forensicGEM™ Cigarette produces high-quality, automatable DNA samples from cigarette butts

DNA Extraction Using forensicGEM™ Cigarette

forensicGEM™ Cigarette is formulated specifically to overcome the inhibition seen in downstream PCR based assays in certain cigarette types. forensicGEM™ Cigarette reliably and robustly delivers PCR ready DNA in less than 25 minutes from a wide range of cigarette brands.

forensicGEM™ is validated and quality tested for forensic applications.
The simplicity of the method maximizes yields of DNA from trace samples.
The kit’s optimized methods and formulations provide DNA in less than 25 minutes.
All buffers and reagents are compatible with common profiling kits.
The methods are closed-tube and hands-off.

With the forensicGEM™ Cigarette kit, technicians can produce high quality DNA from cigarette samples in a limited or high throughput fashion during a largely closed-tube procedure that protects the integrity of the DNA from the cigarette crime samples

See application note for more details on forensicGEM™ Cigarette.

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Workflow

Extraction Method

The following procedure outlines general guidelines. Different sample types will require slight method variations.

Sample Preparation
All manipulations should be performed in a clean room or a PCR hood. Use only certified DNA-free tubes and reagents and wash surfaces likely to come into contact with the samples in 0.05% hypochlorite bleach. Rinse thoroughly with DNA-free water.

Wash the buccal swab in the minimum amount of DNA-free water to cover the swab. Typically, a cotton swab requires 400-500 µl. Use a rolling action against the tube sides and squeeze the swab against the tube to remove as much of the liquid as possible.

1. Remove a 1 cm strip of paper from the cigarette butt and cut into quarters. These can be processed separately as replicates.
2. Cut each sample of paper into smaller pieces (3 mm squares) and place in a thin-walled PCR tube or a 96-well PCR tray.

Extraction Method

1. Add:

89 µl DNA-free water
10 µl of 10x buffer ORANGE
1 µl forensicGEM™

2. Incubate at 75°C for 15 minutes.
3. Incubate at 95°C for 5 minutes. A thermal cycler can be used for this step

Aspirate the extract away from the paper immediately and transfer to a fresh tube.

The sample is now ready for quantification and analysis.

Technical Tips

The forensicGEM™ Cigarette procedure can be automated using most liquid handling robots.
forensicGEM™ Cigarette is a preparative method for DNA extraction - it is not a purification protocol. Its purpose is to lyse cells and to strip the DNA of nucleoproteins. The formulation creates DNA that is sufficiently inhibitor-free to be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.
As would be expected for cigarette butts, yields will vary significantly from sample to sample. The simple and efficient method can yield enough DNA for forensic profiling from most freshly-smoked butts. However, in some cases, a concentration step may be necessary to obtain a profile.
For accurate yield assessment, a qPCR is required. There is insufficient DNA for using estimation methods with fluorescent chelating dyes.
As with any preparative method for nucleic acid extraction, for best results prepare and manage samples at 4ºC, or on ice, before and after extraction.
When storing the sample after extraction, aspirate the supernatant from the precipitated residue and store separately.
forensicGEM™ is stable for 30 days at 4º C; for longer-term storage forensicGEM™ should be stored at -20ºC.
For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.

Typical Results

Gel of GAPDH PCR products

Figure 1. GAPDH PCR products obtained from cigarette butt extractions performed using forensicGEM™ Cigarette. A composite gel has been constructed showing amplified products from a PCR using 5 µl of an extraction derived from one 0.5 x 1 cm piece of cigarette butt paper from a range of cigarette types. Four replicates were amplified for each cigarette type (2 samples from 2 different cigarette butts for each sample type). The amplified product is approximately 850 bp.

DNA Profile

DNA Profile

Figure 2 Typical AmpFlSTR® Identifiler™ PCR Amplification Kit (Applied Biosystems) profiles of two of the cigarette types. 2 µl of extract was used in a standard Identifiler PCR. Top: Rothmans King Size Filter Tip. Bottom: Winfield Extra Mild.

Allele Scores

The proportion of scored alleles using accepted forensic criteria are shown in table 1. Twenty one brands of cigarette were tested.

Cigarette Type	Cigarette Type
Pall Mall Filter	7/15
Pall Mall Menthol	15/15
Marlboro	14.5/15
Marlboro Lites	15/15
Marlboro Lites Menthol	10/15
B&H Extra Mild	15/15
B&H Special Filter	15/15
Winfield Super Mild	15/15
Winfield Extra Mild	15/15
Winfield Menthol	15/15
Winfield Filter	15/15
Camel Filters Generous Flavor	15/15
Rothmans King Size Filter Tipped	15/15
Dunhill Filter	15/15
Lucky Strike Original Red	15/15
State Express 555 Filter Kings	15/15
Peter Stuyvesant Filter King Size	15/15
Holiday Special Filter	14.5/15
Horizon King Size	15/15
Kent USA Charcoal Filter	13.5/15
Mild Seven Charcoal Filter	15/15

Table 1: AmpFlSTR® Identifiler™ (Applied Biosystems) results for cigarette butt extractions.