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Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis.

Author(s): Adamowicz MS, Stasulli DM, Sobestanovich EM, Bille TW

Publication: PLoS One, 2014, Vol. 9, Page e116351

PubMed ID: 25549111 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storage of swabs at 4°C and extraction conditions (incubation time and temperature, with or without agitation, additional incubation steps) on the recovery of DNA from swabs of buccal cell suspensions and swabs of whole blood specimens. Sterile cotton-tipped applicator swabs (20 total) were used to vigorously rub the inner cheek and gum areas of healthy volunteers. Four swabs were cut away from the wooden sticks, placed together in a 2 mL plastic flip-top tube containing Tris buffer, shaken for 10 minutes at 900 rpm to release epithelial cells, and then centrifuged in a DNA IQ Spin Basket to collect any remaining cells. The cell suspensions were separated into 50 µL aliquots and frozen at -20°C until used. Whole blood was collected by an unspecified method, separated into 50 µL aliquots, and frozen until used. Buccal cell suspensions (20 µL) and blood specimens (10 µL) were deposited on swabs in triplicate for each time point and extraction condition and then dried overnight. Swabs used for storage experiments were stored for one week, one month, three months, or six months at 4°C. Swabs were incubated in extraction buffer at 56°C or 65°C for 1, 3, 18, or 24 h with or without agitation. DNA was extracted from the swabs using the QIAamp DNA Investigator kit following manufacturer’s instructions or modified with an additional ‘‘re-suspension’’ step in which the swabs were removed from the extraction buffer, placed in a DNA IQ Spin Basket which was fitted back into the extraction tube, centrifuged for one minute at 13,200 rpm, and then returned to the extraction buffer. This process was repeated every 20 minutes with clean spin baskets for the duration of the incubation (one or three hours). DNA was quantified by real-time qRT-PCR and amplified using the AmpFlSTR Identifiler kit for fragment analysis by capillary electrophoresis.

   Summary of Findings:

   The average DNA yield from 20 µL of the buccal cell suspension was 1.24 ± 0.15 ng/mL versus 0.54 ± 0.06 ng/mL DNA for the same volume of the suspension dried on a cotton swab (P=0.002), indicating that more than 50% of the buccal cell DNA was not recovered from the swabs using the standard extraction protocol. The average DNA yield from 10 µL of blood was 4.24 ± 0.21 ng/mL compared to 0.64 ± 0.33 ng/mL from an equal volume of blood extracted from a cotton swab (P=0.001) which is a loss of over 80%. For each incubation duration, the average recovery of DNA from buccal cells was higher in specimens that were shaken during incubation than those that were not, but the difference was significant only in those incubated for 24 h (0.29 ± 0.04 ng/mL versus 0.11 ± 0.02 ng/mL, P=0.021). The average DNA yield decreased with incubation time (0.54 ± 0.06 ng/mL at 1 h to 0.29 ± 0.04 ng/mL at 24 h, P=0.021) when shaken, but a non-significant trend toward increase in average yield over time was observed when not shaken. In contrast, the yields decreased significantly between specimens incubated for 18 h versus 24 h (P=0.008). Similarly, the average yield of DNA from blood also increased non-significantly when the specimens were shaken.

   DNA yield increased in buccal cell specimens with increasing incubation duration up to 18 h at either temperature with and without shaking (P=0.007 and P=0.001, respectively) with the highest average yield occurring in specimens that were incubated for 18 hours at 65°C with shaking (0.65 ± 0.01 ng/mL). Similar results were observed for blood specimens with the highest average yield found in specimens incubated for three hours at 65°C with shaking (1.47 ± 0.02 ng/mL). Overall, DNA yields were significantly lower for buccal cell and blood specimens incubated for 24 h compared to the other time points (P<0.001 for all). DNA yields from buccal cell and blood specimens increased non-significantly when additional resuspension steps were included in the extraction of specimens incubated for 1 or 3 h of incubation at either temperature (56°C and 65°C) with and without shaking. Analysis by capillary electrophoresis demonstrated no contamination for any of the specimens under any conditions but DNA degradation was observed in when buccal cell and blood specimens were incubated for 24 h, regardless of shaking or temperature. Further, DNA recovery was not affected by storage of buccal cell or blood specimens on swabs for up to six months.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - Buccal

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Capillary electrophoresis-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1 week 1 month 3 months 6 months 0 h
   Analyte Extraction and Purification	Temperature of heat-induced retrieval	56 °C 65 °C
   Analyte Extraction and Purification	Incubation duration/condition	With agitation Without agitation 1 h 3 h 18 h 24 h Additional ‘‘re-suspension’’ step performed every 20 minutes for 1 h Additional ‘‘re-suspension’’ step performed every 20 minutes for 3 h

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