Simple multiplex genotyping by surface-enhanced resonance Raman scattering.
The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.
Graham
Duncan
D
Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, UK. duncan.graham@strath.ac.uk
Mallinder
Benjamin J
BJ
Whitcombe
David
D
Watson
Nigel D
ND
Smith
W Ewen
WE
D003550:Cystic Fibrosis / Q000235:genetics; D004247:DNA / Q000032:analysis*; D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013059:Spectrum Analysis, Raman
D006683:HLA-DQ Antigens; D004247:DNA