A whole-genome analysis of allelic changes in renal cell carcinoma by in-gel competitive reassociation

Abstract

We applied a differential cloning procedure, the in-gel competitive reassociation (IGCR) method, to clone altered genomic sites from the whole genomes of renal cell carcinoma cells. After four rounds of IGCR, we obtained from two patients libraries enriched 1000- and 2500-fold for differential DNA fragments specific to allelic changes in renal cell carcinoma. In these libraries, we found differential fragments of single-copy sequences as well as repetitive sequences. The fragments exhibited allelic loss, restriction-fragment-length polymorphism, size changes, and changes in the copy number, and common allelic losses were also detected in the cancer tissues from several renal cell carcinoma patients. Some of the clones showed changes in the repeat length of microsatellites. One third (seven of 22) of the clones exhibiting these changes were mapped to chromosomes 8 or 9. Decreases in the copy numbers of mitochondrial DNA and satellite I were observed in 13 of 17 and seven of 16 renal cell carcinoma patients, respectively. This suggests that the IGCR method can be used to clone DNA fragments with various structural changes from the whole genomes of cancer tissues.

A Novel Ankyrin Repeat-containing Gene (Kank) Located at 9p24 Is a Growth Suppressor of Renal Cell Carcinoma

“…Localization of an RCC-related Gene at 9p24 -A genome subtraction method, the in-gel competitive reassociation method, which was applied to normal and tumor DNAs, has superiority over conventional methods in detecting deletions of Ͻ1 Mb and also in detecting hemizygous deletions (17)(18)(19)(20). We previously reported 27 potential tumor-related loci using a library obtained by subtraction of tumor tissue DNA from normal tissue DNA of the same patient (14).…”

A comprehensive analysis of loss of heterozygosity caused by hemizygous deletions in renal cell carcinoma using a subtraction library

“…Enrichment of the altered genomic DNA between these tissues was performed using the in‐gel competitive reassociation method. This method has an advantage for comprehensive genomic analysis without using specific probes by providing a high complexity library of clones (10 3 or higher) [9]. The library of clones from a tumor (Patient 1) showed that 35% of the clones showed alteration of genomic DNA between the matched tissues, and the complexity was approximately 1×10 4 .…”

“…Here, we summarized the analysis of a library obtained by subtracting normal tissue DNA with matched tumor DNA to identify the deletions that occurred during tumorigenesis and thus showed preferential enrichment by the IGCR method [9,13–17]. One of the advantages that distinguishes this method from other subtraction methods and those used for comparative genomic analysis [18,19] is that the IGCR method allows a comprehensive and thorough analysis of genomic alterations by maintaining complexity of genomic DNA.…”

“…IGCR clones were obtained as described previously [9]. Briefly, 800 pg of Mse l‐digested normal tissue DNA, which was end‐filled with dATP and biotin‐11‐dUTP, was mixed with 10 μg of MseI‐digested tumor DNA from the same patient and electrophoresed on a Hydrolink gel (AT Biochem, Malvern, PA).…”

“…Genomic DNA was extracted from frozen tissues after proteinase K digestion and phenol‐chloroform extraction. Histopathological classification of the RCCs was mixed (granular > clear) cell type for Patients 1–23 [9] and clear cell type (mostly G1‐G2 grade) for Patients 41–66.…”