PCR-free MDR1 polymorphism identification by gold nanoparticle probes.
Single nucleotide polymorphisms (SNPs) represent the most abundant source of genetic variation in the human genome, and they can be linked to genetic susceptibilities or varied pharmaceutical responses. Established SNP detection techniques are mainly PCR-based, which means that they involve complex, labor-intensive procedures, are easy contaminated, and can give false-positive results. Therefore, we have developed a simple and rapid MS-based disulfide barcode methodology that relies on magnifying the signal from a dual-modified gold nanoparticle. This approach permits direct SNP genotyping of total human genomic DNA without the need for primer-mediated enzymatic amplification. Disulfides that are attached to the gold nanoparticle serve as a "barcode" that allows different sequences to be discerned using MS detection. Specificity is based on two sequential oligonucleotide hybridizations, which include two steps: the first is the capture of the target by gene-specific probes immobilized onto magnetic beads; the second is the recognition of gold nanoparticles functionalized with allele-specific oligonucleotides. The sensitivity of this new method reaches down to the 0.1 fM range, thus approaching that of PCR. The feasability of this SNP identification methodology based on an MS-based disulfide barcode assay was demonstrated by applying it to genomic DNA samples representing all possible genotypes of the SNPs G2677T and C3435T in the human MDR1 gene. Due to its great advantage--the ability to perform SNP typing without the use of PCR--the assay was found to be simple, rapid and robust, and so may be highly suited to routine clinical detection as well as basic medical research.
Yang
Bing
B
School of Medicine, Nanjing University, 22 Hankou Road, Nanjing, 210093, China.
Zhou
Guohua
G
Huang
Lequn Lee
LL
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D006046:Gold / Q000737:chemistry; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015342:DNA Probes; D006046:Gold