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.


Cigarette Identifiler 

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.


 

Cigarette Type

Alleles

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. The proportion of scored alleles using accepted forensic criteria. Twenty one brands of cigarette were tested.

 

forensicGEM Cigarette is a modified kit where the buffer has been formulated to release DNA from epithelial cells but not release the hydrophobic inhibitors associated with smoked cigarette butts.  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.
  • All reagents in the forensicGEM Cigarette kit are QC tested for the presence of gDNA and mtDNA.
  • The simplicity of the method maximizes yields of DNA from trace samples.
  • The kit’s optimized methods and formulations provide DNA in around 10 minutes.
  • All buffers and reagents are compatible with common profiling kits.
  • The methods are closed-tube and hands-off thereby protecting the sample integrity.

Work area preparation

All manipulations should be performed in a clean room and/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. Tweezers, scalpels and scissors should either be disposable certified DNA free or be washed in 1% bleach, rinsed in DNA-free water and baked in and oven at 250°C for 30 minutes.


Method

Wash the buccal swab in the minimum amount of DNA-free water to cover the swab. Typically, a cotton swab requires 300-400 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.

Cigarette method

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 (2 mm squares) and place in a thin-walled PCR tube or a 96-well PCR tray.

3. To each tube add:

  • 44 µl water
  • 5 µl of 10x buffer ORANGE
  • 1 µl forensicGEM

2. Incubate at 75°C for 5 minutes.

3. Incubate at 95°C for 2 minutes.  A thermal cycler can be used for these steps

4. Pipette the DNA into a new tube.  This step should be done immediately.

 The sample is now ready for analysis.


Technical Tips

  • Leaving the paper in the extracted DNA will eventually lead to leaching of inhibitors.
  • forensicGEM™ 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.
  • 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.
  • The forensicGEM™ Cigarette procedure can be automated using most liquid handling robots.

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