Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Mar 11;21(5):1061–1066. doi: 10.1093/nar/21.5.1061

High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

C G Huber 1, P J Oefner 1, E Preuss 1, G K Bonn 1
PMCID: PMC309263  PMID: 8503958

Abstract

DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.

Full text

PDF
1061

Images in this article

1065Image
on p.1065

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  2. Cohen A. S., Najarian D., Smith J. A., Karger B. L. Rapid separation of DNA restriction fragments using capillary electrophoresis. J Chromatogr. 1988 Dec 23;458:323–333. doi: 10.1016/s0021-9673(00)90576-4. [DOI] [PubMed] [Google Scholar]
  3. Eriksson S., Glad G., Pernemalm P. A., Westman E. Separation of DNA restriction fragments by ion-pair chromatography. J Chromatogr. 1986 May 30;359:265–274. doi: 10.1016/0021-9673(86)80080-2. [DOI] [PubMed] [Google Scholar]
  4. Eshaghpour H., Crothers D. M. Preparative separation of the complementary strands of DNA restriction fragments by alkaline RPC-5 chromatography. Nucleic Acids Res. 1978 Jan;5(1):13–21. doi: 10.1093/nar/5.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gordon M. J., Huang X., Pentoney S. L., Jr, Zare R. N. Capillary electrophoresis. Science. 1988 Oct 14;242(4876):224–228. doi: 10.1126/science.242.4876.224. [DOI] [PubMed] [Google Scholar]
  6. Hardies S. C., Wells R. D. Preparative fractionation of DNA restriction fragments by reversed phase column chromatography. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3117–3121. doi: 10.1073/pnas.73.9.3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Heiger D. N., Cohen A. S., Karger B. L. Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields. J Chromatogr. 1990 Sep 7;516(1):33–48. doi: 10.1016/s0021-9673(01)90202-x. [DOI] [PubMed] [Google Scholar]
  8. Huber C. G., Oefner P. J., Bonn G. K. High-performance liquid chromatographic separation of detritylated oligonucleotides on highly cross-linked poly-(styrene-divinylbenzene) particles. J Chromatogr. 1992 May 22;599(1-2):113–118. doi: 10.1016/0021-9673(92)85463-4. [DOI] [PubMed] [Google Scholar]
  9. Jost W., Unger K., Schill G. Reverse-phase ion-pair chromatography of polyvalent ions using oligonucleotides as model substances. Anal Biochem. 1982 Jan 15;119(2):214–223. doi: 10.1016/0003-2697(82)90689-3. [DOI] [PubMed] [Google Scholar]
  10. Kasper T. J., Melera M., Gozel P., Brownlee R. G. Separation and detection of DNA by capillary electrophoresis. J Chromatogr. 1988 Dec 23;458:303–312. doi: 10.1016/s0021-9673(00)90574-0. [DOI] [PubMed] [Google Scholar]
  11. Kato Y., Yamasaki Y., Onaka A., Kitamura T., Hashimoto T., Murotsu T., Fukushige S., Matsubara K. Separation of DNA restriction fragments by high-performance ion-exchange chromatography on a non-porous ion exchanger. J Chromatogr. 1989 Sep 8;478(1):264–268. doi: 10.1016/s0021-9673(01)84395-8. [DOI] [PubMed] [Google Scholar]
  12. Katz E. D., Haff L. A., Eksteen R. Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography. J Chromatogr. 1990 Jul 20;512:433–444. doi: 10.1016/s0021-9673(01)89509-1. [DOI] [PubMed] [Google Scholar]
  13. Kwiatkowski M., Sandström A., Balgobin N., Chattopadhyaya J. Use of reverse phase ion pair chromatography to fractionate and purify DNA fragments and monomeric components of RNA. Acta Chem Scand B. 1984;38(9):721–733. doi: 10.3891/acta.chem.scand.38b-0721. [DOI] [PubMed] [Google Scholar]
  14. Landy A., Foeller C., Reszelbach R., Dudock B. Preparative fractionation of DNA restriction fragments by high pressure column chromatography on RPC-5. Nucleic Acids Res. 1976 Oct;3(10):2575–2592. doi: 10.1093/nar/3.10.2575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maa Y. F., Horváth C. Rapid analysis of proteins and peptides by reversed-phase chromatography with polymeric micropellicular sorbents. J Chromatogr. 1988 Jul 8;445(1):71–86. doi: 10.1016/s0021-9673(01)84509-x. [DOI] [PubMed] [Google Scholar]
  16. Nathakarnkitkool S., Oefner P. J., Bartsch G., Chin M. A., Bonn G. K. High-resolution capillary electrophoretic analysis of DNA in free solution. Electrophoresis. 1992 Jan-Feb;13(1-2):18–31. doi: 10.1002/elps.1150130105. [DOI] [PubMed] [Google Scholar]
  17. Noonan K. E., Beck C., Holzmayer T. A., Chin J. E., Wunder J. S., Andrulis I. L., Gazdar A. F., Willman C. L., Griffith B., Von Hoff D. D. Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7160–7164. doi: 10.1073/pnas.87.18.7160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Usher D. A. Reverse-phase HPLC of DNA restriction fragments and ribooligonucleotides on uncoated Kel-F powder. Nucleic Acids Res. 1979;6(6):2289–2306. doi: 10.1093/nar/6.6.2289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Westman E., Eriksson S., Lås T., Pernemalm P. A., Sköld S. E. Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q. Anal Biochem. 1987 Oct;166(1):158–171. doi: 10.1016/0003-2697(87)90558-6. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES