Biomedical subjects

K A Slentz-Kesler

Publications and source records attributed to K A Slentz-Kesler.

3 recordsLinked to original sources

PubMed2000

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry.

BACKGROUND: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. METHODS: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype. RESULTS: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases. CONCLUSIONS: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.

Chromosomes, Human, Pair 19↗

PubMed2000

A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension.

A rapid, high throughput readout for single-nucleotide polymorphism (SNP) analysis was developed employing single base chain extension and cytometric analysis of an array of fluorescent microspheres. An array of fluorescent microspheres was coupled with uniquely identifying sequences, termed complementary ZipCodes (cZipCodes), which allowed for multiplexing possibilities. For a given assay, querying a polymorphic base involved extending an oligonucleotide containing both a ZipCode and a SNP-specific sequence with a DNA polymerase and a pair of fluoresceinated dideoxynucleotides. To capture the reaction products for analysis, the ZipCode portion of the oligonucleotide was hybridized with its cZipCodes on the microsphere. Flow cytometry was used for microsphere decoding and SNP typing by detecting the fluorescein label captured on the microspheres. In addition to multiplexing capability, the ZipCode system allows multiple sets of SNPs to be analyzed by a limited set of cZipCode-attached microspheres. A standard set of non-cross reactive ZipCodes was established experimentally and the accuracy of the system was validated by comparison with genotypes determined by other technologies. From a total of 58 SNPs, 55 SNPs were successfully analyzed in the first pass using this assay format and all 181 genotypes across the 55 SNPs were correct. These data demonstrate that the microsphere-based single base chain extension (SBCE) method is a sensitive and reliable assay. It can be readily adapted to an automated, high-throughput genotyping system. [Primer sequences used in this study are available as online supplementary materials at www.genome.org.]

DNA, Complementary↗

PubMed1998

Identification and characterization of K12 (SECTM1), a novel human gene that encodes a Golgi-associated protein with transmembrane and secreted isoforms.

The investigation of a DNase-hypersensitive site upstream of the CD7 gene on chromosome 17q25 has led to the discovery of a novel human gene designated K12 (SECTM1, the HGMW assignment). This gene spans approximately 14 kb and encodes a 1.8-kb mRNA detected at the highest levels in peripheral blood leukocytes and breast cancer cell lines. The open reading frame predicts a 248-amino-acid protein with the hydropathic characteristics of a type 1a membrane protein. Western blots show that the K12 protein exists as a cluster of bands around 27 kDa, and extractions using nonionic detergents or high pH conditions demonstrate that it behaves as an integral membrane protein. Immunofluorescence localization studies reveal that K12 is not detectable on the cell surface, but instead is found in a perinuclear Golgi-like pattern and colocalizes with a well-known Golgi marker. In addition, an approximately 20-kDa soluble form of the K12 protein derived from the N-terminal domain is specifically secreted by cells into the culture medium. Immunohistochemical analysis of peripheral blood cells shows that K12 is found in leukocytes of the myeloid lineage, with the strongest staining observed in granulocytes and no detectable expression in lymphocytes. Based on its range of expression, its broad structural characteristics that resemble cytokines and growth factors, and the chromosomal location of the gene in an area already associated with myelogenous leukemias and other malignant neoplasms, this study concludes that K12 is a novel molecule with potential importance in hematopoietic and/or immune system processes.

Antigens, CD7↗