title: LNJHSY Human cleft lip/palate SNP Array
status: Public on Apr 09 2013
contact.city: shenyang
contact.first: lu
contact.last: yongping
contact.email: valensu423@163.com
contact.address: huanggu strict puhe street
contact.country: China
contact.institute: key lab of liaoning province family planning research institution
organism: Homo sapiens
manufacture_protocol: Twenty four oligonucleotide tags function as probe capture on the glass slide were obtained from www.genome.wi.mit.edu, These tags were modified with 3'-amino and synthesized by Nanjing genescript biological technology CO, LTD.
  Tags suspended at a concentration of 30uM in 0.3M carbonate buffer PH 8.0, were spotted with two replicate on the slides using a Nano-Plotter 2.1 arrayer (GeSiM Germany) at a rate of ten hits per spot, in a humidified chamber(60% relative humidity).
  After spotting, the microarrays were incubated at room temperature overnight in a humid chamber, and then broiled at 60C for 1 hour. Microarrays were rinsed in dH2O and dried. Reverse complements of the tags were called com-tags. Each allele-specific primer was composed by a com-tag at the forward and an allele-specific primer sequence backward. For each SNP, two allele-specific primers were designed, which were match with the two alleles of SNPs respectively. In addition, a com-tag labeled with cy3 was synthesized to act as a positive control.
  Multiplex PCR was amplified with the Multiplex PCR Assay Kit (Takara) in 20uL [Multiplex PCR mix2 10uL, Multiplex PCR mix1 0.2uL, 0.2uM primers, 50ng DNA]. In order to remove excess dNTPs and primers, a mix was added into 10ul PCR product, containing 2 units of shrimp alkaline phosphatase (Takara) and 4 units EXO I(Takara) with incubation at 37°C for 30 min followed by 96°C for 10 min. 10ul of treated PCR products was added to 5ul ASPE mix containing 1×Thermopol reaction buffer, 80M of each dATP, dGTP, dTTP (Takara), 40uM Cy3-dCTP (GE), 0.2 unit of a vent exo- DNA polymerase(New England Biolabs), 25nM allele-specific primers.The extension conditions were as follows: an initial 3min at 94°C, 35 cycles of 94°C for 30 second, 60°C for 30 second 72°C for 1 min, and final extension at 72°C for 5minutes. To prepare Cy3 labeled single strand products, 10ul allele-specific extension products were added 10ul mix containing 1.33×SSC, 0.067% SSC, 0.033mg/ml salmon sperm DNA, and 50nM Cy3-labeled com-tag. The mix was denatured at 94°C for 10 min and cooled 5 min on ice. Hybridization was then performed using this hybridization solution in a hybridization chamber at 60°C for 2 hours. Hybridization was followed by two washes in 2×SSC/0.1%SDS (preheated to 50°C) for 5min, proceeded by washing in 0.2×SSC/0.1%SDS for 1 min, then rinsed in dH2O and dried by centrifugation 3 min at 500 r.p.m. Microarrays were scanned at 100-?m resolution, using GenePix 4000B scanner (Axon instruments, USA), the TIFF images were imported into GenePix 6.0 software (Axon Instruments, USA).