What Is Microarraycgh

Genomic instability is a hallmark of cancer development, and in the vast majority of human malignancies overt instability at the chromosomal level exists, frequently giving rise to unbalanced chromosome copy-number changes. For long, the possibilities for detecting these DNA copy-number changes were limited. Karyotyping required living cells, which severely hampered wide-scale clinical application of the technique outside the fields of hematological malignancies and congenital disorders. Fluorescence in situ hybridization allowed the analysis of only a very limited number of targets at a time, whereas DNA cytometry could only disclose the presence of gross genomic aberrations without any further specificity.[1] This situation has dramatically changed with the introduction of comparative genomic hybridization (CGH). Comparative genomic hybridization is a molecular cytogenetic technique that allows the genome-wide analysis of alterations in DNA sequence copy number.[2] Differentially labeled tumor and reference DNAs compete for hybridizing to normal metaphase chromosomes. The ratio between the two labels along the chromosome axis allows the mapping of gains/ amplifications and deletions throughout the whole genome of a tumor (Fig. 1). Because for CGH no cell culturing is required, and even formaldehyde-fixed paraffin-embedded material can be used, this technique has become one of the most popular genome scanning techniques and has contributed to an enormous progress in the analysis and knowledge of chromosomal changes in solid tumors.[3,4] However, CGH is limited by the poor resolution of the metaphase chromosomes. Ge-nomic alterations smaller than 5-10 Mb are not detected.[3] The recently developed CGH microarray technique overcomes this limitation1-5,6-1 as the metaphase chromosomes are replaced by an ordered set of DNA fragments (typically 100-200 kb) of which the precise chromosomal location is known (Fig. 2). In this case the resolution is limited by the insert size and density of the DNAs spotted on the glass slide.[5-7] Thus, microarray-CGH has the ability of detecting small amplicons and deletions that potentially harbor specific oncogenes and tumor suppressor genes. As in conventional CGH, microarray-CGH allows the measurement of DNA copy-number aberrations by means of hybridization of two differentially labeled DNAs (the DNA to be tested against reference DNA). However, in array-CGH, because of the nature of the DNA spotted on the glass slides, it is possible to map the copy-number aberrations directly onto the genomic sequence. The DNA fragments spotted on the slide can be either large-insert genomic clones (cosmids, BACs, or PACs) or cDNA clones.[5,6,8] The last one identifies directly of which genes the chromosomal copy number has changed and allows the parallel comparison of the alterations in DNA copy number and gene expression in the same sample.[8] However, with cDNA clones fluorescent ratios do not reflect the chromosomal copy-number changes as precise, possibly due to cross hybridization effects.[8] Initially, arrays were spotted with fragmented DNA from the BACs/PACs.[5,6] As BACs are single-copy vectors, the yield of DNA obtained in culture is low, and for that reason big culture volumes were needed, and DNA density on the spotted arrays was low. Nowadays, PCR representations of the clones are spotted on the slide. This process fragments the DNA, allows small-scale

Fig. 1 Schematic representation of the comparative genomic hybridization (CGH) technique. Test and reference DNAs are differentially labeled and hybridized to metaphase spreads. After hybridization, the slide is washed and scanned. Images obtained are processed and ratios calculated.

DNA isolations, and allows high spotting concentration, which simplifies the process and enhances sensitivi ty.

Currently, genome-wide CGH arrays comprise generally about 3000 BAC and PAC clones, yielding a resolution of 1 to 1.5 Mb.[9,10] Just very recently, the first results of a 30-kb whole genome tiling path BAC array have been presented.[11] For cDNAs the number of clones is finite and will never be genome covering. Alternative strategies use arrays with spotted oligonucleo-tides (see below).

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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