Analysis of Expression Patterns: The Scope of the Problem, the Problem of Scope

Studies of the expression patterns of many genes simultaneously lead to the observation that even in closely related pathologies, there are numerous genes that are differentially expressed in consistent patterns correlated to each sample type. The early uses of the enabling technology, microarrays, was focused on gathering mechanistic biological insights. The early findings now pose another clear challenge, finding ways to effectively use this kind of information to develop diagnostics

Chromosome specific c-DNA libraries: reduction of unspecific priming events by purification of heteronuclear RNA

Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43249/1/11033_2005_Article_BF00997152.pd

GeneLink: a database to facilitate genetic studies of complex traits

BACKGROUND: In contrast to gene-mapping studies of simple Mendelian disorders, genetic analyses of complex traits are far more challenging, and high quality data management systems are often critical to the success of these projects. To minimize the difficulties inherent in complex trait studies, we have developed GeneLink, a Web-accessible, password-protected Sybase database. RESULTS: GeneLink is a powerful tool for complex trait mapping, enabling genotypic data to be easily merged with pedigree and extensive phenotypic data. Specifically designed to facilitate large-scale (multi-center) genetic linkage or association studies, GeneLink securely and efficiently handles large amounts of data and provides additional features to facilitate data analysis by existing software packages and quality control. These include the ability to download chromosome-specific data files containing marker data in map order in various formats appropriate for downstream analyses (e.g., GAS and LINKAGE). Furthermore, an unlimited number of phenotypes (either qualitative or quantitative) can be stored and analyzed. Finally, GeneLink generates several quality assurance reports, including genotyping success rates of specified DNA samples or success and heterozygosity rates for specified markers. CONCLUSIONS: GeneLink has already proven an invaluable tool for complex trait mapping studies and is discussed primarily in the context of our large, multi-center study of hereditary prostate cancer (HPC). GeneLink is freely available at

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies

Cytogenetic analysis of posterior uveal melanoma

Cytogenetic analysis was performed on short-term cultures of primary tumor samples from seven patients with posterior uveal melanoma. Informative data were obtained from four patients, all of whom had a near-diploid chromosomal number and clonal chromosomal alterations. Analysis of one patient's tumor revealed monosomy 3 as the only cytogenetically distinguishable aberration. Trisomies of chromosome 8 and i(8)(q10) were detected in two other patients in combination with monosomy of chromosome 3. The fourth patient's karyotype displayed two different translocations. One translocation, der(6)t(6;8)(q12;q13.1), resulted in the over-representation of 8q13.1-->qter and a partial monosomy of 6q12-->qter; the other translocation, der(9)t(6;9)(p12;p23), produced a partial trisomy of 6p12-->pter and a partial monosomy of 9p23-->pter. These results support the view that the recurring pattern of chromosomal rearrangements in ocular melanoma is unique from that associated with cutaneous malignant melanoma. Furthermore, these results help confirm that chromosomes 3, 6, and 8 are nonrandomly altered in ocular melanoma.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30919/1/0000589.pd

Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma

Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases have been performed. This work was facilitated by the recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29767/1/0000105.pd

Assignment of the human CRABP-II gene to chromosome 1q21 by nonisotopic in situ hybridization

Two highly conserved forms of cellular retinoic acid binding protein (CRABP-I and CRABP-II) have been described, and one, CRABP-II, is highly expressed in human skin. We have utilized a 10-kb fragment containing the human CRABP-II (hCRABP-II) gene (isolated from a human genomic library) to localize hCRABP-II to human chromosome 1 band q21 by fluorescence in situ hybridization. Localization to 1q was confirmed by hybridization of a hCRABP-II cDNA clone against a human-mouse hybrid cell line containing a t(1;6)(q21;q13) translocation chromosome. The hCRABP-II gene is therefore localized to a band known to contain several other genes that are expressed in the context of epidermal differentiation, including profilaggrin, loricrin, involucrin, and calcyclin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47631/1/439_2004_Article_BF00219171.pd