• Animal cells and systems
• /
• v.5 no.3
• /
• pp.237-241
• /
• 2001

Three STR loci (STRX1[AGAT]$_{n}$, HPRTB[AGAT]$_{n}$ and ARA[AGC]$_{n}$) on X chromosome and three other STR loci (DYS390[CTG(A)T]$_{n}$, DYS392[ATT]$_{n}$ and DYS39［GATA]$_{n}$) on Y chromosome were analyzed in 154 unrelated healthy Korean subjects. Four loci (STRX1, HPRTB, DYS390 and DYS393) were amplified by quadruplex polymerase chain reaction (PCR) using fluorescent labeled primers (FLP). ARA and DYS392 were amplified separately using single PCR, similarly by using FLP. They were then run in an automated DNA sequencer and were analyzed with Genescan software. We found 7 alleles (308-332 bp) in STRX1, 7 alleles (275-299 bp) in HPRTB, 16 alleles (252-315 bp) in ARA, 6 alleles (203-223 bp) in DYS390, 7 alleles (245-263bp) in DYS392 and 5 alleles (116-132 bp) in DYS393. The ＊13(34%), ＊13(5l%), ＊23 (l8%), ＊23(50%), ＊14(39%) and ＊13(40%) alleles were observed to be the highest frequencies of STRX1, HPRTB, ARA, DYS390, DYS392 and DYS393, respectively. The detection of STR loci on sex chromosomes by quadruplex PCR allows simple determination of sex and identification of an individual. individual.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.5
• /
• pp.662-667
• /
• 2020

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

• Analytical Science and Technology
• /
• v.12 no.3
• /
• pp.260-264
• /
• 1999

A simple and rapid procedure, called FoLT-PCR(Formamide Low Temperature-Polymerase Chain Reaction) was applied to amplifying DNA directly from various forensic biological evidences including human blood, saliva, hair root, or semen without any DNA preparative steps. We added washing step with non-ionic detergent, 1% Triton X-100, and used Taq DNA polymerase instead of Tth DNA polymerase to amplify 3 STR loci and gender allele simultaneouly. Optimal concentration of formamide and annealing temperature were determined empirically to 8%(v/v), and $48^{\circ}C$ respectively. We also compared this method with standard PCR.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.5
• /
• pp.2305-2309
• /
• 2012

Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancer cells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin on the immortality of hepatic cancer cells. Cytotoxicity of crocin ($IC_{50}$ 3 mg/ml) in hepatocarcinoma HepG2 cells was determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated through quantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeat amplification protocol (qTRAP). Telomerase activity in 0.5 ${\mu}g$ protein extract of HepG2 cells treated with 3 mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition, i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, were examined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in the presence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in the $T_m$ telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT) gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression of the catalytic subunit of the enzyme.