Virtual genome scan: a tool for restriction landmark-based scanning of the human genome

Abstract

There is substantial interest in implementing technologies that allow comparisons of whole genomes of individuals and of tissues and cell populations. Restriction landmark genome scanning (RLGS) is a highly resolving gel-based technique in which several thousand fragments in genomic digests are visualized simultaneously and quantitatively analyzed. The widespread use of RLGS has been hampered by difficulty in deriving sequence information for displayed fragments and a lack of whole-genome sequence-based framework for interpreting RLGS patterns. We have developed informatics tools for comparisons of sample derived RLGS patterns with patterns predicted from the human genome sequence and displayed as Virtual Genome Scans (VGS). The tools developed allow sequence prediction of fragments in RLGS patterns obtained with different restriction enzyme combinations. The utility of VGS is demonstrated by the identification of restriction fragment length polymorphisms, and of amplifications, deletions, and methylation changes in tumor-derived CpG islands and the characterization of an amplified region in a breast tumor that spanned <230 kb on 17q23.

Figure 1

Whole genome restriction landmark genomic scanning (RLGS) pattern superimposed with a chromosome 1 virtual genome scanning (VGS) profile, both obtained using the NotI/EcoRV/HinfI enzyme combination. (Yellow, draft sequence; blue, finished sequence). Instructions for use of the software are accessed by clicking the Help button. The red spot represents the experimental ALX3 spot described in Table 1. The small rectangle centered on the predicted ALX3 spot (yellow) represents the area encompassing 75% of the predicted-experimental spot pairs, and the larger rectangle encompasses 100% of these pairs.

Figure 2

Close-up sections of restriction landmark genomic scanning (RLGS) patterns containing chromosome 1 derived spots A–C (arrows) with reduced intensity in neuroblastoma STA-NB9 cell line relative to control peripheral blood lymphocytes. The location of matching fragments in virtual genome scanning (VGS) patterns is indicated with black squares. RLGS patterns were obtained using the NotI/EcoRV/HinfI enzyme combination.

Figure 3

(A) Tumor 200 NotI/EcoRV/HinfI restriction landmark genomic scanning (RLGS) profile. Black squares represent predicted spots from chromosome 17 in this section of the RLGS pattern. Arrows point to two amplified fragments specific to Tumor 200 and to the location of their predicted fragments from chromosome 17. The three highly intense spots in the right part of the gel represent rDNA. (B) PCR analysis of TBX2 genomic amplification. Lane 1: MCF7; lane 2, Tumor 200; lane 3, Control DNA. Arrows point to GAPDH (407 bp) and TBX2 (188 bp) PCR products. (C) Extent of the 17q23 amplified region as determined by PCR analysis. Lanes 1– 6 represent multiplexed PCR using GAPDH and primers 1–6, respectively (C, control DNA; T, Tumor 200 DNA). An arrow points to the location of GAPDH (407 bp) PCR product. Positions of primer pairs 1–6 and TBX2 along sequence NT_001445 are drawn. The scale is given in kb.

Figure 4

Semiquantitative RT–PCR analysis of TBX2 gene (188 bp), Hs.269402 UniGene cluster (323 bp) and BE075001 EST (486 bp). Total RNA from Tumor 200 (T), MCF7 (M), and the control HPV11–21 (H) cell line was utilized. GAPDH (407 bp) was coamplified as a control. Numbers given below lanes represent fold intensity for MCF7 and Tumor 200 vs. HPV11–21.