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J Immunol Methods. 1997 Oct 13;208(1):91-101.

Saliva: a convenient source of DNA for analysis of bi-allelic polymorphisms of Fc gamma receptor IIA (CD32) and Fc gamma receptor IIIB (CD16). van Schie RC, Wilson ME. Source Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo 14214-3092, USA. van_schie@sdm.buffalo.edu Abstract Genetic polymorphisms of low-affinity IgG Fc receptors (Fc gamma R) have been found to influence binding of human IgG subclass antibodies, and may influence susceptibility to certain types of infectious and autoimmune diseases. Phenotypic and/or genotypic analyses of Fc gamma R polymorphisms have traditionally employed peripheral venous blood as a source of leukocytes or genomic DNA, respectively. The present study was undertaken to determine whether human salivary DNA is a suitable alternative to DNA extracted from blood for genetic analysis of FC gamma R allelic polymorphisms. Genomic DNA was extracted from whole saliva of 69 healthy adult volunteers using a commercial DNA purification kit. The average quantity of genomic DNA isolated per ml of saliva was 19.2 +/- 14.1 micrograms. To assess intrasubject variation in yield of salivary DNA, ten saliva samples were collected from a single donor over a 3-month period. The average yield of DNA recovered from these samples was 25.2 +/- 13.7 micrograms. Volumes of saliva as small as 100 microliters, as well as saliva samples stored at -70 degrees C for prolonged periods (up to 6 years), provided DNA in amounts sufficient for PCR-based genetic analysis. Two comparative PCR assays were performed using DNA extracted from both peripheral blood and saliva from a number of individuals. The assays were able to detect a single nucleotide substitution (G-->A) in the Fc gamma RIIA gene, as well as two codominant alleles encoding the NA polymorphism in Fc gamma RIIIB, respectively. Furthermore, Fc gamma RIIA and Fc gamma RIIIB genotype results were confirmed by quantitative flow cytometry using specific monoclonal antibodies. Complete concordance was achieved between the typing results of our salivary DNA, and blood DNA-based assays.

Therefore, saliva appears to be an excellent source of DNA for studies of Fc gamma RIIA and Fc gamma RIIIB polymorphisms.

NATIONAL INSTITUTES OF HEALTH

National Institute of Dental Research

FOR RELEASE Wednesday, October 22, 1997

Wayne Little (301) 496-4261

In a study supported by the National Institute of Dental Research, scientists were able to use DNA from saliva to identify individuals who may be at increased risk of certain infectious and autoimmune diseases. The study focused on two genes that play a role in removing bacteria from the body. Dr. Rob van Schie and Mark Wilson at the State University of New York at Buffalo were able to detect person-to-person differences of as little as a single nucleotide, or structural unit, in the genes. This seemingly minor difference in gene structure is known to affect the proper functioning of the immune system. Diseases potentially linked to these genes include childhood respiratory infections, lupus, and juvenile periodontal disease (LJP), a particularly aggressive form of gum disease that strikes young adults. This study is not the first to use saliva as a source of DNA. Forensic scientists can retrieve enough saliva from a postage stamp to identify the person that licked the stamp. Saliva has also been used to test for fragile X syndrome, a rare genetic disorder that causes mental retardation in children who carry the gene. The DNA in saliva comes from many sources, including blood, tissue cells, and nonhuman DNA from bacteria and food particles. Findings: Evidence has shown that adults may also have a genetic marker for periodontal disease and therefore may be candidates for saliva screening. Other notable possibilities would be the genes for Alzheimer's disease, cystic fibrosis, or breast cancer. As the structure of more genes becomes known, it may be possible to test for many genetic disorders from a single sample of saliva.

Cancer Epidemiol Biomarkers Prev. 2002 Oct;11(10 Pt 1):1130-3.

Buccal cell DNA yield, quality, and collection costs: comparison of methods for large-scale studies.
King IB, Satia-Abouta J, Thornquist MD, Bigler J, Patterson RE, Kristal AR, Shattuck AL, Potter JD, White E.

Source Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, 98109-1024, USA.

Cancer Epidemiol Biomarkers Prev 2002 Nov;11(11):1511. Abouta Jessie Satia [corrected to Satia-Abouta Jessie].

Abstract
There is considerable interest in non-invasive and cost-effective methods for obtaining DNA in large-scale studies. In this randomized crossover study of 22 participants, we compared the DNA yield, quality, and associated costs of buccal cell DNA collected using cytobrushes (three brushes per collection) and swish (i.e., mouthwash) in selfadministered procedures. There was a no statistically significant higher yield from the mouthwash compared with cytobrush collections (15.8 microg versus 12.0 microg, respectively; P = 0.53). PCR reactions that required short (0.3 kb) or intermediate (1.1 kb) DNA fragments were 100% successful for DNA from brush and mouthwash, whereas PCRs for reactions that required long fragments (7.8 kb) failed for all of the participants from cytobrush DNA and were 81% successful for DNA from the mouthwash source. The brush collections provided sufficient DNA for an estimated 150225 PCR reactions requiring short and intermediate DNA fragments. The estimated per person costs for buccal brush DNA collections in large studies were less then half (8.50 dollars) those for the mouthwash method (18 dollars). In addition, we tested whether cytobrush instructions to rub cheeks before collection or collect cells only in the morning increased DNA yield and whether repeat brushings of the same cheek reduced DNA yield. These variations resulted in no significant differences in DNA yields. We conclude that the collection of DNA with cytobrushes using simple instructions is cost effective in largescale studies, and yields sufficient quantity and quality of DNA for genotyping

Clin Chim Acta. 2004 May;343(1-2):191-4.

Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva.
Ng DP, Koh D, Choo SG, Ng V, Fu Q.

Source
Department of Community, Occupational and Family Medicine (MD3), Faculty of Medicine, National University of Singapore, 16, Medical Drive, Singapore 117597, Singapore.

Abstract
BACKGROUND:

Saliva is a potentially useful source of genomic DNA for genetic studies since it can be collected in a painless and non-invasive manner. We sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR).
METHODS:

Saliva was collected from healthy volunteers and 2-ml aliquots subjected to different storage conditions: S1--washing of saliva using phosphate-buffered saline (PBS) and extraction of DNA on the same day of collection; S2--washing and centrifugation to yield a pellet, which was stored at-70 degrees C for 1 week prior to DNA extraction; S3--storage of whole saliva at 4 degrees C for 7 days, followed by washing and extraction of DNA; S4-storage at 4 degrees C for 7 days, followed by washing and pellet formation. The pellet was stored at -70 degrees C for 1 month before extraction of the DNA; S5--storage at-70 degrees C for 1 month, followed by washing and extraction of DNA. DNA yield and purity was determined by spectrophotometry at 260 and 280 nm. Twenty nanograms of genomic DNA was used for the polymerase chain reaction, and the resulting PCR band was captured by digital photography and quantified.
RESULTS:

The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction, based on OD(260/280) ratios, was good and comparable. PCR resulted in the presence of a single specific product of the correct size from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions (P<0.05). Compared to S1 samples, PCR bands from conditions S2 and S3 were not as strong, while those amplified from S4 and S5 samples were the weakest. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1-S3. Qualitatively similar results were obtained when the PCR experiment was repeated.

CONCLUSIONS:

Saliva can act as a useful source of genomic DNA, even when stored under less than optimal conditions.

DNA extraction from human saliva deposited on skin and its use in forensic identification procedures:
Braz. oral res. vol.19 no.3 So Paulo July/Sept. 2005
Author(s): Evelyn Anzai Kanto; Mario Hirouki Hirata; Rosario Dominguez Crespo

Hirata; Fabio Daumas Nunes; Rodolfo Francisco Haltenhoff Melani; Rogerio Nogueira Oliveira Saliva is usually deposited on skin found in bite marks, many homicides, assault and other criminal cases. In the present study, saliva obtained from volunteers was deposited on skin and recovered for DNA extraction and typing in order to evaluate its usefulness for practical case investigation and discuss the contribution of forensic dentistry to saliva DNA typing. Twenty saliva samples were collected from different donors and used as suspect samples. Five of these samples were randomly selected and deposited (250 ul) on arm skin. Saliva was collected from skin using the double swab technique. DNA from saliva and skin deposited saliva samples was extracted by the phenol chloroform method. DNA samples were amplified by PCR for DNA typing using a set of 15 STRs. The recovery of DNA from saliva deposited on the skin was 14 to 10 times lower than the DNA quantity from saliva samples. DNA typing was demonstrated in 4 to 5 deposited saliva samples, the likelihood ratios estimated for these samples based on data of the Brazilian population were 1:11, 1:500, 1:159.140 and 1:153.700.123. Results indicated that standardized procedures used for DNA collection and extraction from skin-deposited saliva can be used as a method to recover salivary DNA in criminal cases. However, it is important to observe that DNA recovery in forensic samples can be difficult.

Findings: This study suggested that the analysis of saliva deposited on skin be incorporated into a criminal investigation since it may have great discriminatory power.

Am J Hum Biol. 2007 May-Jun;19(3):319-26.

New saliva DNA collection method compared to buccal cell collection techniques for epidemiological studies.
Rogers NL, Cole SA, Lan HC, Crossa A, Demerath EW.

Source
Lifespan Health Research Center, Department of Community Health, Wright State University School of Medicine, Dayton, Ohio 45420, USA. nikki.rogers@wright.edu Abstract Epidemiological studies may require noninvasive methods for off-site DNA collection. We compared the DNA yield and quality obtained using a whole-saliva collection device (Oragene DNA collection kit) to those from three established noninvasive methods (cytobrush, foam swab, and oral rinse). Each method was tested on 17 adult volunteers from our center, using a random crossover collection design and analyzed using repeated-measures statistics. DNA yield and quality were assessed via gel electrophoresis, spectophotometry, and polymerase chain reaction (PCR) amplification rate. The whole-saliva method provided a significantly greater DNA yield (mean +/- SD = 154.9 +/- 103.05 microg, median = 181.88) than the other methods (oral rinse = 54.74 +/- 41.72 microg, 36.56; swab = 11.44 +/- 7.39 microg, 10.72; cytobrush = 12.66 +/- 6.19, 13.22 microg) (all pairwise P < 0.05). Oral-rinse and whole-saliva samples provided the best DNA quality, whereas cytobrush and swab samples provided poorer quality DNA, as shown by lower OD(260)/OD(280) and OD(260)/OD(230) ratios. We conclude that both a 10-ml oral-rinse sample and 2-ml whole-saliva sample provide sufficient DNA quantity and better quality DNA for genetic epidemiological studies than do the commonly used buccal swab and brush techniques

An Application of Salivary DNA in Twin Research of Chinese Children


Twin Research and Human Genetics Print ISSN: 1832-4274 Volume: 11 | Issue: 5 Cover date: October 2008 Page(s): 546-551

Abstract

Since saliva collection is noninvasive, painless and inexpensive, it may become an alternative to obtain genomic DNA, which is critical to evaluate zygosity and the role of genetic factors in twin research. This study provided a rough description of salivary DNA in Chinese twin children, and presented the DNA yield and quality extracted from saliva in a largescale children sample, which supplied an example for saliva sample using in genetic epidemiology. Three milliliters of saliva was collected from 356 twin children aged 6 to 15, and DNA was extracted by a com mercial DNA isolation kit. The DNA yield and purity was determined by spectrophotometry at 260nm and 280nm. The zygosity determination of the same-sex twins and the assay of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism indicated the quality of salivary DNA. The amount of extracted DNA from three milliliters of saliva was about 34.91g (2.20 ~ 122.04g), average OD260/280 values was 1.84. Saliva DNA is a reliable sample for the determination of twins' zygosity. We conclude that saliva may be a feasible and reliable source of DNA for genetic epidemiology studies, especially for twin research.

Array-Based Whole-Genome Survey of Dog Saliva DNA Yields High Quality SNP Data:
Citation: PLoS ONE 5(5): e10809. doi:10.1371/journal.pone.0010809 February 19, 2010; Accepted: March 14, 2010; Published: May 25, 2010
Author(s): Jennifer S. Yokoyama, Carolyn A. Erdman, Steven P. Hamilton

Genome-wide association scans for genetic loci underlying both Mendelian and complex traits are increasingly common in canine genetics research. However, the demand for high-

quality DNA for use on such platforms creates challenges for traditional blood sample ascertainment. Though the use of saliva as a means of collecting DNA is common in human studies, alternate means of DNA collection for canine research have instead been limited to buccal swabs, from which dog DNA is of insufficient quality and yield for use on most highthroughput array-based systems. We thus investigated an animal-based saliva collection method for ease of use and quality of DNA obtained and tested the performance of saliva-extracted canine DNA on genome-wide genotyping arrays. Extractions yielded high concentrations (~125 ng/ul) of high-quality DNA that performed equally well as blood-extracted DNA on the Illumina Infinium canine genotyping platform, with average call rates >99%. Concordance rates between genotype calls of saliva- versus blood-extracted DNA samples from the same individual were also >99%. Additionally, in silico calling of copy number variants was successfully performed and verified by PCR. Conclusions/Significance The use of saliva-obtained samples for genome-wide association studies in canines, highlighting an alternative means of collecting samples in a convenient and non-invasive manner.

Genomic DNA Purification from Cigarette Butts and Buccal Swabs Using the DNA IQ System
Author(s): Caroline Turner , Rita Weispfenning , Eric Vincent , Kim berly Huston and Joseph Bessetti , Promega Corporation They described experiments examining the ability of the DNA IQ System to isolate DNA from cigarette butts and cigarette butt paper in the presence and absence of a proteinase K digestion.

In addition they described a protocol for DNA isolation from buccal swabs in the absence of a centrifugation step. DNA isolation from buccal swabs using the DNA IQ System involves a 30 minute incubation followed by centrifugation. By increasing the incubation time in Incubation Buffer without proteinase K to 120 minutes and including a vortex mixing before and after incubation, the DNA IQ System was capable of isolating sufficient DNA for amplification without centrifugation These methods simplify the pre-treatment steps for these samples and demonstrated that centrifugation of the lysate following initial incubation may not be required for successful DNA isolation. Elimination of centrifugation steps make complete processing of these sample types on automated workstations possible. Findings: DNA isolated from cigarette paper was more successful in amplification than DNA from the cigarette filter. The partial profile from sample 2A suggests that DNA isolation and amplification from cigarette filter is possible with further optimization.