After DNA fragments exit capillary, plot {electropherogram} shows relative dye concentration versus time expressed as frame number. In electropherograms, small peaks {pull-up peak} can appear under main dye peaks. Incorrect dyes, dye contamination, or capillary or fluid-property changes after spectral calibration can cause pull-up peaks.
In direct methods, mRNA-AAAAA + reverse transcriptase + Oligo-dT Primer + dNTPs + Cy3 and Cy5 or SymJAZ dye-dNTP -> Dye-cDNA {DNA labeling}.
In random priming methods, mRNA + reverse transcriptase + T7-T20-24 + MuLV -> DNA/RNA + hydrolysis -> cDNA first strand + Bst DNA polymerase + ligase + pN8-9 -> T7-ds cDNA + dye-UTP + T7 RNA polymerase + IVT -> labeled cRNA.
In RNase H methods, mRNA + reverse transcriptase -> DNA/RNA + RNase H -> DNA/RNA + DNA polymerase + ligase -> T7-ds cDNA + dye-UTP + T7 RNA polymerase + IVT -> labeled cRNA.
purposes
DNA labeling can measure labeled-cRNA dye incorporation, reverse-transcriptase conversion, fluorescence-specific activity, minimum RNA, maximum RNA, IVT amplification, total amplification, and length.
controls
Control reagents aid spot finding, image analysis, and signal quantification. Array probes monitor spotting, labeling, hybridization, printing, attachment, and features. Labeling controls monitor enzyme activity, target stability, and dye incorporation during labeling protocols. Hybridization controls monitor mixing, stringency, and washing during array-hybridization protocol. Printing and attachment controls monitor array manufacturing, probe attachment, and lot-to-lot variability. Feature controls normalize signal variability.
4-Genetics-Recombinant DNA-DNA Sequencing
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Date Modified: 2022.0225