Studies On Determination Of DNA By Resonance Light-scattering (RLS) Method And Their Applications

The resonance light-scattering (RLS) spectra characteristics of small organic molecules and deoxyribonucleic acid (DNA) systems have been studied on the foundation of numerous references and conditions of the laboratory. On the basis of the studies, we have established four new determination methods of DNA by means of resonance light-scattering (RLS) technique in this paper, This paper consists of four chapters.In the first chapter,the spectral characteristics of RLS, the effective factors, the mechanistic and optimum conditions of the reaction system of Methyl Green(MG)-fish sperm DNA(fsDNA) have been investigated. In the buffer of Tris(hydroxymethyl)aminometh- ane-HCl(Tris-HCl) at pH 10.0, methyl green exhibited five resonance scattering peaks at 292, 339, 389, 468, 534 nm, with the addition of fsDNA the intensity was greatly enhanced. There was linear relationship between the enhanced intensity and fish sperm deoxyribonucleic acids concentration at 339nm. Linear equation of fish sperm deoxyribonucleic acid was △I= -22.6602 + 56.1966C(C: μg/mL). Based on the above results a sensitive and simple method was established for determination of deoxyribonucleic acid. The linear range of the calibration graph is 400~1800μg/L with a correlation coefficient of 0.9990.The detection limit is 20.6μg/L.This method is simple, rapid and has been applied to the determination of DNA in mixed samples with satisfactory results. In the second chapter, the spectral characteristics of RLS, the effective factors, the mechanistic and optimum conditions of the reaction system of fuchsin basic-DNA(FB-DNA) have been investigated and a new method for the determination of yDNA was established by RLS technique. The resonance light-scattering(RLS) of fuchsin basic is greatly enhanced by yeast deoxyribonucleic acid(yDNA)in the pH range of 6.75～7.25 in the buffer of Tris-HCl. The enhanced intensity of RLS at 594nm is proportional to the concentration of yDNA in the range of 200～1600μg/L with a correlation coefficient of 0.9997, The linear equation of yDNA isΔI =-21.99+236.5C(C:μg/mL).The detection limit is 26.5μg/L.This method is simple, rapid and has been applied to the determination of DNA in synthetic samples with satisfactory results.In the third chapter, the resonance light scattering(RLS) spectra of Azure Ⅰ and yeast deoxyribonucleic acid(yDNA) has been studied. The RLS of Azure Ⅰ was greatly enhanced by yeast deoxyribo- nucleic acid in pH range of 9.5～10.5 in the buffer of Tris-HCl. There are resonance-light-scattering peaks at 299nm, 355nm, 400nm, 570nm, and 630nm. The enhanced intensity of RLS at 355nm was proportional to the concentration of deoxyribonucleic acid. So a method of RLS for the determination of deoxyribonucleic acid was established. The linear equation of yDNA isΔI= -96.62+606.6C(C:μg/mL). The linear range of the calibration graph is 200～600μg/L. The detection limit is 11.2μg/L. This method is simple, rapid and has been applied to the determination of DNA in mixed samples with satisfactory results.In the fourth chapter, a new method for determination of nucleic acid has been established. In hexamethylene tetramine (HMTA) buffer (pH6.75), alizarin red S (ARS) and yeast deoxyribonucleic acids (DNA) react with cetyltrimethylammonium bromide (CTMAB) and form large particles, which results in significant enhancement of RLS intensities. The spectral characteristics of RLS, the effective factors and optimum conditions, mechanistic of the reaction have been investigated. Under the optimum conditions the enhanced RLS intensity was proportional to the concentration of DNA in the range 25 to 800μg/mL for yeast DNA(yDNA). The linear equation isΔI= 57.632+412.76C(C:μg/mL), The correlation coefficient is 0.9991, the detection limits is 11.3μg/L.This method is simple, rapid and has been applied satisfactorily to determination of yDNA in synthetic samples.