Standard mixture for analysis of GC content of DNA with nuclease P1

Introduction

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 chromatography^1-3). 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.

Methods

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.

Packaging unit and product code

1 kit	GC Analysis Sta: 3 tubes
	Nuclease P1：400units
Product code: 7160

References

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.