Navigation Links
Oragene is compatible with TaqMan SNP genotyping

J. Chartier and H.C. Birnboim
DNA Genotek Inc., Ottawa, ON, Canada

DNA collected with Oragene works well with TaqMan SNP Genotyping Assays. SNPs in the thymidylate synthetase and apolipoprotein E genes were reliably detected.


Introduction
Single-nucleotide polymorphisms (SNPs) are highly abundant, and are estimated to occur at 1 out of every 1,000 bases in the human genome (ref. 1). In addition to diagnostic applications, SNPs are useful as markers in population genetics and evolutionary studies (ref. 2). The TaqMan 5 nuclease assay is a widely-used SNP genotyping technology from Applied Biosystems. The purpose of this study was to evaluate the compatibility of Oragene DNA Self-Collection Kits with TaqMan SNP Genotyping Assays.


Materials and Methods
DNA collection
Saliva was collected from 25 donors using Oragene kits. DNA was purified from 200 L aliquots of Oragene/saliva samples using the ethanol precipitation protocol supplied with the kits. Purified DNA was redissolved in 200 L of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA was quantified using a fluorimeter and SYBR Green I dye (Molecular Probes) according to the F/D Protocol (ref. 3).

SNP genotyping
Validated TaqMan SNP Genotyping Assays were obtained from Applied Biosystems. The assays are described in Table 1. The probes were labeled with FAM or VIC dye at the 5 end and a minor-groove binder and non-fluorescent quencher at the 3 end. The reaction components for the allelic discrimination reactions were set up according to Table 2. Although it is recommended to use 1 to 20 ng of DNA per reaction (ref. 4), 1 L of DNA was added to each 25 L PCR reaction without adjusting the concentrations. SNP genotyping reactions were performed on a Rotor-Gene 3000 real-time quantitative thermal cycler (Corbett Research) using the cycling conditions in Table 3.


Results
DNA quantification
The purifi ed DNA from 25 samples had a median concentration of 19.1 ng/L and ranged from 1.2 to 53.4 ng/L. Consequently, a number of the genotyping reactions used more than the 20 ng of DNA template recommended by the manufacturer.

Allelic discrimination plots
Figure 1 shows the allelic discrimination plot for the thymidylate synthetase assay and Figure 2 shows the plot for the apolipoprotein E assay. The plots are presented as the signal (average fluorescence between cycles 41 and 45) minus the background (average fluorescence between cycles 21 and 25).


Discussion and Conclusions
TaqMan assays are widely used for SNP genotyping. As with other molecular genetic techniques, the quality and purity of DNA is important for reliable results. Our findings indicate that DNA samples collected and purified using Oragene kits are suitable for allelic discrimination using TaqMan probes. Clear discrimination between different genotypes is evident in Figures 1 and 2.

Although it is recommended to use 1 to 20 ng of DNA per reaction, this study used DNA amounts ranging from 1.2 to 53.4 ng and all 25 samples gave clear, interpretable results. It was possible to perform the reactions without first determining the amount of DNA. Thus, the step of adjusting the DNA concentrations co uld be avoided. In summary, DNA collected with Oragene is suitable for SNP genotyping with TaqMan assays.


References
1. Sachidanandam, R. et al. (2001). A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature. 409, 928933.

2. Schork, N. J., Cardon, L. R. & Xu, X. (1998). The future of genetic epidemiology. Trends Genet. 14, 266272.

3. Technical Bulletin: DNA Quantification Using the Fluorescence/DNase (F/D) Assay, v1.2. DNA Genotek. Nov 29, 2004.

4. TaqMan SNP Genotyping Assays Protocol. Part Number 4332856 Rev. B. Applied Biosystems. June, 2004.


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. DNA Stability with Oragene
2. Oragene and Whole Genome Amplification with GenomiPhi
3. DNA Yield with Oragene
4. Oragene and DNA sequencing with ABI PRISM
5. The iCycler iQ Detection System for TaqMan Assays, Rev B
6. Amplification Efficiency of TaqMan Gene Expression Assays
7. Microfluidic analysis of multiplex PCR products for the genotyping of Helicobacter pylori
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/11/2017)... ... October 11, 2017 , ... At its national board ... Suneel I. Sheikh, the co-founder, CEO and chief research scientist of Minnesota-based Advanced ... for membership in ARCS Alumni Hall of Fame . ASTER Labs is ...
(Date:10/11/2017)... 2017  VMS BioMarketing, a leading provider of patient support ... Nurse Educator (CNE) network, which will launch this week. The ... health care professionals to enhance the patient care experience by ... other health care professionals to help women who have been ... ...
(Date:10/10/2017)... SAN DIEGO, CALIF. (PRWEB) , ... October 10, 2017 , ... ... website as part of its corporate rebranding initiative announced today. The bold new ... broaden its reach, as the company moves into a significant growth period. , It ...
(Date:10/10/2017)... Calif. , Oct. 10, 2017 SomaGenics ... from the NIH to develop RealSeq®-SC (Single Cell), expected ... for profiling small RNAs (including microRNAs) from single cells ... Program highlights the need to accelerate development of approaches ... "New techniques for measuring levels ...
Breaking Biology Technology:
(Date:5/6/2017)... 5, 2017 RAM Group , ... new breakthrough in biometric authentication based on a ... properties to perform biometric authentication. These new sensors are ... created by Ram Group and its partners. This sensor ... supply chains and security. Ram Group is a ...
(Date:4/18/2017)... 18, 2017  Socionext Inc., a global expert in SoC-based imaging ... server, the M820, which features the company,s hybrid codec technology. A ... Tera Probe, Inc., will be showcased during the upcoming Medtec Japan ... at the Las Vegas Convention Center April ... Click here for ...
(Date:4/13/2017)... , April 13, 2017 According to a ... Identity Authentication, Identity Analytics, Identity Administration, and Authorization), Service, Authentication Type, Deployment ... the IAM Market is expected to grow from USD 14.30 Billion in ... Rate (CAGR) of 17.3%. ... MarketsandMarkets Logo ...
Breaking Biology News(10 mins):