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Optimization of the Sample Preparation Method for DNA Sequencing

Keywords: Hi-Di Form amide , BDT cycle sequencing kit v3.1 , PCR product

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This study reports on the modification and optimization of the sample preparation method for analyzing nucleotide sequence in plant species by capillary electrophoresis. The experimental study was aimed at developing possible ways of increasing the throughput of the method. The optimization conditions modified were purification of the PCR product, concentration adjustment of cycle sequencing kit BDT v3.1 from half to quarter reactions for economic analysis. Concentrations of the tamplet, primer and program of thermocycler were also optimized. Finally the volume of Hi-Di form amide was adjusted for the required sequencing. The accuracy of analysis was greater than 99.99%. The modified method enables quality sequencing results with higher average signal intensities compared to the standard recommended method. With the optimized electrophoresis conditions reported here, greater than 700 base pairs can be analyzed within 75 min using POP7. It was found that deviation from the recommended method consistently delivers higher signal-to-noise ratios and signal intensities and longer Phred20 read lengths. It is more reproducible than that of other methods and enables rapid, more cost effective robustness for nucleotide sequencing in the samples of plant origin received from research laboratory sources.


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