Development of HyBeacon® probes for specific mRNA detection using body fluids as a model system

HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA pr...

Full description

Saved in:
Bibliographic Details
Published in:Molecular and cellular probes 2018-04, Vol.38, p.51-59
Main Authors: Stafford-Allen, Beccy, Dawnay, Nick, Hanson, Erin K., Ball, Glyn, Gupta, Ambika, Blackman, Stephen, French, David J., Duxbury, Nicola, Ballantyne, Jack, Wells, Simon
Format: Article
Language:English
Subjects:
Base Sequence
Biomarkers - blood
Body fluid
Body Fluids - metabolism
DNA - analysis
Forensic
Humans
HyBeacons
Models, Biological
Molecular Probes - chemistry
mRNA
Polymorphism, Single Nucleotide - genetics
RNA, Messenger - analysis
RNA, Messenger - genetics
RT-PCR
Sensitivity and Specificity
Tissue Donors
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA profiling. This work explores their use for the identification of expressed gene sequences through mRNA profiling. The use of mRNA is becoming increasingly common in forensic casework to identify body fluids on evidence items, as it offers higher specificity and fewer false positives than current chemical presumptive testing methods. The work presented here details the development of a single-step one-tube RT-PCR assay to detect the presence of body fluids of forensic interest (saliva, blood, seminal fluid, vaginal fluid and menstrual blood) using HyBeacon® probes and melt curve analysis. Each assay shows a high degree of specificity to the target body fluid mRNA suggesting there is no requirement to remove genomic DNA prior to analysis. Of the five assays developed, four were able to detect between 10 and 100 copies of target cDNA, the fifth 1000 copies of target. The results presented here demonstrate that such an approach can be optimised for non-expert users and further areas of work are discussed. •HyBeacon-based RT-PCR assays developed for five forensically-relevant body fluids.•Assays were shown to be specific to target body fluids.•Assays have the ability to differentiate between mRNA target and gDNA.
ISSN:0890-8508
1096-1194
DOI:10.1016/j.mcp.2017.11.007