Abstract
BackgroundThe detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids. This selective removal can be performed using a magnetic bead-based solid phase with covalently immobilized capture probes. One of the requirements to attain efficient host background nucleic acids subtraction is the capture probe characteristics.FindingsIn this study we investigate how various capture probe characteristics influence the subtraction efficiency. While the primary focus of this report is the impact of probe length, we also studied the impact of probe conformation as well as the amount of capture probe attached to the solid phase. The probes were immobilized on magnetic microbeads functionalized with a phosphorous dendrimer. The subtraction efficiency was assessed by quantitative real time PCR using a single-step capture protocol and genomic DNA as target. Our results indicate that short probes (100 to 200 bp) exhibit the best subtraction efficiency. Additionally, higher subtraction efficiencies with these probes were obtained as the amount of probe immobilized on the solid phase decreased. Under optimal probes condition, our protocol showed a 90 - 95% subtraction efficiency of human genomic DNA.ConclusionsThe characteristics of the capture probe are important for the design of efficient solid phases. The length, conformation and abundance of the probes determine the capture efficiency of the solid phase.
Highlights
The detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids
We focused on the effects of the length, conformation and the amount of probe on the selective capture of human genomic DNA using a previously developed magnetic bead based solid phase that enables capture of genomic targets in a single step [17,18,19]
The results indicated that the Klenow probes showed the best subtraction efficiency with less experimental variation, while the Sequenase probes showed similar subtraction efficiency with higher variations
Summary
We have investigated the effect of probe length, conformation and amount on the solid phase capture efficiency of human genomic DNA. Details on the magnetic bead bases solid support preparation, hybridization capture assays (Figure S1) and primer sequences and PCR conditions (Table S1). Competing interests There are two pending patent applications, one for preparation and functionalized of magnetic beads and one for genomic DNA subtraction protocol, that are related to this article. Both MJA, and BL are listed as inventors of these two patent applications. Authors' contributions MJA participated in the concept development, characterization and fabrication the solid phase, and drafting the manuscript. BL participated in the concept development, performing quantitative real time PCR analysis, and drafting the manuscript
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.
