Chapter 3
Purification of Nucleic Acids From Eukaryotic Cells
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
lntroduction
Essential to most molecular biology techniques is the analysis of DNA and
RNA. In general, it is difficult to prepare both RNA and high-molecularweight DNA from the same extract, because optimum conditions are mutually exclusive.
Ultimately, recognition of the optimum conditions for a particular purification procedure is dependent on the amount of starting material, the
nature of the nucleic acids to be isolated and on their subsequent use.
This chapter starts with the description of the most common purification
method for nucleic acids, the extraction with phenol/chloroform. Subsequently, modifications to improve yield, purity and concentration are discussed. The second part covers methods for isolation of genomic DNA and
RNA from complex mixtures of molecules such as celllysates.
Subprotocol 1
Extraction of Nucleic Acids
Principles of phenol extraction. The standard way to prepare nucleic acids
for use in enzymatic reactions is to extract first the DNA or RNA sample
with phenol:chloroform:isoamyl alcohol (PCA) {25:24:1). Phenolleads to
denaturation of proteins, which will subsequently accumulate at the interphase, whereas the hydrophilic nucleic acids will remain in the aqueous
(phenol containing) phase. The mixture of different organic solvents makes
deproteinization more efficient. Furthermore, phenol alone does not comHans Gerd Nothwang, Correspondence to Friedhelm Hildebrandt, Universitäts-Kinderklinik, Mathildenstrasse 1, Freiburg, 79106, Germany (phone +49-761-270-4301;
fax +49-761-270-4533; e-mail hildebra@kkl200.ukl.uni-freiburg.de)
Purification of Nucleic Acids From Eukaryotic Cells
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
lntroduction
Essential to most molecular biology techniques is the analysis of DNA and
RNA. In general, it is difficult to prepare both RNA and high-molecularweight DNA from the same extract, because optimum conditions are mutually exclusive.
Ultimately, recognition of the optimum conditions for a particular purification procedure is dependent on the amount of starting material, the
nature of the nucleic acids to be isolated and on their subsequent use.
This chapter starts with the description of the most common purification
method for nucleic acids, the extraction with phenol/chloroform. Subsequently, modifications to improve yield, purity and concentration are discussed. The second part covers methods for isolation of genomic DNA and
RNA from complex mixtures of molecules such as celllysates.
Subprotocol 1
Extraction of Nucleic Acids
Principles of phenol extraction. The standard way to prepare nucleic acids
for use in enzymatic reactions is to extract first the DNA or RNA sample
with phenol:chloroform:isoamyl alcohol (PCA) {25:24:1). Phenolleads to
denaturation of proteins, which will subsequently accumulate at the interphase, whereas the hydrophilic nucleic acids will remain in the aqueous
(phenol containing) phase. The mixture of different organic solvents makes
deproteinization more efficient. Furthermore, phenol alone does not comHans Gerd Nothwang, Correspondence to Friedhelm Hildebrandt, Universitäts-Kinderklinik, Mathildenstrasse 1, Freiburg, 79106, Germany (phone +49-761-270-4301;
fax +49-761-270-4533; e-mail hildebra@kkl200.ukl.uni-freiburg.de)