A new method for the preparation of oligonucleotide microarray for gene expression detection was found. The key techniques and standards of quality controlling for preparation of oligonucleotide microarray was explore...A new method for the preparation of oligonucleotide microarray for gene expression detection was found. The key techniques and standards of quality controlling for preparation of oligonucleotide microarray was explored using gene of human 23kD highly protein and Luciferase and mouse cytokine-associated genes. By the using of a software system MProbe, oligonucleotide probes were designed and BLAST. All the probes have a very high specificity, i.e. except target sequence, the similarity between the probe and non-target sequences is less than 70% and the hairpin structure are not exist in all probes.All the probes have the same length 40. GC contents in all probes are in a narrow scope (from 45% to 55%). All the probes are modified with amino at 5′ or 3′ terminal. The satisfied images with good sensativity and very high specificity were obtained by the using of the methods above and also using of positive and negative controls and some internal controls(house keeping gene) to quantitate and balance expression of genes. High specificity, good sensativity and stablity have been verified by three continuous experiments using the oligonucleotide microarray to study gene expression profile of normal mouse breast grand tissue .The oligonucleotide microarray for expression detection prepared using our method have high specificity, good sensativity and stablity et al . It may be a more advanced method for analysis of gene expression profile.展开更多
Optimization for the technological processes of fabricating oligonucleotide microarray by the molecular stamping method is studied in this note. Three factors that affect the pressing coupling reactions of the nucleos...Optimization for the technological processes of fabricating oligonucleotide microarray by the molecular stamping method is studied in this note. Three factors that affect the pressing coupling reactions of the nucleosides are focused on: the stability of the chemical activities of the reaction solutions, the contamination of the remain of the reactive nucleotides among the different spots on the chip, and the influence of the capping reaction on the hybridization result. The experiments show that the acetonitrile solution of tetrazole and nucleoside monomer could maintain sufficient reactive activity for more than 10 h. An effective method has been used and proved to eliminate the residual reactive nucleosides on chip with small molecules containing hydroxylgroup. Finally, the capping step-a regular step in theconventional DNA chemical synthesis can be neglected in our on-chip DNA synthetic process, which would not affect its hybridization results.展开更多
The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute respiratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first sub...The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute respiratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first submitted coronavirus strain, according to the sequence of TOR2 (GENEBANK Accession: AY274119). These primers were synthesized and printed into a microarray with 12×12 spots. RNAs were extracted from the throat swab and gargling fluid of SARS patients and reverse-transcripted into the double strand cDNAs. The cDNAs were prepared as restricted cDNA fragments by the restriction display (RD) technique and labeled by PCR with the Cy5-universal primer. The labeled samples were then applied to the oligo microarray for hybridization. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The scanning result showed that samples of SARS patients were hybridized with multiple SARS probes on the microarray, and there is no signal on the negative and blank controls. These results indicate that the genome of SARS coronavirus can be detected in parallel by the 60mer oligonucleotide microarray, which can improve the positive ratio of the diagnosis. The oligo microarray can also be used for monitoring the behavior of the virus genes in different stages of the disease status.展开更多
The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute res-piratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first su...The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute res-piratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first submit-ted coronavirus strain, according to the sequence of TOR2 (GENEBANK Accession: AY274119). These primers were synthesized and printed into a microarray with 12×12 spots. RNAs were extracted from the throat swab and gargling fluid of SARS patients and reverse-transcripted into the double strand cDNAs. The cDNAs were prepared as re-stricted cDNA fragments by the restriction display (RD) technique and labeled by PCR with the Cy5-universal primer. The labeled samples were then applied to the oligo microar-ray for hybridization. The diagnostic capability of the mi-croarray was evaluated after the washing and scanning steps. The scanning result showed that samples of SARS patients were hybridized with multiple SARS probes on the microar-ray, and there is no signal on the negative and blank controls. These results indicate that the genome of SARS coronavirus can be detected in parallel by the 60mer oligonucleotide mi-croarray, which can improve the positive ratio of the diagno-sis. The oligo microarray can also be used for monitoring the behavior of the virus genes in different stages of the disease status.展开更多
文摘A new method for the preparation of oligonucleotide microarray for gene expression detection was found. The key techniques and standards of quality controlling for preparation of oligonucleotide microarray was explored using gene of human 23kD highly protein and Luciferase and mouse cytokine-associated genes. By the using of a software system MProbe, oligonucleotide probes were designed and BLAST. All the probes have a very high specificity, i.e. except target sequence, the similarity between the probe and non-target sequences is less than 70% and the hairpin structure are not exist in all probes.All the probes have the same length 40. GC contents in all probes are in a narrow scope (from 45% to 55%). All the probes are modified with amino at 5′ or 3′ terminal. The satisfied images with good sensativity and very high specificity were obtained by the using of the methods above and also using of positive and negative controls and some internal controls(house keeping gene) to quantitate and balance expression of genes. High specificity, good sensativity and stablity have been verified by three continuous experiments using the oligonucleotide microarray to study gene expression profile of normal mouse breast grand tissue .The oligonucleotide microarray for expression detection prepared using our method have high specificity, good sensativity and stablity et al . It may be a more advanced method for analysis of gene expression profile.
基金This work was supported by the 863 National High Technology Research Development Program (Grant Nos. 103-13-05-01 and 101-01-01)+1 种基金the 973 National Key Fundamental Research Project (Grant No. G1998051204)the National Science Foundation of China (Gra
文摘Optimization for the technological processes of fabricating oligonucleotide microarray by the molecular stamping method is studied in this note. Three factors that affect the pressing coupling reactions of the nucleosides are focused on: the stability of the chemical activities of the reaction solutions, the contamination of the remain of the reactive nucleotides among the different spots on the chip, and the influence of the capping reaction on the hybridization result. The experiments show that the acetonitrile solution of tetrazole and nucleoside monomer could maintain sufficient reactive activity for more than 10 h. An effective method has been used and proved to eliminate the residual reactive nucleosides on chip with small molecules containing hydroxylgroup. Finally, the capping step-a regular step in theconventional DNA chemical synthesis can be neglected in our on-chip DNA synthetic process, which would not affect its hybridization results.
文摘The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute respiratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first submitted coronavirus strain, according to the sequence of TOR2 (GENEBANK Accession: AY274119). These primers were synthesized and printed into a microarray with 12×12 spots. RNAs were extracted from the throat swab and gargling fluid of SARS patients and reverse-transcripted into the double strand cDNAs. The cDNAs were prepared as restricted cDNA fragments by the restriction display (RD) technique and labeled by PCR with the Cy5-universal primer. The labeled samples were then applied to the oligo microarray for hybridization. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The scanning result showed that samples of SARS patients were hybridized with multiple SARS probes on the microarray, and there is no signal on the negative and blank controls. These results indicate that the genome of SARS coronavirus can be detected in parallel by the 60mer oligonucleotide microarray, which can improve the positive ratio of the diagnosis. The oligo microarray can also be used for monitoring the behavior of the virus genes in different stages of the disease status.
文摘The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute res-piratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first submit-ted coronavirus strain, according to the sequence of TOR2 (GENEBANK Accession: AY274119). These primers were synthesized and printed into a microarray with 12×12 spots. RNAs were extracted from the throat swab and gargling fluid of SARS patients and reverse-transcripted into the double strand cDNAs. The cDNAs were prepared as re-stricted cDNA fragments by the restriction display (RD) technique and labeled by PCR with the Cy5-universal primer. The labeled samples were then applied to the oligo microar-ray for hybridization. The diagnostic capability of the mi-croarray was evaluated after the washing and scanning steps. The scanning result showed that samples of SARS patients were hybridized with multiple SARS probes on the microar-ray, and there is no signal on the negative and blank controls. These results indicate that the genome of SARS coronavirus can be detected in parallel by the 60mer oligonucleotide mi-croarray, which can improve the positive ratio of the diagno-sis. The oligo microarray can also be used for monitoring the behavior of the virus genes in different stages of the disease status.