Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments

Abstract

Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas.

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

“…5'-TAATACGACTCACTATAGGG-3', SP6 primer: 5'-ATTTAGGTGACACTATAGAA-3'), then run on a 1% agarose gel electrophoresis Khan et al 2016;Fu et al 2017).…”

“…One particularly useful approach, based on initial RAPD sequencing, is Sequence Characterized Amplified Region (SCAR) analysis, which has the advantage of generating markers that are usually very stable and which therefore provides a high level of authenticity in genetic identification Khan et al 2016;Fu et al 2017). By using the SCAR markers, molecular analysis has been simplified to a PCR reaction using PCR primers designed from the sequences of RAPD amplicons (Kumla, et al 2012;Fu et al 2015).…”

Time series expression pattern of key genes reveals the molecular process of esophageal cancer

“…It was finally concluded that CPLX2 is a novel clinical biomarker for LNET [26]. In the study of breast cancer diagnostic markers, Fu et al [27] found that changes in gene expression such as DPEP1 may lead to cancer progression. DPEP1 has been identified as a prognostic gene for colorectal cancer.…”

Diagnostic value of a combination of next-generation sequencing, chorioretinal imaging and metabolic analysis: lessons from a consanguineous Chinese family with gyrate atrophy of the choroid and retina stemming from a novel OAT variant

“…Subsequently, a product with 555 bp products was PCR-amplified using gDNA as a template 22. Conventional Sanger sequencing using primer OAT-M212L was conducted to validate suspected pathogenic variants, and a 3500 DX ABI Genetic Analyzer was used for cosegregation analysis of family members.…”