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Biotechnology
  Year: 2008 | Volume: 7 | Issue: 3 | Page No.: 514-522
DOI: 10.3923/biotech.2008.514.522
Detection of DNA Damage, Molecular Apoptosis and Production of Home-Made Ladder by Using Simple Techniques
S.E. Hassab El-Nabi and Y.A. Elhassaneen

Abstract:
Simple, sensitive and rapid techniques were modified for detection of total genomic damage of DNA (TGD-DNA), Electrophoretic pattern of RNA and molecular apoptosis. The techniques were applied in both cell culture and solid tissues. In cell culture, the cells were lysated directly in the gel wells and mixed with loading buffer. In solid tissues, 5-10 mg were squeezed and lysated in ependorf tubes, then 1 mg of lysated tissues was loaded in the gel wells and mixed with loading buffer. This technique elaborated the DNA damage as smear or apoptotic bands. Detection of molecular apoptosis by gel electrophoresis was modified in our laboratory, where, salt out extraction method of DNA was applied using small amount of tissues (5-10 mg), squeezed by blue tips and lysated quickly by lysing buffer. Mixture of loading mix was developed which has a great effect on the stability and resolution of apoptotic bands. The developed technique is very simple, fast and economic to prepare DNA apoptotic ladder from liver, spleen and human leukocytes. Induction of apoptosis was done chemically by methotraxate in liver, spleen and thymus of rats, or naturally by postmortem changes in liver of rat and storage blood of human. Some factors were studied for stability and resolution of apoptotic ladder such concentrations of SDS in lysing buffer, effects of temperatures, glycerol and our loading mix. Concentration of SDS has no effect on the intensity of apoptotic bands in extracted DNA, while in cell or tissue lysate, the apoptotic bands appeared sharp at low concentrations of SDS (0.5-1%). After preparation of DNA and addition of TE buffer the best temperature that keeps on the stability of DNA was 37 °C. Our loading mix and glycerol protects apoptotic bands from degradation at room temperature or -20 °C. The stability of home made apoptotic ladder was stable for 6 month or more at room temperature.
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How to cite this article:

S.E. Hassab El-Nabi and Y.A. Elhassaneen, 2008. Detection of DNA Damage, Molecular Apoptosis and Production of Home-Made Ladder by Using Simple Techniques. Biotechnology, 7: 514-522.

DOI: 10.3923/biotech.2008.514.522

URL: https://scialert.net/abstract/?doi=biotech.2008.514.522

 
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