PURIFICATION AND QUANTITATIVE-ANALYSIS OF NUCLEIC-ACIDS BY ANION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

被引:37
作者
HENNINGER, HP
HOFFMANN, R
GREWE, M
SCHULZESPECKING, A
DECKER, K
机构
[1] ALBERT LUDWIGS UNIV,INST BIOCHEM,HERMANN HERDER STR 7,D-79104 FREIBURG,GERMANY
[2] ALBERT LUDWIGS UNIV,HAUTKLIN,D-79104 FREIBURG,GERMANY
来源
BIOLOGICAL CHEMISTRY HOPPE-SEYLER | 1993年 / 374卷 / 08期
关键词
BETA-ACTIN; DNA; LIPOPOLYSACCHARIDE; REVERSE TRANSCRIPTION; POLYMERASE CHAIN REACTION; TUMOR NECROSIS FACTOR-ALPHA;
D O I
10.1515/bchm3.1993.374.7-12.625
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
A novel and reliable high-performance liquid chromatography (HPLC) method is described for the purification and quantification of double-stranded DNA. The nucleic acids may be obtained by polymerase chain reaction (PCR) or as restriction fragments from enzymatic cleavage, the separated products are devoid of contaminating material like agarose, ethidium bromide or non-specific DNA sequences. Because of the non-destructive nature of this HPLC procedure, the purified DNA is optimally suited for cloning experiments. The DNA separation by HPLC has major advantages when combined with reverse transcription (RT)PCR. This is exemplified by analysis of the TNF-alpha mRNA obtained from endotoxin-elicited rat liver macrophages. If the standard procedure of Northern blotting is compared with the combination of RT-PCR and quantification of the PCR products by HPLC, it is obvious that the dynamic changes of tumor necrosis factor (TNF)-alpha mRNA synthesis are at least as precisely reflected with the RT-PCR/HPLC combination. The latter method is presented as a reliable and powerful tool for quantitative studies on gene expression.
引用
收藏
页码:625 / 634
页数:10
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