
Direct amplification of mitochondrial DNA for genetic detection of MELAS syndrome from whole blood
- Geon Park Jong Yeol Kim Jin Lim Ok Yeon Jeoung Sook Jin Jang Young Jin Park Dae Soo Moon
- 조선대학교 의학연구원
- The Medical Journal of Chosun University
- 제34권 제2호
- 2009.06
- 108 - 111 (4 pages)
Background: The amplification of mitochondrial DNA is very important for genetic diagnosis of mitochondrial myopathy, Encephalopathy, Lactic acidosis, and Stroke like episodes (MELAS) syndrome. However, the DNA extraction that is an essential step for amplification of DNA is a labor-intensive and sample-consuming step. Herein, we performed direct amplification of mitochondrial DNA from whole blood without DNA extraction and purification 니sing a direct PCR buffer system (AnyDirect; BioQuest, Seoul, Korea) and a general Taq DNA polymerase (AnyTaq; BioQ니est). Material and Methods: The performance of the direct PCR buffer system was evaluated in terms of amplificability, the allowable sample volume, the upper allowable concentration limits of PCR inhibitors (hemoglobin and K2 EDTA) and storage conditions (temperature and duration) from one healthy male volunteer values (hemoglobin 16.0 g/dL, white blood cells 5,100 cells/ul and platelets 160,000 cells/uL), 100 WB selected at random, 5 WB left alone at room temperature over one year, 10 WB storaged at +4°C, 20 WB storaged at -20°C and 4 WB requested with tests for genetic detection of MELAS. For detection of hot mitochondrial mutation spot (m.3243A>G) in MELAS, we amplified the interesting part (358bp) in MTTL1. To compare amplificability of conventional PCR and direct PCR using same PCR condition, DNA of 140 samples were extracted using a commercial DNA extraction kit (Qiagen, Valencia, CA, USA). The genetic sequences should be obtained only from the patients requested with tests for genetic detection of MELAS. Results: Amplificability of conventional PCR and direct PCR using same PCR condition was same at 100% from 140 cases. Direct amplification can be performed from samples that were stored for over one year at room temperature. All samples storaged at +40C or -20°Cover three years were successfully amplifiable. When direct PCR used the volunteer s WB with 1.8 mg/mL K2 EDTA, the percent of allowable sample volume was 2 pL (10%) in a total PCR reaction volume of 20 |jl_. The upper allowable concentration limits of hemoglobin and K2 EDTA were 21 g/dL (Hematocrit 70%) and 5.4 mg/mL using 0.5 pL WB in a total volume of 20 \jL, respectively. Conclusion: Direct amplification of mitochondrial DNA without DNA isolation performed with AnyDirect b니ffer is a simple, rapid and economic infrastructural tool for detection of mutation in mitochondrial DNA.
Introduction
Materials and Methods
Results
Discussion