RESUMO
We describe a contact printing approach for microarrays that uses fused-silica capillary tubes with tapered tips for printing pins and a pressure/vacuum system to control pin loading, printing, and cleaning. The printing process is insensitive to variable environmental factors such as humidity, and the small diameter of the pins allows routine printing from 1536 well source plates. Pin load capacity, 0.2 microL in the current system, is adjustable by controlling pin length. More than 2000 spots can be printed per 0.2-microL pin load (<100 pl/spot), and densities of >12,000 spots/cm(2) are readily achievable. Solutions with a wide range of viscosities and chemical properties can be printed. The system can print tens of thousands of different solutions at high speed, due to the ability to use large numbers of pins simultaneously, and can produce a large number of replicate arrays since all of the solution picked up by the pins is available for deposition.
Assuntos
Análise em Microsséries/instrumentação , Dióxido de Silício/química , Cromossomos Artificiais Bacterianos/química , Cromossomos Artificiais Bacterianos/genética , DNA/genética , DNA/isolamento & purificação , Humanos , Análise em Microsséries/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligonucleotídeos/química , Oligonucleotídeos/genética , VácuoRESUMO
BACKGROUND: Array-based comparative genomic hybridization (aCGH) enables genome-wide quantitative delineation of genomic imbalances. A high-resolution contig array was developed specifically for chromosome 8q because this chromosome arm is frequently altered in many human cancers. METHODS: A minimal tiling path contig of 702 8q-specific bacterial artificial chromosome (BAC) clones was generated with a novel computational tool (BAC Contig Assembler). BAC clones were amplified by degenerative oligonucleotide primer (DOP) polymerase chain reaction and subsequently printed onto glass slides. For validation of the array DNA samples of gastroesophageal and prostate cancer cell lines, and chronic myeloid leukemia specimens were used, which were previously characterized by multicolor fluorescence in situ hybridization and conventional CGH. RESULTS: Single and double copy gains were confidently demonstrated with the 8q array. Single copy loss and high-level amplifications were accurately detected and confirmed by bicolor fluorescence in situ hybridization experiments. The 8q array was further tested with paraffin-embedded prostate cancer specimens. In these archival specimens, the copy number changes were confirmed. In fresh and archival samples, additional alterations were disclosed. In comparison with conventional CGH, the resolution of the detected changes was much improved, which was demonstrated by an amplicon of 0.7 Mb and a deletion of 0.6 Mb, both spanned by only six BAC clones. CONCLUSIONS: A comprehensive array is presented, which provides a high-resolution method for mapping copy number alterations on chromosome 8q.
