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:10/10/2017)... ... 10, 2017 , ... San Diego-based team building and cooking events company, Lajollacooks4u, ... The bold new look is part of a transformation to increase awareness, appeal ... growth period. , It will also expand its service offering from its signature gourmet ...
(Date:10/10/2017)... Oct. 10, 2017 SomaGenics announced the receipt ... to develop RealSeq®-SC (Single Cell), expected to be the ... RNAs (including microRNAs) from single cells using NGS methods. ... need to accelerate development of approaches to analyze the ... "New techniques for measuring levels of mRNAs in ...
(Date:10/9/2017)... , ... October 09, 2017 , ... At its national ... Christopher Stubbs, a professor in Harvard University’s Departments of Physics and Astronomy, has been ... a member of the winning team for the 2015 Breakthrough Prize in Fundamental physics ...
(Date:10/7/2017)... 6, 2017  The 2017 Nobel Prize in ... Jacques Dubochet, Joachim Frank and ... cryo-electron microscopy (cryo-EM) have helped to broaden ... biology community. The winners worked with systems manufactured ... produce highly resolved, three-dimensional images of protein structures ...
Breaking Biology Technology:
(Date:4/4/2017)... YORK , April 4, 2017   EyeLock ... today announced that the United States Patent and Trademark ... patent broadly covers the linking of an iris image ... same transaction) and represents the company,s 45 th ... latest patent is very timely given the multi-modal biometric ...
(Date:3/30/2017)... , March 30, 2017 Trends, opportunities ... (physiological and behavioral), by technology (fingerprint, AFIS, iris recognition, ... recognition, and others), by end use industry (government and ... immigration, financial and banking, and others), and by region ... , Asia Pacific , and ...
(Date:3/24/2017)... March 24, 2017 The Controller General of Immigration ... Abdulla Algeen have received the prestigious international IAIR Award for ... Continue Reading ... ... Deputy Controller Abdulla Algeen (small picture on the right) have received the ...
Breaking Biology News(10 mins):