• Bulletin of the Korean Chemical Society
• /
• v.27 no.4
• /
• pp.519-523
• /
• 2006

Poly(dimethyl siloxane) (PDMS) has been employed as a microchip material for DNA separation in microfluidic condition. Different sieving molecules such as cellulose derivatives having glucose building block (methyl cellulose (MC), hydroxyethyl cellulose (HEC), and hydroxypropyl methyl cellulose (HPMC)) and polyethylene oxide (PEO) having linear (ring-opened ethylene oxide) unit were used and their performance was compared in terms of separation efficiency and resolution. In general, PEO showed better separation performance than cellulose derivatives probably due to the nature of linear shape polymer conformation. It was possible to perform at least 15 consecutive running with 1.2% PEO at the electric field strength around 200 V/cm. Fast analysis of the standard $\Phi$X 174 RF DNA/Hae III (less than 130s) was obtained with the number of the theoretical plate around 250,000/m. Our PMDS microchip was applied to the measurement of CAG repeat number, which is related to male infertile disease.

• Journal of Genetic Medicine
• /
• v.10 no.1
• /
• pp.33-37
• /
• 2013

Purpose: Myotonic dystrophy 1 (DM1, OMIM 160900) is an autosomal-dominant muscular disorder caused by an expansion of CTG repeats in the 3' UTR of the DMPK gene. Variable expansions of CTG repeats preclude the accurate determination of repeat size. We tried to show the clinical and analytical validity of the application of Southern blotting after long-range PCR was demonstrated in Korean DM1 patients. Materials and Methods: The Southern blotting of long-range PCR was applied to 1,231 cases with clinical suspicion of DM1, between 2000 and 2011. PCR was performed using genomic DNA with forward 5'-CAGTTCACAACCGCTCCGAGC-3' and reverse 5'-CGTGGAGGATGGAACACGGAC-3' primers. Subsequently, the PCR fragments were subjected to gel electrophoresis, capillary transfer to a nylon membrane, hybridization with a labeled (CAG)10 probe. The correlation between clinical manifestations and the CTG repeat expansions were analyzed. Results: Among a total of 1,231 tested cases, 642 individuals were diagnosed with DM1 and the range of the detected expansion was 50 to 2,500 repeats; fourteen cases with mild DM1 ($75{\pm}14$ repeats), 602 cases with classical DM1 ($314{\pm}143$ repeats), and 26 cases with congenital DM1 ($1,219{\pm}402$ repeats). The positive and negative predictive values were 100%. The age at test requested and the CTG repeat numbers were inversely correlated (R=-0.444, P<0.01). Conclusion: This study indicates that Southern blotting after long-range PCR is a reliable diagnostic method DM1.

• Journal of Ginseng Research
• /
• v.46 no.4
• /
• pp.572-584
• /
• 2022

Background: Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of trinucleotide CAG repeat in the Huntingtin (Htt) gene. The major pathogenic pathways underlying HD involve the impairment of cellular energy homeostasis and DNA damage in the brain. The protein kinase ataxia-telangiectasia mutated (ATM) is an important regulator of the DNA damage response. ATM is involved in the phosphorylation of AMP-activated protein kinase (AMPK), suggesting that AMPK plays a critical role in response to DNA damage. Herein, we demonstrated that expression of polyQ-expanded mutant Htt (mHtt) enhanced the phosphorylation of ATM. Ginsenoside is the main and most effective component of Panax ginseng. However, the protective effect of a ginsenoside (compound K, CK) in HD remains unclear and warrants further investigation. Methods: This study used the R6/2 transgenic mouse model of HD and performed behavioral tests, survival rate, histological analyses, and immunoblot assays. Results: The systematic administration of CK into R6/2 mice suppressed the activation of ATM/AMPK and reduced neuronal toxicity and mHTT aggregation. Most importantly, CK increased neuronal density and lifespan and improved motor dysfunction in R6/2 mice. Conversely, CK enhanced the expression of Bcl2 protected striatal cells from the toxicity induced by the overactivation of mHtt and AMPK. Conclusions: Thus, the oral administration of CK reduced the disease progression and markedly enhanced lifespan in the transgenic mouse model (R6/2) of HD.

• Clinical and Experimental Reproductive Medicine
• /
• v.34 no.3
• /
• pp.179-188
• /
• 2007

Objectives: Many neurological diseases are known to be caused by expansion of trinucleotide repeats (TNRs). It is hard to diagnose the alteration of TNRs with single cell level for preimplantation genetic diagnosis (PGD). In this study, we describe methods optimized for PGD of TNRs related diseases such as Huntington's disease (HD), spinocerebellar ataxia 3 (SCA3) and fragile X syndrome (FXS). Methods: We performed the preclinical assays with heterozygous patient's lymphocytes by single cell PCR strategy. Fluorescent semi-nested PCR and fragment analysis using automatic genetic analyzer were applied for HD and SCA 3. Whole genome amplification with multiple displacement amplification (MDA) method and fluorescent PCR were carried out for FXS. Amplification and allele drop-out (ADO) rate were evaluated in each case. Results: The fluorescent semi-nested PCR of single lymphocyte showed 100.0% of amplification and 14.0% of ADO rate in HD, and 94.7% of amplification and 5.6% of ADO rate in SCA3, respectively. We could not detect the PCR product of CGG repeats in FXS using the fluorescent semi-nested PCR alone. After applying the MDA method in FXS, 84.2% of amplification and 31.3% of ADO rate were achieved. Conclusions: Fluorescent semi-nested PCR is a reliable method for PGD of HD and SCA3. The advanced MDA method overcomes the problem of amplification failure in CGG repeats of FXS case. Optimization of methods for single cell analysis could improve the sensitivity and reliability of PGD for complicated single gene disorders of TNRs.