Temperature gradient gel electrophoresis

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Negative staining of an ethidium bromide -stained DGGE gel.

The temperature gradient gel electrophoresis (TGGE, English temperature gradient gel electrophoresis ) and the denaturing gradient gel electrophoresis (DGGE, denaturing gradient gel electrophoresis ) are gel electrophoretic methods for separating charged biomolecules . You are using a temperature gradient or a chemical gradient across the length of the polyacrylamide gels . The TGGE and DGGE are used for separation of nucleic acids such as DNA or RNA also, and more rarely for proteins used. Alternative methods are e.g. B. DNA sequencing , SSCP , heteroduplex EMSA and denaturing HPLC .

Temperature gradient gel electrophoresis

The TGGE is a gel electrophoresis with a temperature difference over the length of the gel. At a certain temperature , double-stranded DNA melts into two single strands. The melting point is from the DNA sequence and its base pairing dependent and can be approximately calculated . The speed of migration of the DNA in the gel drops significantly from the melting temperature. The gradient can be applied parallel to the electrophoretic migration direction ( English parallel TGGE ) or orthogonally (English perpendicular TGGE ) to it. The orthogonally oriented gradients allow the identification of the melting point and the optimal temperature range for the sharpest possible separation. In the case of temperature gradients running parallel to the electrophoresis, separation takes place on the basis of different melting points and only initially according to the molar mass . This allows both mutations and secondary structures to be recognized. A variant of TGGE uses hybridization to heteroduplexes beforehand in order to amplify the differences in electrophoretic mobility.

Denaturation gradient gel electrophoresis

In DGGE, the gel contains a chemical gradient of a chaotrope . The abolition of secondary structures takes place through the increasingly denaturing concentration of the chaotrope. Denaturation usually does not take place continuously over the gradient, but in discrete steps. This allows mutations such as SNP to be compared in two DNA sequences. The disadvantage of chemical gradients is their poor reproducibility and, in the case of heteroduplexes, an occasional fuzzy separation.

history

The DGGE was developed from 1979 by Leonard Lerman and Stuart Fischer at SUNY Albany . The separation of proteins by DGGE was first described by Thomas E. Creighton at the MRC in Cambridge. The TGGE was developed from 1981 by Thatcher and Hodson as well as Roger Wartell .

Individual evidence

  1. ↑ G. Muyzer: DGGE / TGGE a method for Identifying genes from natural ecosystems. In: Current Opinion in Microbiology. Volume 2, Number 3, June 1999, ISSN  1369-5274 , pp. 317-322, doi : 10.1016 / S1369-5274 (99) 80055-1 , PMID 10383868 .
  2. ↑ CN Hestekin, AE Barron: The potential of electrophoretic mobility shift assays for clinical mutation detection. In: Electrophoresis. Volume 27, number 19, October 2006, ISSN  0173-0835 , pp. 3805-3815, doi : 10.1002 / elps.200600421 , PMID 17031787 .
  3. ↑ M. Salimullah, K. Hamano, M. Tachibana, K. Inoue, K. Nishigaki: Efficient SNP analysis enabled by joint application of the muTGGE and heteroduplex methods. In: Cellular & molecular biology letters. Volume 10, Number 2, 2005, ISSN  1425-8153 , pp. 237-245, PMID 16010289 .
  4. ↑ R. Fodde, M. Losekoot: mutation detection by denaturing gradient gel electrophoresis (DGGE). In: Human mutation. Volume 3, Number 2, 1994, ISSN  1059-7794 , pp. 83-94, doi : 10.1002 / humu.1380030202 , PMID 8199599 .
  5. ↑ SG Fischer, LS Lerman: Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. In: Cell. Volume 16, Number 1, January 1979, ISSN  0092-8674 , pp. 191-200, PMID 369706 .
  6. ↑ SG Fischer, LS Lerman: Separation of random fragments of DNA according to properties of their sequences. In: Proceedings of the National Academy of Sciences . Volume 77, Number 8, August 1980, ISSN  0027-8424 , pp. 4420-4424, PMID 6254023 , PMC 349855 (free full text).
  7. ↑ SG Fischer, LS Lerman: DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. In: Proceedings of the National Academy of Sciences . Volume 80, Number 6, March 1983, ISSN  0027-8424 , pp. 1579-1583, PMID 6220406 , PMC 393645 (free full text).
  8. ↑ TE Creighton, D. Shortle: Electrophoretic characterization of the denatured states of staphylococcal nuclease. In: Journal of molecular biology. Volume 242, Number 5, October 1994, ISSN  0022-2836 , pp. 670-682, doi : 10.1006 / jmbi.1994.1616 , PMID 7932723 .
  9. ^ DR Thatcher, B. Hodson: Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis. In: The Biochemical journal. Volume 197, Number 1, July 1981, ISSN  0264-6021 , pp. 105-109, PMID 6797412 , PMC 1163059 (free full text).
  10. ↑ RM Wartell, SH Hosseini, CP Moran: Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis. In: Nucleic acids research. Volume 18, Number 9, May 1990, ISSN  0305-1048 , pp. 2699-2705, PMID 2339057 , PMC 330754 (free full text).
  11. ↑ J. Zhu, RM Wartell: The relative stabilities of base pair stacking interactions and single mismatches in long RNA measured by temperature gradient gel electrophoresis. In: Biochemistry. Volume 36, Number 49, December 1997, ISSN  0006-2960 , pp. 15326-15335, doi : 10.1021 / bi9716783 , PMID 9398261 .