forensicGEM™ Saliva is specifically developed for extracting DNA from buccal swabs

DNA Extraction Using forensicGEM™ Saliva

forensicGEM™ Saliva is a kit specifically developed for extracting DNA from buccal swabs.

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 30 minutes.
All buffers and reagents are PCR compatible with common profiling kits.
The methods are closed-tube and hands-off.

With the forensicGEM™ Saliva kit, technicians can produce high quality DNA samples with high-throughput automation during a closed-tube procedure that protects the integrity of the DNA from the saliva samples.

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Workflow

Extraction Method

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. In a thin-walled PCR tube add (agitate suspension prior to adding the following):

89 µl of the swab eluate
10 µl of 10x buffer BLUE
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

The sample is now ready for quantification and analysis.

Technical Tips

The forensicGEM™ Saliva procedure can be automated using most liquid handling robots.
forensicGEM™ Saliva is a preparative method for DNA extraction - it is not a purification protocol. Its purpose it to lyse cells and strip the DNA of nucleoproteins. The formulation creates DNA that can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.
There is no concentration step in the procedure and so the final concentration is dependent on: 1) The vigor with which the swab was taken. 2) The type of swab. 3) The volume of buffer used to wash the swab. We recommend using the smallest amount of buffer required when washing the swab in order to maximize the DNA concentration.
forensicGEM™ extracted DNA is largely single-stranded because of the heat step. If double-stranded DNA is required, the 95° step can be omitted and standard purification procedures can be used.
For accurate yield assessment, a qPCR is recommended. The DNA produced by forensicGEM™ is approximately 90% single-stranded. If standard fluorescent chelating dyes are to be used for quantification, this factor should be taken into consideration. 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.

Typical Results

DS Profile

Figure 1: Typical AmpFlSTR® Identifiler™ PCR Amplification Kit (Applied Biosystems) profile from a buccal swab of a male subject. 2 µl of extract was used in a standard Identifiler PCR.

Yield of DNA

Table 1
	Quantiblot®	Quantifiler®
TOTAL YIELD: ng per µl ¼ swab	175 ± 101.5	235 ± 88.9
CONC. in extract ng /µl	1.8 ± 1.02	2.4 ± 0.89

Table 1:The yields were calculated for forensicGEM™ using both a blot-based system, ABI Quantiblot® and a qPCR method, Quantifiler®. Sample size = ¼ of a swab. Final Volume forensicGEM™ extraction = 100 µl

On this basis a complete swab would be expected to yield ~ 940 ng of DNA.
Large variation in yields is common with buccal swabs; samples are expected to vary between 500 and 2000 ng. QIAGEN reports similar yields for the QIAamp® 96 DNA Swab BioRobot Procedure.

Quantifiler® Traces

Traces Graph

Figure 2: Blue: Examples of Quantifiler® traces from forensicGEM™ buccal swab extracts. Four extractions are shown. Red: Standards generated using the manufacturer's DNA

Yield of DNA

DNA Yield Graph

Figure 3: Yields from buccal swabs (four individuals) using forensicGEM™ and other extraction methods. Error bars are one standard deviation.