Internal Validation of Reduced Polymerase Chain Reaction Volume for the Amplification of Nonquantified DNA Extracts from Reference Samples

Abstract

Introduction:

The present study was conducted to validate the reduced PCR reaction volume of Globalfiler^™ amplification kit for the amplification of non-quantified DNA from standard reference samples.

Methods:

For comparison purpose, the quantified and non-quantified DNA extracts were amplified using manufacturer's recommended PCR reaction volume of 25 μl and experimental volume of 10 μl. The amplified products were electrophoresed on Applied Biosystems^™ 3500 genetic analyser following the run conditions mentioned in the Applied Biosystems^™ Global Filer^™ user guide. GeneMapper ID-X software V1.4 was used for sample analysis.

Results:

Data analysis and subsequent statistical calculations indicated considerable difference in the peak height intensities, however quality of the profiles remained unaffected. The application of reduced volume PCR reaction for the amplification of standard reference samples would save not only the cost of the analysis but the time of analysis as well without compromising the quality of the profiles obtained.