Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations

被引:34
作者
Meierhofer, D [1 ]
Mayr, JA [1 ]
Ebner, S [1 ]
Sperl, W [1 ]
Kofler, B [1 ]
机构
[1] Paracelsus Private Med Univ Salzburg, Dept Paediat, A-5020 Salzburg, Austria
关键词
DHPLC; MtDNA; mutation; heteroplasmy;
D O I
10.1016/j.mito.2005.06.001
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injections of fragments yielded straightforward interpretation of results with a detection limit down to 1 % mtDNA heteroplasmy. Furthermore, collection and re-amplification of low degree heteroduplex peak-fractions allowed sequence analysis of mtDNA mutations down to the detection limit of the DHPLC method. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples. (c) 2005 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
引用
收藏
页码:282 / 296
页数:15
相关论文
共 23 条
[1]   Mutation screening of the mitochondrial genome using denaturing high-performance liquid chromatography [J].
Biggin, A ;
Henke, R ;
Bennetts, B ;
Thorburn, DR ;
Christodoulou, J .
MOLECULAR GENETICS AND METABOLISM, 2005, 84 (01) :61-74
[2]   Qualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography [J].
Conley, YP ;
Brockway, H ;
Beatty, M ;
Kerr, ME .
BRAIN RESEARCH PROTOCOLS, 2003, 12 (02) :99-103
[3]  
Danielson PB, 2003, CROAT MED J, V44, P447
[4]   Clinical features of diabetic patients with 0.01-0.1% heteroplasmy A3243G mutation in leukocyte mitochondrial DNA [J].
Iwase, M ;
Gotoh, D ;
Urata, M ;
Kang, D ;
Hamasaki, N ;
Yoshinari, M ;
Fujishima, M .
DIABETES RESEARCH AND CLINICAL PRACTICE, 2001, 54 (03) :215-217
[5]   Frequency of mitochondrial transfer RNA mutations and deletions in 225 patients presenting with respiratory chain deficiencies [J].
Jaksch, M ;
Kleinle, S ;
Scharfe, C ;
Klopstock, T ;
Pongratz, D ;
Müller-Höcker, J ;
Gerbitz, KD ;
Liechti-Gallati, S ;
Lochmuller, H ;
Horvath, R .
JOURNAL OF MEDICAL GENETICS, 2001, 38 (10) :665-673
[6]   Lowe level mosaicism detectable by DHPLC but not by direct sequencing [J].
Jones, AC ;
Sampson, JR ;
Cheadle, JP .
HUMAN MUTATION, 2001, 17 (03) :233-234
[7]   Gram type-specific broad-range PCR amplification for rapid detection of 62 pathogenic bacteria [J].
Klausegger, A ;
Hell, M ;
Berger, A ;
Zinober, K ;
Baier, S ;
Jones, N ;
Sperl, W ;
Kofler, B .
JOURNAL OF CLINICAL MICROBIOLOGY, 1999, 37 (02) :464-466
[8]  
LaBerge GS, 2003, CROAT MED J, V44, P281
[9]   Bioenergetics of mitochondrial diseases associated with mtDNA mutations [J].
Lenaz, G ;
Baracca, A ;
Carelli, V ;
D'Aurelio, M ;
Sgarbi, G ;
Solaini, G .
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 2004, 1658 (1-2) :89-94
[10]   Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography [J].
Liu, MR ;
Pan, KF ;
Li, ZF ;
Wang, Y ;
Deng, DJ ;
Zhang, L ;
Lu, YY .
WORLD JOURNAL OF GASTROENTEROLOGY, 2002, 8 (03) :426-430