Standard mixture for analysis of GC content of DNA with nuclease P1
GC content of DNA is an indicator essential for classifying and identifying microorganisms. It has been measured by indirect methods, such as dye-buoyant-density and melting temperature methods. On the other hand, a novel method was almost simultaneously developed by three Japanese research groups, in which DNA is enzymatically decomposed into four kinds of deoxynucleotide, followed by direct measurement of GC content by high-performance liquid chromatography1-3). This method is applicable to heat-dried dead cells, as well as viable cells, and has various advantages, such as unnecessity of expensive equipment and skilled technicians.
The kit consists of nuclease P1 that decomposes test DNA into four kinds of deoxynucleotide and a standard deoxynucleotide (equimolar) mixture for high-performance liquid chromatography, providing simple and convenient measurements of GC content of DNA.
|1.||Preparation of DNA (EDTA free).
DNA concentration: 1 mg/ml distilled water
|2.||Heat treatment at 100℃ for 5 minutes|
|3.||Rapid cooling in ice water|
|4.|| 100 μl of DNA (100 μg) solution
*Enzyme solution: 100 μl (0.2 units)
(*: Nuclease P1 (2 units/ml))
|5.||Incubated at 50℃ for 1 hour|
|6.||After HPLC analysis of the equimolar standard mixture, the above enzymatically decomposed solution was analyzed.|
|1 kit||GC Analysis Sta: 3 tubes|
|Product code: 7160|
1. Kumagai M et al. (1988). Nucleic Acids Research Symposium series. 19. 65.
2. Noguchi T et al. (1988). Agric. Biol Chem. 52. 2355.
3. Kaneda T et al. (1986). J.Microbiol Methods. 4. 229.