-specific primer extension and genotyping results by allele-specific primer extension: (a) A pair of allele-specific primers with different 3’-ends that are complementary to each mutant or variable SNP allele is immobilized on a microscope slide as small circular arrays (125–150 uM in diameter). The DNA templates are amplified by multiplex PCR. During PC, T7 RNA polymerase promoter is inserted into the 5’-end of the DNA fragments. For genotyping, the PCR products are added to the primer arrays together with ribonucleotides (rNTPs) and a T7 RNA polymerase to generate multiple RNA targets by reverse transcription of each PCR product. Simultaneously with reverse transcription, fluorescent CY5-labeled dNTPs are incorporated into the target molecules in allele-specific primer extension reactions. The fluorescent signals on the microscope slides are quantified using a confocal fluorescence scanner, and the data are interpreted with a custom-designed software. (b) Carrier frequencies of 31 mutations of the ‘Finnish disease heritage’ determined in 2,100 population samples from four geographical regions of Finland.

Article Snippet: We used a microarray format on microscope slides for SNP genotyping with fluorescence detection using four dideoxy nucleotides (ddNTPs) labeled with Tamra (tetramethyl-6-carboxyrhodamine) in four separate reaction mixtures (NEN Life Science Products).

Techniques: Mutagenesis, Microscopy, Amplification, Multiplex Assay, Reverse Transcription, Labeling, Fluorescence, Software

Journal: Upsala Journal of Medical Sciences

Article Title: From early methods for DNA diagnostics to genomes and epigenomes at high resolution during four decades – a personal perspective

doi: 10.48101/ujms.v129.11134

Figure Lengend Snippet: Principle of four color tag-array minisequencing of single nucleotide polymorphisms: (a) Each minisequencing primer contains a unique 5’-tag sequence for capture of the extended primers by an immobilized 3’-complementary (c-tag) oligonucleotide in an ‘array-of-array’ configuration on a microscope slide. C-tag oligonucleotides have been immobilized on CodeLink Activated microarray slides (Motorola) by mediation of a 3’-NH2 group using a ProSys 5,510 spotter (Cartesian). The SNPs to be genotyped are amplified by multiplex PCR, after which cyclic minisequencing reactions in solution are performed with four fluorescently labeled ddNTPs in which the extended detection primers anneal immediately adjacent to each polymorphic SNP position. After genotyping, the fluorescent signals are measured using a ScanArray 5,000 instrument (Perkin Elmer Life Sciences), and the genotypes are assigned using the QuantArray ® analysis software of the instrument. (b) Images obtained by scanning a microscope slide at four wavelengths from one individual genotyped by WGA for a panel of 45 SNPs using tag-array minisequencing. Result from primers in both DNA polarities at duplicate positions is shown. The images from genomic DNA (WGA) and primer extension preamplification (PEP) and MDA products are shown in three vertical rows. The fluorescent labels used for the four dideoxy-dNTPs are indicated above the horizontal rows of the subarrays. The obtained signals are reproduced with an artificial rainbow scale with blue as low and white as saturated signal.

Article Snippet: We used a microarray format on microscope slides for SNP genotyping with fluorescence detection using four dideoxy nucleotides (ddNTPs) labeled with Tamra (tetramethyl-6-carboxyrhodamine) in four separate reaction mixtures (NEN Life Science Products).

Techniques: Sequencing, Microscopy, Microarray, Amplification, Multiplex Assay, Labeling, Software

Journal: RSC Advances

Article Title: A surface-enhanced Raman scattering-based approach for rapid and highly sensitive quantitative analysis of 3-carboxy-4-methyl-5-propyl-2-furanpropionate and indole-3-acetic acid in saline, human serum and uremic serum of patients with chronic kidney disease

doi: 10.1039/d0ra06123a

Figure Lengend Snippet: Comparison of the performance of the modified SERS vs. different methods of detection of IAA

Article Snippet: AuNP-modified SERS substrates in a microscope slide format (RAM-SERS-Au-5) were purchased from Ocean Optics.

Techniques: Modification, Electrophoresis, Colorimetric Assay