Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5'-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleo-tide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.
The phosphorous amide approach is of the highest coupling efficiency among the DNA in situ synthesis approaches at present. The strict anhydrous condition is required in the process of DNA in situ synthesis, and the DNA synthesis in the molecular stamp method must be performed in the anhydrous glove box. Because various volatile reagents remain in the glove box, the reagent gases invade into the coupling reaction system so that the coupling efficiency is decreased. This decrease is caused by the pyridine contained in the oxidizing agent. An improved oxidizing agent system of I 2/Ac 2O/AcOH/THF for the synthesis of oligodeoxy-nucleotides was reported. Using this oxidizing agent, 16-mer oligodeoxynucleotide probes were successfully synthesized on the CPG(controlled pore size glass) or the modified glass slide and a single step coupling efficiency of 98.2% was achieved. The synthesized DNA probes were hybridized with fluorescence and gold-labeled target oligodeoxynucleotides respectively. The fluorescence intensity and gray level for the synthesized probes in the I 2/Ac 2O/AcOH/THF oxidizing agent were similar to those in the traditional phosphorous amide approach possessing a water- and pyridine-containing oxidizing agent I 2/H 2O/Pyridine/THF. The reported oxidizing agent will make the contact printing approach to in situ synthesized DNA microarrays more feasible by eliminating the decrease of coupling efficiency which is because the cross reagent interfering arises from pyridine and water.
Mutations in cardiac troponin I （cTnI） gene were assessed based on gene-chip technology.Special probes were designed to fabricate the low-density gene-chip,which could detect the mutations in exons 3,5,7,and 8 of the cTnI gene simultaneously.For each exon,two oligonucleotide sequences labeled with fluorescein at the 5＇-end were designed,one （oligonucleotide Ⅰ） simulating the wild type and the other （oligonucleotide Ⅱ） simulating the mutant.Oligonucleotides Ⅰ and Ⅱ were mixed together to simulate the heterozygote.After optimizing the hybridization protocols,the fabricated gene-chip could detect the mutations in the exons of the cTnI gene with relative high sensitivity and specificity.The fully complementary probe gave a fluorescent signal almost 50% stronger than that of the one-base mismatched one,which is in accordance with the result from a theoretical estimate. An applicable special gene-chip is available to investigate and diagnose familial hypertrophic cardiomyopathy （FHCM） after further improvement.
