title: Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection.
abstract: Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (Tm) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique Tm when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective Tm values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.
authors: Department of Translational Medicine, School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian 361005, China.
authors: Yiqun
authors: Y
authors: Liao
authors: Xiaobo
authors: X
authors: Wang
authors: Chao
authors: C
authors: Sha
authors: Zhongmin
authors: Z
authors: Xia
authors: Qiuying
authors: Q
authors: Huang
authors: Qingge
authors: Q
authors: Li
mesh_terms: D005456:Fluorescent Dyes; D060005:Genotyping Techniques; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes