Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis

被引:19
作者
vanderSchans, MJ
Kuypers, AWHM
Kloosterman, AD
Janssen, HJT
Everaerts, FM
机构
[1] UNIV NIJMEGEN HOSP,DEPT HEMATOL,NL-6500 HB NIJMEGEN,NETHERLANDS
[2] MINIST JUSTICE,FORENS SCI LAB,RIJSWIJK,NETHERLANDS
关键词
polymerase chain reaction; DNA;
D O I
10.1016/S0021-9673(97)00142-8
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Capillary electrophoresis has become a powerful analytical tool for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. The use of replaceable polymer solutions increases the lifetime of the capillary, improves the repeatability of the migration times and enables pressure injection. When pressure injection is used, rather than electrokinetic injection, it is assured that a representative part of the sample is introduced into the capillary. The possible lower resolution, which is a side effect of the pressure injection, can be made up for by an increase of selectivity. Using fluorescent labels, it is possible to detect DNA with a fluorescence detector under both native conditions in double-stranded (ds) form, and under denaturing conditions in single-stranded (ss) form. When DNA is separated in its ss form, as is necessary for DNA sequencing, we observe an increased selectivity compared to separation of that sample in its ds form. In this work, we exploited this increased selectivity for the analysis of denatured PCR products. It was found that DNA separated in the ss form yields superior separation; that is, a given analysis can be achieved with the same resolution in a shorter separation time compared to dsDNA. Therefore, it is advisable to separate DNA in the ss form if high resolution, size dependent separation is required. The enhanced resolution achieved with DNA migrating in the ss form enabled the separation of allelic ladders of short tandem repeats with a difference of 4 base pairs in the 200 base pair range, with separation times no longer than 6 min.
引用
收藏
页码:255 / 264
页数:10
相关论文
共 44 条
[1]   RAPID TYPING OF VARIABLE NUMBER OF TANDEM REPEAT LOCUS IN THE HUMAN APOLIPOPROTEIN-B GENE FOR DNA DIAGNOSIS OF HEART-DISEASE BY POLYMERASE CHAIN-REACTION AND CAPILLARY ELECTROPHORESIS [J].
BABA, Y ;
TOMISAKI, R ;
SUMITA, C ;
MORIMOTO, I ;
SUGITA, S ;
TSUHAKO, M ;
MIKI, T ;
OGIHARA, T .
ELECTROPHORESIS, 1995, 16 (08) :1437-1440
[2]   The effects of polymer properties on DNA separations by capillary electrophoresis in uncross-linked polymer solutions [J].
Barron, AE ;
Sunada, WM ;
Blanch, HW .
ELECTROPHORESIS, 1996, 17 (04) :744-757
[3]   CAPILLARY ELECTROPHORESIS OF DNA IN UNCROSS-LINKED POLYMER-SOLUTIONS [J].
BARRON, AE ;
SOANE, DS ;
BLANCH, HW .
JOURNAL OF CHROMATOGRAPHY A, 1993, 652 (01) :3-16
[4]   A TRANSIENT ENTANGLEMENT COUPLING MECHANISM FOR DNA SEPARATION BY CAPILLARY ELECTROPHORESIS IN ULTRADILUTE POLYMER-SOLUTIONS [J].
BARRON, AE ;
BLANCH, HW ;
SOANE, DS .
ELECTROPHORESIS, 1994, 15 (05) :597-615
[5]   SEQUENCE-DEPENDENT MIGRATION BEHAVIOR OF DOUBLE-STRANDED DNA IN CAPILLARY ELECTROPHORESIS [J].
BERKA, J ;
PARIAT, YF ;
MULLER, O ;
HEBENBROCK, K ;
HEIGER, DN ;
FORET, F ;
KARGER, BL .
ELECTROPHORESIS, 1995, 16 (03) :377-388
[6]  
BUTLER JM, 1994, BIOTECHNIQUES, V17, P1062
[7]   APPLICATION OF DUAL INTERNAL STANDARDS FOR PRECISE SIZING OF POLYMERASE CHAIN-REACTION PRODUCTS USING CAPILLARY ELECTROPHORESIS [J].
BUTLER, JM ;
MCCORD, BR ;
JUNG, JM ;
LEE, JA ;
BUDOWLE, B ;
ALLEN, RO .
ELECTROPHORESIS, 1995, 16 (06) :974-980
[8]   QUANTITATION OF POLYMERASE CHAIN-REACTION PRODUCTS BY CAPILLARY ELECTROPHORESIS USING LASER FLUORESCENCE [J].
BUTLER, JM ;
MCCORD, BR ;
JUNG, JM ;
WILSON, MR ;
BUDOWLE, B ;
ALLEN, RO .
JOURNAL OF CHROMATOGRAPHY B-BIOMEDICAL APPLICATIONS, 1994, 658 (02) :271-280
[9]   CAPILLARY ELECTROPHORESIS OF DNA - POTENTIAL UTILITY FOR CLINICAL DIAGNOSES [J].
FELMLEE, TA ;
ODA, RP ;
PERSING, DA ;
LANDERS, JP .
JOURNAL OF CHROMATOGRAPHY A, 1995, 717 (1-2) :127-137
[10]   HIGH-SPEED DNA-SEQUENCING BY USING MIXED POLY(ETHYLENE OXIDE) SOLUTIONS IN UNCOATED CAPILLARY COLUMNS [J].
FUNG, EN ;
YEUNG, ES .
ANALYTICAL CHEMISTRY, 1995, 67 (13) :1913-1919