Academic journal article
By Lauk, Christiane; Schaaf, Jorg
Forensic Science Communications , Vol. 9, No. 1
The use of nexttec clean columns compared with a silica-based system for extracting DNA from postage stamps is reported. Pieces of stamps can be added directly to the extraction solution. Undesired substances such as proteins are retained on the column matrix while the DNA passes through the column in a single centrifugation step. In an attempt to accelerate the isolation of DNA from stamps to make this procedure suitable for routine laboratory work, we optimized a preparative technique for DNA extraction with this new surface modification, which leads to a high binding capacity for proteins and an almost inert behavior with respect to nucleic acids. The quality of the DNA obtained from stamps with nexttec columns was compared with that from a silica-based system by short tandem repeat (STR) genotyping using a multiplexed polymerase chain reaction (PCR) system. The DNA extracted using the nexttec method is suitable for analysis by the PCR-based typing system. The result is a tremendous saving of time and waste during nucleic acid purification without any loss of quantity or quality in DNA yield of amplification products.
Human identification systems based on polymerase chain reaction (PCR) have become the method of choice for forensic DNA casework. These PCR systems have made possible the analysis of samples containing low quantities of DNA (Balogh et al. 2003; Gill et al. 2000; Hopkins et al. 1994). The development of multiplex short tandem repeat (STR) primer systems allows simultaneous amplification and separation of up to 16 STR loci in a single reaction and provides the possibility of obtaining a DNA profile from almost any source of biological material. However, forensic genetic laboratories have to analyze different types of biological material (saliva, blood, sperm, or epidermal cells) that are present on a wide range of supports (e.g., biological tissues, clothes, buccal swabs, stamps, bottles, or cigarette butts) (Fridez et al. 1996; Sinclair et al. 2000; D. J. Walsh et al. 1992; P. S. Walsh et al. 1991).
To date, many DNA extraction protocols have been described. Some methods seem to be efficient in removing inhibitors but may reduce the amount of recovered DNA. Others that recover substantial amounts of DNA may be relatively inefficient in removing inhibitors.
Thus, in forensic casework, the isolation of DNA represents the crucial step in obtaining a complete DNA profile. However, genomic DNA extraction is still a rate-limiting and time-consuming step in forensic science.
Here we present the use of a column system as a fast and sensitive method for DNA extraction from postage stamps. Nexttec's system (nexttec is a registered trademark of nexttec GmbH, Leverkusen, Germany) for purification of nucleic acids is based on new, specially designed chromatographic supports. These supports are composed of silica particles with an optimized porous structure that is coated with a thin polymer film. The polymer selectively binds different substances with high affinity. Therefore, the final sorbent has three characteristics: it has a high binding capacity for many biomacromolecules (especially proteins), it is nearly inert with respect to nucleic acids, and it has a high retention capability for low-molecular-weight compounds.
A lysate applied to this column quickly enters the sorbent layer because of the hydrophilic surface. During a short incubation period, small molecules (such as metabolites and salts) enter the pores, and macromolecules are adsorbed by the polymer. After low-speed centrifugation, purified DNA is contained in the flow-through.
Our main focus was to increase the efficiency and sensitivity of the DNA extraction compared with a silica column system and make it suitable for routine laboratory work.
Materials and Methods
The recovery efficiency of three methods of DNA extraction as shown in the table below was determined by applying known amounts of purified DNA. …