The goal of this study was to determine the integrity of human genomic DNA isolated from saliva-soaked cotton spit wads that had been preserved at -20°C for 11 years. During the years 1996–2000, 783 spit wad samples were collected from an ADHD study population (Vermont Family Study). An available commercial kit, the QIAamp DNA Blood Midi Kit, was used to extract human genomic DNA from spit wads (Qiagen, Inc., Valencia, CA.) along with certain modifications.
The efficiency of a commercially available kit in extracting and purifying human genomic DNA from saliva drenched cotton spit wads are described in this technique study. It was discovered that the DNA extracted from spit wads was of sufficient amount and quality to be used in subsequent genetic research. The yield of genomic DNA from buccal cells extracted from spit wads was extremely varied, ranging from 1 g to 80 g. The range in DNA yield is similar to other research using buccal cytobrushes, which reported yields ranging from 0.5 gs to 12.66 gs. They generated a mean of 17.3 gs 11.9 gs of genetic material in our sample of 783 people.
They also present an estimate of DNA quality extracted from Vermont Family Study samples using an A260/A280 ratio to distinguish between DNA purity and protein contamination. DNA is typically regarded to be of good quality when the A260/A280 ratio is between 1.70 and 2.0. The purity of genomic DNA from saliva saturated cotton spit wads was more than satisfactory, as measured by spectrophotometer readings, with a mean A260/A280 ratio of 1.937 0.226 for the 783 samples. They imposed a 50 ng/l concentration cut-off for each sample prior to being kept at -20°C because the DNA was kept for future genetic studies. Microcon YM-100 centrifugal filter units were used to concentrate samples with concentrations below this threshold. Surprisingly, the purity of these samples rose after they were concentrated through the filter unit, as determined by an A260/A280 ratio. After the concentration of samples dropping below the 50 ng/l cutoffs, the adjusted mean was 1.965 0.124. During the concentration step, any pollutants or impurities are most likely eliminated, boosting the purity of the genomic DNA prep.
PCR success using primers specific for the 5HTTLPR polymorphism was also used to determine the quality of genomic DNA. The genomic DNA template for this PCR reaction had a 98.3 percent success rate (770/783). This showed the gel displaying the fragments detected from a PCR reaction using 5HTTLPR specific primers.
This blog outlines a unique, cost-effective, and time-efficient method for collecting genomic DNA from participants in genetic investigations. Using the commercially available QIAamp DNA Blood Midi Kit from Qiagen, Inc., researchers should be able to collect good quality and abundant genomic DNA from saliva drenched cotton spit wads using this procedure. Furthermore, DNA can be extracted in under 3 hours, and numerous samples can be processed at the same time, saving time and money.
The resulting DNA output, which ranged from about 1 g to a total of 80 g (mean 17.3 gs 11.9 gs), was more than adequate for genetic analysis. Human genomic DNA obtained from spit wads had A260/A280 ratios continuously within the generally acceptable range of 1.7–2.0, with the lowest purity being 1.70 and a mean value of 1.937 0.226 for the 783 samples. As demonstrated by the amplification of the serotonin-transporter-linked polymorphic region, 5 HTTLPR, the DNA was likewise acceptable for PCR procedures. The 5HTTLPR polymorphism in the promoter region of the serotonin transporter gene (HTT, SLC6A4, or SERT) consists of two alleles that have been extensively investigated. After PCR using primers, 770 of the 783 samples (98.3%) yielded fragments of the expected size.
It can be concluded that a DNA extraction kit can be used to obtain high quality human genomic DNA suitable for PCR and genotyping from 11-year-old saliva saturated cotton spit wads (Elhi et al., 2008).
Elhi, E. A., Nelson, T. J., Hudziak, J. J., & Davies, G. E. (2008). Using a commercially available DNA extraction kit to obtain high quality human genomic DNA suitable for PCR and genotyping from 11-year-old saliva saturated cotton spit wads. BMC Research Notes, (133). https://doi.org/10.1186/1756-0500-1-133