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.

Keywords: RAMP; genomic instability; high-GC primer; random amplified polymorphic DNA (RAPD); sequence-characterized amplified region (SCAR).

Figure 1. Improved RAPD PCR from breast cancer tissues and their adjacent tissues

(A) High-GC primer FY-10. (B) High-GC primer FY-13. (C) High -GC primer FY-31. Five pairs of genomic DNA from breast cancer tissues and their adjacent or surrounding normal tissues were subjected to improved RAPD amplification (high-GC RAMP-PCR). Lanes 1, 3, 5, 7 and 9 are DNA from breast cancer tissues (see Table 1). Lanes 2, 4, 6, 8 and 10 are their matched DNA from adjacent tissue. The blue arrows indicate bands that were excised for DNA cloning. Lane “M” shows the DL2000 DNA molecular weight marker (bp).

Figure 2. Molecular cloning for RAPD products

(A) Agarose gel electrophoresis for RAPD DNA fragments 10, 13 and 31 derived from high-GC primers FY-10, FY-13 and FY-31, respectively. “T” indicates “pGM-T vector”. (B) Enzymatic identification of positive clones from RAPD fragment 10. (C) Enzymatic identification of positive clones from RAPD fragment 13. (D) Enzymatic identification of positive clones from RAPD fragment 31. Lane 1 contains undigested plasmid DNA; lane 2 contains plasmid DNA after EcoRI digestion. Clones 10-1, 13-4 and 31-2 (blue text color) were selected for Sanger sequencing. Lane “M” contains the DL2000 DNA molecular weight marker (bp).

Figure 3. Results of Sanger-sequencing of the cloned DNA fragments

(A) The sequence of clone 10-1. (B) The sequence of clone 13-4. (C) The sequences of clone 31-2.

Figure 4. The Human genome locations of clones 10-1 and 13-4 with their partial cDNAs of DEPEP1 and RNF40, respectively

Figure 5. The sequence of clone 13-4 aligns with RNF40 cDNA

Depicted is the BLAST output showing alignment of the clone 13-4 and RNF40 cDNA sequences. The sequence of clone 13-4 showed 583bp (Plus strand) identity with the cDNA of RNF40 (minus strand).

Figure 6. Genomic DNA amplification of SCAR markers BC10-1, BC13-4 and BC31-2 in breast cancer patients

(A) SCAR marker BC10-1 in five pairs of genomic DNA from breast cancer tissues and their adjacent tissues. (B) SCAR marker BC31-2 in five pairs of genomic DNA from breast cancer tissues and their adjacent tissues. Lanes 1, 3, 5, 7 and 9 contain DNA from breast cancer tissues (see Table 1). Lanes 2, 4, 6, 8 and 10 contain their matched adjacent tissue DNA. Blue arrows indicate the amplified band, whereas the stars “*” indicate the internal control. (C) Real-time PCR for SCAR marker BC10-1. (D) Real-time PCR for SCAR marker BC13-4. “Cancer”, breast cancer tissues; “Adjacent”, normal tissues adjacent to or surrounding the breast tumor; “Ctrl”, normal women blood DNA; “**”p value ≤ 0.05.

Figure 7. Analysis of DPEP1 and PHKG2 expression in breast cancer cell lines and tissues

(A) DPEP1 gene expression in breast cancer cell lines. (B) DPEP1 gene expression in invasive ductal carcinoma specimens. (C) PHKG2 gene expression in breast cancer cell lines. “MDA231”, MDA-MB-231 cells; “MDA435”, MDA-MB-435 cells.