Quantification of protein interaction kinetics in a micro droplet.

Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

Preliminary study on the loss of heterozygosity at 17p13 in gastric and colorectal cancers.

AIM: To evaluate the role of p53 in the development and progression of colorectal cancer and gastric carcinoma by analyzing the loss of heterozygosity (LOH) at 17p13.1 and 17p13.3. METHODS: LOH at the p53 gene locus and 17p13.3 were examined in 22 cases of gastric carcinoma and 14 cases of colorectal cancer by Southern blot analysis. RESULTS: Of the 22 gastrocarcinoma cases, 12 (54%) were heterozygous and LOH was detected in 6 (50%) of the 12 informative cases. In the 14 colorectal cancer cases, 10 (71%) were heterozygous, and LOH was detected in 6 (60%) of the 10 informative cases. CONCLUSION: LOH at the p53 gene locus is a frequent event in multiple step carcinogenesis progression. The high frequency of LOH at 17p13.3 suggests that there may be another tumor suppresser gene in that chromosome region.

[Cloning, sequencing and chromosome location of Sry gene of Muntjak munticus vaginalis by DOP-PCR and microdissection].

We isolated 1, Y1, Y2 chromosomes of the male M.m vaginalis by microdissection and amplified the DNA copies by DOP-PCR. Using the DOP-PCR products of 1, Y1, Y2 chromosomes of M.m vaginalis as template respectively and the primers designed within human SRY HMG box, the Sry gene of the male M.m vaginalis was amplified, and it was cloned and sequenced. It is proved that Y2 is the real sex chromosome in the male M.m vaginalis at molecular level for the first time, and Sry was localized on Y2 chromosome.

[Studies of meiotic origin of the extra chromosome 21 in Down syndromes detected by using (GT)n polymorphic DNA markers].

The polymorphics of two pericentric (GT)n sequences on the long arm of human chromosome 21 have been analyzed after PCR amplification, PAGE and Ag-staining for the first time in 50 Chinese Han people, and were used to detect meiotic origin of the extra chromosome 21 in Down syndromes. Six and 5 alleles were found in Chinese Han people for D21S215 and D21S120, respectively, with observed heterozygosities of 0.68 and polymorphic information content PIC, 0.67 and 0.65. For 17 Down syndromes whose parental origin of the extra chromosome 21 were known, meiotic origin of the extra chromosome 21 were determined in 16 cases, with 7 and 4 maternal meiosis I and II nondisjunction, 2 and 3 paternal meiosis I and II, respectively. The possible biological significance of the study on origin of the extra chromosome 21 has been discussed.