OPTIMIZATION OF CYCLE SEQUENCING DNA BASE WITH REAGENTS BIGDYE PGEM USING DNA SEQUENCER GENETIC ANALYSIS AUOTOMATIS ABI PIRSM 310HARUMI YUNIARTI, BAMBANG CHOLIS S AND ASTRI RINANTI NUGROHO
This study aims to optimize the process cycle sequencing of DNA bases by dilution reagent BigDye (pGEM-BGT) using Automatic DNA sequencer ABI Prism 310 Genetic Analyzer based on fluorescence. With the concentration of volume variation method Reagents 2μL, 4μL and 8μL, c/ycling cycle duration and annealing temperature. Injected through a capillary tube automatically. Fluorescence detection of DNA fragments after electrophoresis process in the capillary tube is considered a method of DNA sequencing analysis simplification. Capillary electrophoresis instrument can be interpreted from the results elektroferogram display based on the length sequences, noisy, elektroferogram display, signal intensity and similarity with the reagent comparison. The accuracy of the sequencing results is influenced by several stages of the process of sequencing is the isolation, purification, amplification, characterization, etc. Big Dye treatment 2iL volume generated by the application program Phydit low similarity (95%) when compared with standard pGEM. Elektroferogram peak results show relatively low sequence (noisy) which is caused by a large pile of weak signal. However, the length of sequences that read relatively good, for long sequences of 400 bp range, as well as treatment for BigDye 4μL (99.55%). At 8iL obtained the highest similarity (99.74% at 25x cycle cycling duration and annealing temperature 500C) so it is more properly used to sequence length } 3000 bp. Quality and a good level of confidence shown by the display peaks at elektroferogram are clearer when edited using a program Chromas Lite V2.1. Process cycle sequencing of DNA bases with BigDye diluted reagent specific results, fast, precise, and accurate. In addition this technique does not require special skills for users and reduces the complexity of the sequencing process.
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