Optimization of Recovery and PCR Amplification of DNA from Stamps and Envelopes
Charlotte J. Word and Scott Gregory
Cellmark Diagnostics; 20271 Goldenrod Lane; Germantown, MD 20876
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Data from validation studies, casework samples and anecdotal information from many laboratories indicate that licked stamps and envelope flaps are often problematic for the recovery of DNA in a sufficient amount for DNA analysis. In addition, when DNA is recovered from licked stamps or envelopes, it often cannot be amplified using the polymerase chain reaction (PCR) with the commercially available PCR kits used in forensic DNA analysis. A study was undertaken to examine four commonly used DNA extraction protocols to determine which procedure provides: (1) the optimal recovery of DNA from stamps and envelopes, and, (2) DNA-containing extracts that can be readily amplified with several PCR kits used in forensic DNA testing.
These studies were done with stamps containing a known amount of saliva as well as with stamps and envelopes that had been received through the mail and stored for several months or years. The four extraction procedures that were used are: (1) Chelex®1 extraction where the stamp or envelope flap was left in the Chelex® throughout the procedure, (2) Chelex® extraction where the stamp or envelope flap was left in for the initial incubation in water and then removed for the rest of the procedure, (3) organic extraction2 where the stamp or envelope flap was incubated overnight in Gill buffer and then removed prior to phenol/chloroform extraction, and, (4) steaming apart the stamp or envelope flap, swabbing of the DNA, and organic extraction [as in (3)] of the swab. The amount of DNA recovered was determined using the QuantiBlot® Human DNA Quantitation Kit. Attempts were made to amplify the DNA without any additional treatment using the AmpliType® PM + DQA1 PCR Amplification and Typing Kit, the AmpliFLP
Ô D1S80 PCR Amplification Kit, the CTT GenePrintÔ STR Multiplex System with the GenePrintÔ Sex Determination System (Amelogenin), and the AmpFISTRÔ Blue PCR Amplification Kit. DNA was recovered from stamps and envelope flaps using all four extraction procedures, although the amount of DNA recovered using the steaming/swabbing technique was less than that recovered using the other three procedures. Inhibition of the PCR was observed in many samples of DNA isolated using either of the Chelex® extraction procedures and in most of the samples isolated using the organic extraction procedure. In contrast, the majority of the samples isolated using the swabbing/steaming technique amplified readily.Studies were undertaken to overcome the inhibition of PCR amplification seen with many of the DNA extracts. The approach taken was to concentrate and wash extracts using Microcons® 100 filters, or to add various reagents to the amplification reaction, such as bovine serum albumin (BSA), spermidine, or additional Taq polymerase. The addition of BSA was the only treatment that routinely gave rise to amplification products suitable for analysis and interpretation. To attempt to identify the cause of the inhibition, studies were done to examine the degree of degradation of the DNA, uninhibited and inhibited DNA extracts were mixed and amplified, and stamp components were tested for inhibition of the PCR. The results of these studies and those listed above will be presented.
REFERENCES
1 Walsh, P.S., Metzger, D.A. and Higuchi, R. 1991, BioTechniques 10:506-513.
2 Walsh, D.J., et al., 1992. J.For. Sci 37:387-395.
Funded by Technical Support Working Group.
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