Abstract
Genomic DNA (gDNA) obtained from whole blood samples is a critical element for genomic research and clinical diagnosis. PCR efficiencies of the targeted genes like HLA-A, -B, -C, DPB1 and DRB1 using such isolated gDNAs were variable in spite of having similar amounts of gDNA taken for PCR. We addressed such PCR variabilities by normalizing the gDNA’s using an internal control of human coagulation factor XIII that was found to be variable with all samples and did not correlate with the observed A260 nm readings. The PCR and Q-PCR methodologies for the human coagulation factor XIII have been optimized, and the advantages of normalizing gDNA preparations based on F13 copy numbers have been discussed. This method will serve as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples for the next-generation sequencing purposes, and in forensic labs with limited sample availability.
Highlights
Preparation of pure human genomic DNA from whole blood in appreciable quantities is critical for basic science research, genetics, metagenomics, and clinical diagnosis
For next-generation sequencing (NGS), the primary requirement is equimolar pooling of the amplicons for unambiguous human leucocyte antigen (HLA) typing
From the HLA polymerase chain reaction (PCR) optimization experiments carried in laboratory, it was clear that the PCR signals of various genes of HLA were not constant in spite of taking an equal concentration of genomic DNA (gDNA) for every target based on A260 nm readings
Summary
Preparation of pure human genomic DNA (gDNA) from whole blood in appreciable quantities is critical for basic science research, genetics, metagenomics, and clinical diagnosis. It has been reported that there is a steep rise in the average primer-dimer rate and PCR cross-overs with increasing numbers of PCR cycles at DNA concentrations below 30 ng/ml, especially for applications of human leucocyte antigen (HLA) typing [5,6]. We realized that alternate methods to determine accurate concentrations of gDNAs would be immensely useful for metagenomics studies and other applications. We detailed out experiments carried out on the use of human coagulation factor XIII (F13) gene as an internal control (IC) for accurate quantification of gDNA and demonstration of the usefulness of this approach in HLA typing
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
