Genome wide instability scanning in chewing-tobacco associated oral cancer using inter simple sequence repeat PCR

Genomic instability plays a major role in cancer, facilitating tumour progression and tumour heterogeneity. Inter simple sequence repeat PCR (ISSR-PCR) is a sensitive tool for detection of whole genome scanning. In fifteen oral cancer patients, using tumor tissue and adjacent normal tissue DNA, we investigated genomic instability regions using ISSR-PCR assay. The genomic fragments were cloned, sequenced and identified. Two-anchored dinucleotide repeat primers, (CA)(8)A/GG and (CA)(8)A/GC/T, were used in the study. About 40-50 fragments were observed on polyacrylamide gel electrophoresis, with 25 distinct fragments of less than 2 kb. The electrophoretic pattern highlighted several distinct fragments in tumor adjacent normal tissues. The distinct fragments of 258, 325, 430, 440, 600 and 900 bp sizes using (CA)(8)A/GG primer, and 300, 475, 675 and 800 bp using (CA)(8)A/GC/T primers, in the normal tissues showed partial (>50%) or complete loss in multiple tumor tissues. These fragments were eluted from the gel, cloned in pMos Blue vector and subjected to nucleotide sequencing. Insilico analysis defined the specific genomic sequences, given as follows: RP11-399D2 () on chromosome (chr)4; RP1-39J2 (), NKp44RG () and RP11-518I13 () on chr6; NC-T-2 () on chr7; RP11-586K2 () and RP11-495O10 () on chr8; RP11-101K10 () on chr9; R-794A8 () on chr14; and RP11-679B19 () on chr16. The sequences of our clones have been submitted to NCBI gene bank, accession numbers to , and . The Genomic Instability Index was calculated and ranged from 6% to 28.5% (median 12%) in the oral cancer samples, excluding one case where genomic instability was not observed. Thus, our results indicate presence of widespread genomic alterations in chewing-tobacco associated oral cancers.