title: Development of a PCR/ligase detection reaction/nanogold-based universal array approach for the detection of low-abundant DNA point mutations.
abstract: The aim of this study was to investigate the feasibility of combining PCR and ligase detection reaction (LDR) with a novel nano-gold-based universal array for the detection of low abundance point mutations from fetal DNA in maternal plasma samples. The sequence with the target point mutation was first amplified by PCR and then used as a template for LDR in which the upstream specific primer contains a tag sequence at the 5'-end. After hybridization to the probes of a universal array containing anti-tag sequences, the ligated products were bound to streptavidin-labeled nano-gold particles and the hybridization signals were amplified by silver staining. The PCR/LDR/universal array was first tested for sensitivity with nano-gold-based detection, and then this system was applied to detect the low abundance specific mutation IVS2 654(C→T) of the β-globin gene in a model using maternal plasma samples. The nano-gold-based method unambiguously identified a single mutation at a sensitivity of 1:1000. This approach was applied to detect the paternally inherited IVS2 654(C→T) mutation from thirty maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. The PCR/LDR/nano-gold-based universal array is able to detect low-abundance point mutations with high sensitivity.
authors.affiliation: Department of Obstetrics and Gynecology, Research Institute of Surgery, Daping Hospital, Third Military Medical University, 10 Changjiangzhilu, Daping, Yuzhong District, Chongqing 400042, People's Republic of China.
authors.forename: Ping
authors.initials: P
authors.lastname: Yi
authors.forename: Weiping
authors.initials: W
authors.lastname: Lu
authors.forename: Jianxin
authors.initials: J
authors.lastname: Guo
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Zhuqin
authors.initials: Z
authors.lastname: Chen
authors.forename: Jian
authors.initials: J
authors.lastname: Han
authors.forename: Li
authors.initials: L
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005333:Fetus; D006046:Gold / Q000737:chemistry*; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008297:Male; D053768:Metal Nanoparticles; D008969:Molecular Sequence Data; D009035:Mothers; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D055544:beta-Globins / Q000235:genetics
chemical_list: D055544:beta-Globins; D006046:Gold; D004247:DNA; D008025:Ligases