Multiplex Single Nucleotide Polymorphism Genotyping Utilizing Ligase Detection Reaction Coupled Surface Enhanced Raman Spectroscopy

Abstract

Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high specificity. We present the first multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The platform has the advantage in that the diagnostic peaks of Raman are more distinct than fluorescence, and in theory, a clinically significant number of markers can be multiplexed in a single sample using different SERS reporters. Here we report LDR-SERS multiplex SNP genotyping of K-Ras oncogene alleles at 10 pM detection levels, optimization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Genomic DNA from typed cells lines was obtained and scored for the K-Ras genotype. These advances are significant as we have further developed our new SNP genotyping platform and have demonstrated the ability to correlate genotype ratios directly to diagnostic Raman peak signal intensity.

Enzyme-free detection and quantification of double-stranded nucleic acids

“…SERRS is a method with greater resolution than fluorescence, because Raman peaks are sharper. Consequently several SERRS-labeled oligonucleotides can be simultaneously detected if the absorption maxima of the tags are close enough to the laser excitation wavelength [25][26][27][28][29][30]. Maximum absorption of HEX and R6G is at 535 and 524 nm, respectively, and can therefore be simultaneously measured with an argon laser tuned at 514.5 nm [27,29,31,37].…”

“…Several SERRS-labeled oligonucleotides can be simultaneously detected [25][26][27][28][29][30]. A variety of molecules can be detected by SERRS if their absorption maxima are close enough to the laser excitation wavelength.…”

“…The fluorescence of the SERRS-label is quenched by the metallic surface enabling the amplified Raman fingerprint of the molecule to be observed. A wide range of labels can be detected by SERRS, and the sharpness of Raman peaks gives SERRS high multiplexing capacity [25][26][27][28][29][30]. SERRS has been developed for DNA detection since 1997 [24,31] and has become a promising enzyme-free tool for specific detection and quantification of single-stranded DNA on either micro-chips [30,32,33] or magnetic micro-beads [34,35].…”

Detection of Single Nucleotide Polymorphisms by a Gold Nanowire‐on‐Film SERS Sensor Coupled with S1 Nuclease Treatment

“…[16,17,21,22] To develop disease diagnostic sensors, it is important to verify that the sensor can identify SNPs in clinical practice. Clinical tests demonstrated that the NW-on-film sensor successfully identified mutations in the relevant DNAs in Avellino corneal dystrophy (ACD) and Wilson disease (WD).…”

A graphene-based platform for single nucleotide polymorphism (SNP) genotyping

“…With the development of genetic therapy, clinical diagnosis and molecular biology, SNP is regarded as not only a genetic marker in the study of cancer-related drug metabolism or reactivity, but also a fundamental tool in the identification of inherited disease-causing genes (Imyanitov et al, 2004;McCarthy and Hilfiker, 2000;Sidransky, 2002;Sauna, 2007). Therefore, great efforts have been devoted to developing technique methods for screening SNP, such as allele specific oligonucleotide hybridization (Ding et al, 2010;Liu et al, 2005;Liu and Lin, 2007), endonuclease digestion (Gaylord et al, 2005;Li and Liu, 2009), primer extension (Duan et al, 2009a;Litos et al, 2009;Nelson et al, 1996), oligonucleotide ligation (Huh et al, 2009;Lowe et al, 2010;Xue et al, 2009), nonenzymatic ligation (Xu et al, 2001), DNA-specific redox indicators and conjugated mediators (Drummond et al, 2003;Kelley et al, 1999), in combination with fluorescence (FL) (Duan et al, 2007;Guo et al, 2010;Liu et al, 2009;Wang and Liu, 2007), electrochemistry (Kerman et al, 2004;, chemiluminescence (Liu et al, 2006), colorimetry (He et al, 2010a,b;Lee et al, 2010), and mass spectrometry (Mattes and Seitz, 2001) as signal readout.…”