A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag)

<p>Abstract</p> <p>Background</p> <p>Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence.</p> <p>Results</p> <p>A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size.</p> <p>Conclusions</p> <p>BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs.</p

SNP analysis of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene by a fluorescence-adapted SSCP method

BACKGROUND: Single-nucleotide polymorphisms (SNPs) are considered to be useful polymorphic markers for genetic studies of polygenic traits. Single-stranded conformational polymorphism (SSCP) analysis has been widely applied to detect SNPs, including point mutations in cancer and congenital diseases. In this study, we describe an application of the fluorescent labeling of PCR fragments using a fluorescent-adapted primer for SSCP analysis as a novel method. METHODS: Single-nucleotide polymorphisms (SNPs) of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene were analyzed using a fluorescence-adapted SSCP method. The method was constructed from two procedures: 1) a fluorescent labeling reaction of PCR fragments using fluorescence-adapted primers in a single tube, and 2) electrophoresis on a non-denaturing polyacrylamide gel. RESULTS: This method was more economical and convenient than the single-stranded conformational polymorphism (SSCP) methods previously reported in the detection of the labeled fragments obtained. In this study, eight SNPs of the IHRP gene were detected by the fluorescence-adapted SSCP. One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases. Six SNPs of the IHRP were associated with two haplotypes. CONCLUSIONS: The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs

Development of Genome-wide Simple Sequence Repeat Markers Using Whole-genome Shotgun Sequences of Sorghum (Sorghum bicolor (L.) Moench)

Simple sequence repeat (SSR) markers with a high degree of polymorphism contribute to the molecular dissection of agriculturally important traits in sorghum (Sorghum bicolor (L.) Moench). We designed 5599 non-redundant SSR markers, including regions flanking the SSRs, in whole-genome shotgun sequences of sorghum line ATx623. (AT/TA)n repeats constituted 26.1% of all SSRs, followed by (AG/TC)n at 20.5%, (AC/TG)n at 13.7% and (CG/GC)n at 11.8%. The chromosomal locations of 5012 SSR markers were determined by comparing the locations identified by means of electronic PCR with the predicted positions of 34 008 gene loci. Most SSR markers had a similar distribution to the gene loci. Among 970 markers validated by fragment analysis, 67.8% (658 of 970) markers successfully provided PCR amplification in sorghum line BTx623, with a mean polymorphism rate of 45.1% (297 of 658) for all SSR loci in combinations of 11 sorghum lines and one sudangrass (Sorghum sudanense (Piper) Stapf) line. The product of 5012 and 0.678 suggests that ∼3400 SSR markers could be used to detect SSR polymorphisms and that more than 1500 (45.1% of 3400) markers could reveal SSR polymorphisms in combinations of Sorghum lines

Fatty acid synthase: a metabolic enzyme and candidate oncogene in prostate cancer

BACKGROUND: Overexpression of the fatty acid synthase (FASN) gene has been implicated in prostate carcinogenesis. We sought to directly assess the oncogenic potential of FASN. METHODS: We used immortalized human prostate epithelial cells (iPrECs), androgen receptor-overexpressing iPrECs (AR-iPrEC), and human prostate adenocarcinoma LNCaP cells that stably overexpressed FASN for cell proliferation assays, soft agar assays, and tests of tumor formation in immunodeficient mice. Transgenic mice expressing FASN in the prostate were generated to assess the effects of FASN on prostate histology. Apoptosis was evaluated by Hoechst 33342 staining and by fluorescence-activated cell sorting in iPrEC-FASN cells treated with stimulators of the intrinsic and extrinsic pathways of apoptosis (ie, camptothecin and anti-Fas antibody, respectively) or with a small interfering RNA (siRNA) targeting FASN. FASN expression was compared with the apoptotic index assessed by the terminal deoxynucleotidyltransferase-mediated UTP end-labeling method in 745 human prostate cancer samples by using the least squares means procedure. All statistical tests were two-sided. RESULTS: Forced expression of FASN in iPrECs, AR-iPrECs, and LNCaP cells increased cell proliferation and soft agar growth. iPrECs that expressed both FASN and androgen receptor (AR) formed invasive adenocarcinomas in immunodeficient mice (12 of 14 mice injected formed tumors vs 0 of 14 mice injected with AR-iPrEC expressing empty vector (P < .001, Fisher exact test); however, iPrECs that expressed only FASN did not. Transgenic expression of FASN in mice resulted in prostate intraepithelial neoplasia, the incidence of which increased from 10% in 8- to 16-week-old mice to 44% in mice aged 7 months or more (P = .0028, Fisher exact test), but not in invasive tumors. In LNCaP cells, siRNA-mediated silencing of FASN resulted in apoptosis. FASN overexpression protected iPrECs from apoptosis induced by camptothecin but did not protect iPrECs from Fas receptor-induced apoptosis. In human prostate cancer specimens, FASN expression was inversely associated with the apoptotic rate (mean percentage of apoptotic cells, lowest vs highest quartile of FASN expression: 2.76 vs 1.34, difference = 1.41, 95% confidence interval = 0.45 to 2.39, Ptrend = .0046). CONCLUSIONS: These observations suggest that FASN can act as a prostate cancer oncogene in the presence of AR and that FASN exerts its oncogenic effect by inhibiting the intrinsic pathway of apoptosis