PMC:549431 / 5357-9502 JSONTXT

{"project":"2_test","denotations":[{"id":"15722479-12697903-76827383","span":{"begin":206,"end":208},"obj":"12697903"},{"id":"15722479-12697903-76827384","span":{"begin":342,"end":344},"obj":"12697903"},{"id":"15722479-12697903-76827385","span":{"begin":1018,"end":1020},"obj":"12697903"},{"id":"15722479-12697903-76827386","span":{"begin":1604,"end":1606},"obj":"12697903"},{"id":"15722479-9685507-76827387","span":{"begin":2236,"end":2238},"obj":"9685507"}],"text":"MATERIALS AND METHODS\n\nDNA sample selection\nA series of 120 DNA samples derived from biopsies of lymphoma patients were previously screened for all possible ATM mutations using oligonucleotide microarrays (30). Here, we have selected a total of 68 samples that showed robust amplification signals in all 62 coding exons for further analysis (30). A total of 17 unique mutations, each in a one-to-one mixture with wild-type sequence, occurred once in these samples. The impact of any given mutation in a single sample is minimal given that 67 other samples with wild-type sequences in the region encompassing a given mutation are included in this analysis. Several single nucleotide polymorphisms (SNPs) were present multiple times: 735 C/T, 2572 T/C and 4258 C/T in two samples; 3161 C/G in four samples; and 5557 G/A in five samples. Likewise, these SNPs have a minimal effect on our global analyses given the large number of samples and bases interrogated in this study.\n\nTarget preparation\nAs previously described (30), individual ATM coding exons were amplified from genomic DNA using primers containing T3 and T7 RNA polymerase tails, pooled, and then in vitro transcribed using T3 or T7 RNA polymerase to create biotin-labeled sense and antisense strand targets, respectively. Fluorescein-labeled reference target was made using genomic DNA from an unaffected individual. Reference and test sample targets were fragmented, diluted in hybridization buffer [3 M TMA-Cl (tetramethylammonium chloride), 1× TE, pH 7.4, 0.001% Triton X-100] and hybridized to the ATM microarrays as described previously (30). Afterwards, the microarray was stained with a phycoerythrin–streptavidin conjugate and digitized hybridization images from both reference and test targets were acquired using the Gene Array Scanner (Hewlett Packard, Palo Alto, CA) equipped with the appropriate emission filters.\n\nData analysis\nCustom software was used to quantify hybridization signals for each probe and subtract background hybridization signals. We exclusively focused on raw data from the biotin-labeled test targets since they provide approximately seven times the hybridization signal of the fluorescein-labeled wild-type reference target in this system (28). This enhanced signal provides greater sensitivity toward detecting weak hybridization.\nFor each sample, for each base and for each potential type of mutation (i.e. substitution, one or two base deletion or one base insertion), the specificity was calculated as the ratio of the PM probe hybridization signal of the wild-type target to their cognate insertion, deletion or single base substitution probes on each strand. The logarithm of these ratios was plotted as a function of the position within the gene. To illustrate the special patterns and to smooth out random variation, running averages of data from 10 bases were used. To capture the variability, at each base, the sample-to-sample standard deviation was again calculated using data derived from a running average of 10 bases for each sample.\nTo estimate the mean hybridization specificity for each type of mutation, the geometric mean (i.e. the antilog of the average of the logged ratios) over all bases and over all specimens was calculated (Table 1). To further examine the variability of the specificity ratios, the coefficient of variation (cv) was calculated in two ways. The cv is the ratio of the standard deviation divided by the mean; it is useful for understanding the amount of variability relative to the magnitude of the mean or typical value. For the intra-sample cv, the cv was calculated for each of the 68 samples (using the running average of 10 at each ATM base) and the average of the 68 coefficient of variations was taken. For the inter-sample cv, at each of the bases, the cv was calculated using the 68 samples, and the average of the coefficient of variations was taken. For both calculations, the moving average of 10 was used, instead of the original value, since the goal was to understand how the specificity varied over bases and across samples, rather than to estimate the experimental (or measurement) error.\n"}