Programmable DNA-responsive microchip for the capture and release of circulating tumor cells by nucleic acid hybridization

The detection and analysis of circulating tumor cells （CTCs） from patients＇ blood is important to assess tumor status; however, it remains a challenge. In the present study, we developed a programmable DNA-responsive microchip for the highly efficient capture and nondestructive release of CTCs via nucleic acid hybridization. Transparent and patternable substrates with hierarchical architectures were integrated into the microchip with herringbone grooves, resulting in greatly enhanced cell-surface interaction via herringbone micromixers, more binding sites, and better matched topographical interactions. In combination with a high-affinity aptamer, target cancer cells were specifically and efficiently captured on the chip. Captured cancer cells were gently released from the chip under physiological conditions using toehold-mediated strand displacement, without any destructive factors for cells or substrates. More importantly, aptamercontaining DNA sequences on the surface of the retrieved cancer cells could be further amplified by polymerase chain reaction （PCR）, facilitating the detection of cell surface biomarkers and characterization of the CTCs. Furthermore, this system was extensively applied to the capture and release of CTCs from patients＇ blood samples, demonstrating a promising high-performance platform for CTC enrichment, release, and characterization.

Improved Nucleic Acid Spot Hybridization Technique for Detection of Potato Spindle Tuber Viroid(PSTVd)

Potato spindle tuber viroid(PSTVd)disease is one of the major diseases that threatens potato production.Therefore,an advanced,rapid and sensitive detection technology is needed to detect the disease for better control.In order to establish an easier nucleic acid spot hybridization(NASH)method,some studies were tried as the followings:(1)the pre-hybridization step of nucleic acid spot hybridization(NASH)was omitted compared with ordinary way;(2)RNA extraction(phenol extraction and Ames buffer extraction)methods were compared;(3)fixed RNA by UV lamp and oven compared with UV cross-linker;(4)hybridized the RNA in shaking incubator and so on.The results showed that RNA extracted by Ames buffer was more effective than by the phenol extraction method.Besides,the result of hybridization without pre-hybridization step was better than that with 1.5 h of pre-hybridization.The more important discovery was that the shaking incubator could replace the hybridization oven and the ordinary UV lamp could replace the UV cross-linker.After a long term repeated research and testing,a new hybridization system that could rapidly detect the PSTVd by improved NASH technique merely using common instruments and equipment was established.

LOCALIZATION OF TYPE I AND TYPE Ⅲ PROCOLLAGEN mRNAs IN BREAST SCIRRHOUS CARCINOMA BYIN SITU HYBRIDIZATION

Scirrhous carcinoma is charactertzed by reinarkable amount of collagen fibrils, mainly type I and type III collagens. The origin of collagens is still under debate.cDNA fragments of type I and type III procollagens were subcloned into Gemini pGEM vectors to synthesize the 35Slabeled cRNA probes. By in situ hybridization, we have found the fibroblasts surrounding the tumor cells and cords contained abundent type I and type III procollagen mRNAs which decreased with the distance of fibroblasts from the tumor cells. In all freshly prepared tissues, the tumor cells also contained significant pro α1 (I) and pro α1 (III) mRNAs, but no or little pro α2 (I) mRNA. The results indicated that type I and type III collagens in human scirrhous carcinoma of breast are mainly produced by fibroblasts. Tumor cells also perticipate in the disposition of collagen fibrils, probably type I trimer and type III collagens in accordance with what was observed in biochemical

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Detection of Gene Alteration for Color Vision Defects by Polymerase Chain Reaction

According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...

Covalent Attachment of DNA to Glass Supports Using A New Silane Coupling Agent and Chemiluminescent Detection

A new kind of silane coupling agent, N- (β-aminoethyl ) - γ-aminopropyl triet hoxysilane, was used for DNA direct attachment on the surfaces of glass supports, then the immobilized DNA was hybridized with horseradish peroxidase (HRP)-labeled probe, and detected by using enhanced chemiluminescent method. In comparison with γ-aminopropyl triethoxysilane, the detection limits (S/N) of DNA were 10 pg and 75 pg respectively. Several experimental conditions of DNA attachING to glass supports were investigated, and the system of hybridization of nucleic acid on the surfaces of glass supports was developed.