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Forensic Science Communications January 2007 – Volume 9 – Number 1 
Technical Note

A New Approach for the Extraction of DNA from Postage Stamps

Christiane Lauk
Chief Executive Officer
Dr. Lauk and Dr. Breitling Company
Wildberg, Germany

Jörg Schaaf
Executive Chief Officer
Dr. Lauk and Dr. Breitling Company
Wildberg, Germany

Abstract | Introduction | Materials and Methods | Results and Discussion | Conclusion | Acknowledgments | References

Abstract

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.

Introduction

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

Recovery efficiency

The recovery efficiency of three methods of DNA extraction as shown in the table below was determined by applying known amounts of purified DNA.

Table 1: DNA Test Kits Used

Types of Kits Spacing Material Manufacturer
QIAamp DNA Mini Kit Qiagen, Hilden, Germany
Genomic DNA Isolation Kit for Tissue and Cells 7 mm nexttec, Leverkusen, Germany
Genomic DNA Isolation Kit for Tissue and Cells 14 mm nexttec, Leverkusen, Germany

One sample of 8-µg genomic DNA (1 µg/µL) from the K562 cell line (ATCC, LGC Promochem GmbH, Welsel, Germany) was incubated in 200 µL of QIAamp buffer ATL with 20 µL of proteinase K (20 mg/mL) of the QIAamp DNA Mini Kit (Qiagen), and two samples of the same amount of DNA were incubated in 100 µL of nexttec lysis buffer overnight at 56 °C.

One sample lysed with nexttec buffer was placed on a prepared nexttec clean column with 7 mm of spacing material, and the second was placed on a nexttec clean column with 14 mm of spacing material. The spacing material is the sorbent involved in binding macromolecules such as proteins. The sample columns were incubated for 3 minutes at room temperature. The columns were then centrifuged at 700 g for 1 minute. The flow-through (almost 100 µL) contained the DNA.

Two hundred µL of QIAamp buffer AL was added to the Qiagen sample. The sample was incubated at 70 °C for 10 minutes, and 210 µL of absolute alcohol (99.8%, Roth, Karlsruhe, Germany) was added. This mixture was transferred to a QIAamp spin column and centrifuged at 6,000 g for 1 minute. The column was washed with 500 µL of high-salt buffer AW1 and AW2 containing ethanol at 6,000 g centrifugation for 1 minute. The column was centrifuged at 16,000 g for 3 minutes to dry the membrane completely. The DNA was eluted in 100 µL of elution buffer (AE buffer, Qiagen, preheated to 70 °C) by centrifugation of the column at 16,000 g for 1 minute.

The DNA from these three extractions had to be precipitated because of the nexttec buffer, which affects spectrophotometric quantifications.

One hundred µL of the DNA solutions prepared by nexttec or Qiagen and a separate control DNA solution (80 ng/µL K562, ATCC, LGC Promochem GmbH, Germany) were precipitated by 1.75 µL of glycogen (carrier for precipitation of nucleic acids, Roche), 11.25 µL of sodium acetate (3 M, pH 6, Roth) and 282.5 µL of absolute ethanol (99.86%, Roth). The solution for the blank value was treated the same way: 1.75 µL of glycogen, 11.25 µL of sodium acetate (3 M, pH 6), and 281.25 µL of absolute ethanol (99.86%) were added to 100 µL of TE buffer.

All precipitates were washed once with 200 µL of ethanol (70%) and dissolved in 400 µL of TE buffer. For quantitating the amount of DNA, spectrophotometric readings were taken at wavelengths of 260 and 280 nm.

Extractions from Stamps

DNA was extracted from nine stamps using a Qiagen QIAamp DNA Mini Kit and a Genomic DNA Isolation Kit for Tissue and Cells supplied by nexttec. Postage stamps (55 cents, Germany, bird series, 2004) always licked once by the same person were used as starting material for all experiments. The stamps were stuck to a white paper (80 g/m2) and stored approximately 24 hours at room temperature. No attempt was made to separate the stamps from the paper. Nine 20- by 5-mm sample areas of the stamps were cut into small pieces. The DNA was extracted in three different ways. Six samples involved incubation in 80 µL of nexttec lysis buffer, and three samples involved incubation in 180 µL of QIAamp buffer ATL with 20 µL of proteinase K (20 mg/mL) of the silica-based system. All samples were incubated overnight at 56 °C.

Three samples of the nexttec lysates were placed on prepared nexttec clean columns with 7 mm of spacing material, and the other three nexttec lysates were placed on prepared nexttec clean columns with 14 mm of spacing material. Both methods involved incubation for 3 minutes at room temperature. The columns were centrifuged at 700 g for 1 minute. The flow-through (almost 80 µL) contained the purified DNA.

For each of the three Qiagen samples, the lysate (200 µL) was transferred to a QIAshredder column (Qiagen) and centrifuged at 13,500 g for 5 minutes. Two hundred µL of QIAamp buffer AL was added to the flow-through. These samples were incubated at 70 °C for 10 minutes, and 210 µL of ethanol (99.8%) was added. Each of these mixtures was transferred to a QIAamp spin column and centrifuged at 6,000 g for 1 minute. The columns were washed twice with 500 µL of high-salt AW1 and AW2 buffer containing ethanol at 6,000 g centrifugation for 1 minute. The columns were centrifuged at 16,000 g for 3 minutes to dry the membrane completely. The DNA was eluted with 80 µL of AE buffer (preheated to 70 °C) by centrifugation at 16,000 g for 1 minute.

The DNA extracts were amplified using the AmpFℓSTR SEfiler PCR Amplification Kit (Applied Biosystems, Foster City, California). All amplifications were carried out in an Eppendorf Mastercycler gradient (Eppendorf, Hamburg, Germany).

The amplifications were performed in a 25-µL final reaction volume containing 10 µL of AmpFℓSTR SEfiler Reaction Mix Set (Applied Biosystems), 1 µL of AmpliTaq Gold DNA Polymerase (5U, Applied Biosystems), 5 µL of AmpFℓSTR SEfiler Primer Set (Applied Biosystems), 4 µL of DNA extract, and 5 µL of H2O bidest. The amplification conditions were 95 °C for 11 minutes; followed by 28 cycles of 94 °C for 1 minute, 59 °C for 1 minute, and 72 °C for 1 minute; and a final step of 60 °C for 45 minutes.

Amplification reaction mixtures (1 µL) were combined with 10 µL of Hi-Di Formamide (Applied Biosystems) and the internal lane standard GeneScan-500 LIZ (0.5 µL, Applied Biosystems). The capillary electrophoresis was carried out using an ABI PRISM 3100 Genetic Analyzer and ABI PRISM 3100 Data Collection Software 3.0 (Applied Biosystems). Fragment sizes were determined using ABI PRISM GeneScan Analysis Software Version 3.7. Allele designations were assigned using the allelic ladders of the Genotyper Software Version 3.6 (Applied Biosystems). An allele was scored when its peak height was greater than 20 percent of the peak height of the most prominent allele at a given locus.

Results and Discussion

Recovery efficiency

After precipitation, the DNA (8 µg) extracted by nexttec or Qiagen and the control DNA solution were dissolved in 400 µL of TE buffer, and concentrations were determined by readings at wavelengths of 260 and 280 nm (Table 2).

Table 2: Spectrophotometric Determination of the Amount of DNA Extracted via the Various Methods

  OD*260 nm OD280 nm Yield (ng/µL) Standard
Deviation
(OD260 nm)
Control DNA Solution
(20 ng/µL)
0.038 0.021 18.5 0.00829
Qiagen Method 0.013 0.007 6.5 0.00519
nexttec Method (7 mm) 0.024 0.013 12 0.00463
nexttec Method (14 mm) 0.024 0.013 12 0.00450
*Optical density

For the control DNA (20 ng/µL), 18.5 ng/µL was the determined concentration. The efficiency of recovery after precipitation was 92 percent.

Using the Qiagen method and precipitation, only about 32.5 percent of the applied DNA was recovered. Using the nexttec methods and precipitation, 60 percent of the known DNA concentration was detected. There were no significant differences between the nexttec clean columns with fill heights of 7 mm and 14 mm.

Extractions from Stamps

After precipitation, the DNA extracted by nexttec or Qiagen was determined by readings at wavelengths of 260 and 280 nm. Using the nexttec clean column with a fill height of 7 mm, 27.7 ng DNA with standard deviation of 6.68 per stamp piece was determined. Approximately the same amount of DNA (26.8 ng 6.34) could be detected by using the nexttec clean column with a fill height of 14 mm. In comparison, after extraction by the silica-based system, 16.7 ng DNA with standard deviation of 8.17 was detected.

However, in forensic casework, the quality of DNA extract is also important for getting strong amplification. Different inhibitors could lead to inefficient amplification, resulting, for example, in false homozygosity.

Therefore, the nexttec method must be checked for its efficiency in retaining undesired substances. The inhibition of the amplification in the PCR is often characterized by weak or unbalanced bands of the simultaneously amplified DNA fragments.

In our routine work, the genomic DNA extraction protocol from stamps using the Qiagen method provides sufficient yield and purity of DNA, ensuring strong amplification and high sensitivity. In all samples analyzed for evaluation, allelic characterization was performed without any problems (Figure 1).

Figure 1 (PDF) | Figure 1 (HTML): Typical STR profile with allele designation and peak height values for genomic DNA extracted from stamps using the Qiagen method. The horizontal axis represents the values in base pairs (bp), and the vertical axis represents the values in relative fluorescent units (RFU). The 6-FAM channel (blue, top) shows the loci D3S1358, vWA, D16S539, and D2S1338; the VIC channel (green, second) contains the loci Amelogenin, D8S1179, and SE33; the NED channel (yellow, third) shows the loci D19S433, THO1, and FGA; the PET channel (red, bottom) contains the loci D21S11 and D18S51. The peaks of about 103 bp in blue, yellow, and red channels are bleed-throughs. This figure is in Adobe Portable Document Format. To view it, you will need to have the Adobe Acrobat Reader plug-in installed on your computer. The Reader can be downloaded at no cost from http://www.adobe.com/products/acrobat/readermain.html.

The data evaluated with nexttec clean columns were identical to the control samples previously genotyped using the silica-based DNA extraction method.

However, in all of the samples, the nexttec clean column method with a fill height of 7 mm produced a higher yield of amplification products than the silica method, probably because of the higher DNA amount in the amplification setup (Figure 2).

Figure 2 (PDF) | Figure 2 (HTML): Typical STR profile with allele designation and peak height values for genomic DNA extracted from stamps using the nexttec column with a fill height of 7 mm. This figure is in Adobe Portable Document Format.

All samples analyzed with DNA isolated by the nexttec clean column method with a fill height of 14 mm resulted in a higher yield of amplification products than the nexttec method with a fill height of 7 mm. Of the three presented methods, the data in Figure 3 demonstrate the strongest amplification and the highest sensitivity.

Figure 3 (PDF) | Figure 3 (HTML): Typical STR profile with allele designation and peak height values for genomic DNA extracted from stamps using the nexttec column with a fill height of 14 mm. This figure is in Adobe Portable Document Format.

Figure 4 summarizes the proportion of successfully typed DNA profiles from the three different DNA extraction methods. The multiplex PCR system showed that the DNA extracted by the silica-based method and that extracted by nexttec columns resulted in the same allelic characterization.

The nexttec extraction method with a fill height of 14 mm led to the strongest amplification. The increase in the peak heights compared with the Qiagen method is probably due to the higher amount of DNA in the amplification setup. The increase in the peak heights of the nexttec column with a fill height of 14 mm compared with the nexttec column with a fill height of 7 mm might be caused by a higher quality of the DNA. The nexttec extraction method with a fill height of 14 mm may be more efficient in removing inhibitors without reducing the amount of recovered DNA.

Figure 4 (PDF) | Figure 4 (HTML): The proportion of typed allelic peaks from the three different DNA extraction methods for 12 loci of the AmpFℓSTR SEfiler PCR Amplification Kit, arranged according to increasing fragment lengths. The graph shows the average values for the heterozygous loci and the half of the values of the homozygous loci. The average relative fluorescence unit (RFU) values from the three different DNA extractions and the standard deviations were also plotted.

Conclusion

We present the use of the nexttec column system as a fast and highly sensitive method for DNA extraction from stamps in forensic casework.

Thus, a true one-step purification for nucleic acids is realized. Instead of the usual binding, washing, and final elution steps, DNA on nexttec clean columns is purified during a single passage through the column. The result is a tremendous saving of time and waste during nucleic acid purification.

We suggest that the protocol presented here might be successfully used in practical casework for the isolation of DNA from stamps and other forensic materials, although many different types of biological material might be more complex than our controlled laboratory situation. The simplicity of the nexttec single-step preparative method generates reliable and reproducible results with very high sensitivity. This method promises strong advantages compared with silica-based protocols.

Acknowledgments

The authors thank Jens Sperveslage, nexttec GmbH, Leverkusen, Germany, for providing the different nexttec columns. Special thanks to Dr. Wolfgang Staiber, Institute for Genetics, University of Hohenheim, Stuttgart, Germany, for his technical support regarding spectrophotometric determination of DNA concentrations.

References

Balogh, M. K., Burger, J., Bender, K., Schneider, P. M., and Alt, K. W. STR genotyping and mtDNA sequencing of latent fingerprint on paper, Forensic Science International (2003) 137:188–195.

Fridez, F. and Coquoz, R. PCR DNA typing of stamps: Evaluation of the DNA extraction, Forensic Science International (1996) 78:103–110.

Gill, P., Whitaker, J., Flaxman, C., Brown, N., and Buckleton, J. An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Science International (2000) 112:17–40.

Hopkins, B., Williams, N. J., Webb, M. B. T., Debenham P. G., and Jeffreys, A. J. The use of minisatellite variant repeat-polymerase chain reaction (MVR-PCR) to determine the source of saliva on a used postage stamp, Journal of Forensic Sciences (1994) 39:526–531.

Sinclair, K. and McKechnie, V. M. DNA extraction from stamps and envelope flaps using QIAamp and QIAshredder, Journal of Forensic Sciences (2000) 45:229–230.

Walsh, D. J., Corey, A. C., Cotton, R. W., Forman, L., Herrin, G. L. Jr., Word, C. J., and Gamer, D. D. Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva, Journal of Forensic Sciences (1992) 37:387–395.

Walsh, P. S., Metzger D. A., and Higuchi, R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, Biotechniques (1991) 10(4):506–513.