Automated extraction and amplification of DNA from whole blood using a robotic workstation and an integrated thermocycler

被引:28
作者
Smit, ML
Giesendorf, BAJ
Heil, SG
Vet, JAM
Trijbels, FJM
Blom, HJ
机构
[1] Univ Nijmegen St Radboud Hosp, Dept Paediat 424, NL-6500 HB Nijmegen, Netherlands
[2] Univ Nijmegen St Radboud Hosp, Dept Clin Chem 564, NL-6500 HB Nijmegen, Netherlands
关键词
automation; DNA analysis; homocysteine;
D O I
10.1042/BA20000043
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Growing knowledge of the genetic basis of inheritable diseases has resulted in a rapidly increasing demand for DNA mutation analysis, Current methods are reliable and suitable for low-throughput mutation analyses, but are unable to cope with the increasing demand for genetic analyses, necessitating the development of new, fully automated and reliable methods, We developed a semi-automated method for DNA mutation analysis by integrating a thermocycler into a robotic pipetting workstation. DNA was extracted from 84 samples of 10 mu l of EDTA-treated whole blood, using magnetic beads within 2 h, Directly after isolation, the DNA was automatically transferred to an integrated thermocycler for amplification. Our semi-automated method proved to be reliable and robust, showing unambiguously interpretable PCR signals without occurrence of contamination, It is also faster than conventional manual methods. Only a brief manual intervention is required to remove and refit the seal of the PCR plate. This semi-automated assay is a step forward in the development of fully automated assays for DNA mutation analysis.
引用
收藏
页码:121 / 125
页数:5
相关论文
共 20 条
[1]   Rapid isolation of PCR-ready DNA from blood, bone marrow and cultured cells, based on paramagnetic beads [J].
Deggerdal, A ;
Larsen, F .
BIOTECHNIQUES, 1997, 22 (03) :554-557
[2]  
ENGBERSEN AMT, 1995, AM J HUM GENET, V56, P142
[3]   Disorders of homocysteine metabolism [J].
Fowler, B .
JOURNAL OF INHERITED METABOLIC DISEASE, 1997, 20 (02) :270-285
[4]   A CANDIDATE GENETIC RISK FACTOR FOR VASCULAR-DISEASE - A COMMON MUTATION IN METHYLENETETRAHYDROFOLATE REDUCTASE [J].
FROSST, P ;
BLOM, HJ ;
MILOS, R ;
GOYETTE, P ;
SHEPPARD, CA ;
MATTHEWS, RG ;
BOERS, GJH ;
DENHEIJER, M ;
KLUIJTMANS, LAJ ;
VANDENHEUVEL, LP ;
ROZEN, R .
NATURE GENETICS, 1995, 10 (01) :111-113
[5]  
Giesendorf BAJ, 1998, CLIN CHEM, V44, P482
[6]  
HEIL SG, 1998, NED TIJDSCHR KLIN CH, V23, P58
[7]   Separation of DNA sequencing fragments using an automated capillary electrophoresis instrument [J].
Karger, AE .
ELECTROPHORESIS, 1996, 17 (01) :144-151
[8]   Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency [J].
Kluijtmans, LAJ ;
Wendel, U ;
Stevens, EMB ;
van den Heuvel, LPWJ ;
Trijbels, FJM ;
Blom, HJ .
EUROPEAN JOURNAL OF HUMAN GENETICS, 1998, 6 (03) :257-265
[9]   Thermolabile methylenetetrahydrofolate reductase and factor V Leiden in the risk of deep-vein thrombosis [J].
Kluijtmans, LAJ ;
den Heijer, M ;
Reitsma, PH ;
Heil, SG ;
Blom, HJ ;
Rosendaal, FR .
THROMBOSIS AND HAEMOSTASIS, 1998, 79 (02) :254-258
[10]  
Kluijtmans LAJ, 1996, AM J HUM GENET, V58, P35