Surface-enhanced Raman scattering based ligase detection reaction.
Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced Raman scattering (SERS) to the ligase detection reaction (LDR). The reaction uses two LDR primers, one of which contains a Raman enhancer and the other a reporter dye. In LDR, one of the primers is designed to interrogate the SNP. When the SNP being interrogated matches the discriminating primer sequence, the primers are ligated and the enhancer and dye are brought into close proximity enabling the dye's Raman signature to be detected. By detecting the Raman signature of the dye rather than its fluorescence emission, our technique avoids the problem of spectral overlap which limits number of reactions which can be carried out in parallel by existing systems. We demonstrate the LDR-SERS reaction for the detection of point mutations in the human K-ras oncogene. The reaction is implemented in an electrokinetically active microfluidic device that enables physical concentration of the reaction products for enhanced detection sensitivity and quantization. We report a limit of detection of 20 pM of target DNA with the anticipated specificity engendered by the LDR platform.
Huh
Yun Suk
YS
Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York 14853, USA.
Lowe
Adam J
AJ
Strickland
Aaron D
AD
Batt
Carl A
CA
Erickson
David
D
D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D011905:Genes, ras; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods*; D008958:Models, Molecular; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D013059:Spectrum Analysis, Raman / Q000379:methods*
D004247:DNA