JURNAL KEDOKTERAN YARSI 30.
: 49-57 .
p-ISSN : 0854-1159.
e-ISSN: 2460-9382 TaqMan Genotyping Assay Method for Single Nucleotide Polymorphisms (SNP.
detection in promoter region of N-Acetyltransferase 2 (NAT.
Kinasih Prayuni1.
Rika Yuliwulandari1,2 1Genomic Research Centre.
Universitas YARSI Jakarta 2Department of Pharmacology.
Faculty of Medicine.
YARSI University.
Jakarta Corresponding author: rika.
yuliwulandari@yarsi.
KEYWORDS
AT-DILI.
NAT2 gene promoter.
TaqMan
genotyping assay
ABSTRACT
The N-acetyltransferase 2 (NAT.
polymorphism in coding region has been studies intensively.
However, there are limited studies for promoter region of NAT2 gene.
Several reported study showed that the promoter region polymorphism of NAT2 gene is genotyped by PCR-sequencing In this paper, we describe TaqMan based assays for the NAT2 polymorphism genotyping in promoter region with following SNP:
rs4646243 [T>C], rs4646244 [T>A], rs4646267 [A>G], rs4345600 [A>G], and rs4646246 [A>G].
Our result showed a good separation cluster, trailing cluster and some mix cluster.
TaqMan genotyping assay has shown a sensitivity and specificity to detect polymorphism in NAT2 promoter region.
INTRODUCTION
Human Nacetyltransferase (NAT.
responsible for the acetylation of numerous arylamine and hydrazine xenobiotics including drugs and carcinogens which consequently relates to drug effects or toxicity level (Walraven et al.
The NAT2 enzyme is encoded by NAT2 gene located in human chromosome 8 (Fuselli et al.
Polymorphism at the N-acetylation level is an example of a pharmacogenetic trait that implies on inter-individual differences in response to xenobiotics (Hein et al.
Previous showed that NAT2 polymorphism in coding region can be categorized into rapid, intermediate and slow groups based on the acetylation rate.
Several studies have shown that the slowacetylator NAT2 phenotype is strongly associated with a higher risk of AT-DILI consistently across different ethnic groups (Ohno et al.
Fountain et al.
Khalili et al.
Yuliwulandari et al.
A previous study on NAT2 in individuals within the Javanese and Sundanese ethnic groups of Indonesia reveals that slow-acetylator NAT2 variants are frequently observed (Yuliwulandari et al.
Another study in Indonesia population also reveals that slow acetylator allele has a strong association with AT-DILI (Yuliwulandari et al.
However, most studies focusing on NAT2 have JURNAL KEDOKTERAN YARSI 30.
: 49-57 .
p-ISSN : 0854-1159.
e-ISSN: 2460-9382 been based on the genotyping of nucleotide variants in the coding region and ignore the potential effects of the role of promoter polymorphisms in NAT2 Promoter region is the regulator region for gene expression and initiate transcription of a particular gene (Li & Zhang 2014.
De Vooght et al.
However, the best of our knowledge, the genotyping method for NAT2 promoter region was using sequencing Therefore, we tried to find another approach to genotype single SNP with affordable price and good The TaqMan discrimination assay is a very popular and widely used medium-throughput genotyping format: the assays use two short minor groove binder (MGB) hydrolysis probes, each annealing to one allelic variant of the genotyped single nucleotide polymorphism (SNP) amplified within a short PCR product (Malkki & Petersdorf 2012.
Shen et al.
The TaqMan discrimination is supported by most of real-time PCR platforms, however their proprietary software solutions utilize mainly the endpoint analyses, largely fluorescence data collected over the whole course of a PCR run (Konopac et In the present paper, we describe TaqMan based assays for the NAT2 polymorphism in promoter region, which illustrate the general principle of TaqMan based genotyping.
METHOD
Sample Collection Samples was obtained from Sample bank of YARSI Research Institute.
Universitas YARSI.
All of protocol in this study was approved by Universitas YARSI Ethics Committee.
DNA Extraction Genomic DNA .
DNA) was extracted using the QIAamp Blood mini (Qiage.
manufacturerAos instructions.
Isolated gDNA was analyzed by 0.
8% agarose gel electrophoresis to evaluate DNA DNA quantity was assessed by using the Infinite 200 Pro NanoQuant (TECAN).
SNP genotyping in NAT2 promoter SNP genotyping was performed with TaqManA SNP genotyping assays (Applied Biosystems In.
to detect the specific NAT2 promoter region that contain the following SNPs: rs4646243 [T>C], rs4646244 [T>A], rs4646267 [A>G], rs4345600 [A>G], and rs4646246 [A>G] (Yuliwulandari et al.
SNP genotyping was performed on Real-time PCR thermal cycler LightCycler 480 (Roch.
in 384 well plates pre-loaded with 20x TaqMan genotyping Assays in 5 AAL reaction TaqMan (Life Technologie.
, nuclease free water (Promeg.
, and 10 ng DNA template.
PCR program the following cycling conditions at 95AC for 10 min, followed by 40 cycles at 92 AC for 15 s and at 60 AC for 1 min.
The amplification analysis discrimination with automated allele calling settings for the Light Cycler Software 4.
0 (Roch.
RESULT
An allelic discrimination cluster plot is shown in Fig.
1 to Fig.
5 from SNP amplification in promoter region.
The cluster plot diagram was analyzed using EndPoint Genotyping Analysis JURNAL KEDOKTERAN YARSI 30.
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p-ISSN : 0854-1159.
e-ISSN: 2460-9382 on Light Cycler Software 4.
0 software (Roch.
A fluorescent signal from only the VIC dye indicates homozygosity for Allele A.
the presence of only FAM dye fluorescence indicates homozygosity for Allele B, and the presence of both fluorescent signals indicates Allele A and Allele B (A/B) heterozygosity.
Ideally, a cluster plot shows one, two, or three clusters and the no template controls (NTC.
The points in each cluster are grouped closely together and each cluster is located well away from the other clusters.
Based on TaqMan Assay manual (Applied Biosystem 2.
, allele A will be located on lower right corner.
Allele B will be located on upper left corner and allele A/B will be located approximately on midway between allele A and allele B No templete control (NTC) always located on the bottom left corner, as well as no-amplification Meanwhile undetermined samples or cluster will be located anywhere outside the region of fix A cluster plot from rs4646243 (Fig.
, rs4646244 (Fig.
, rs4646267 (Fig.
, and rs4646246 (Fig.
showed a good separation.
Good separation means the NTC are distant from any clusters and the separation of the three clusters is clear.
However, the cluster in rs4646243 (Fig.
and rs4646267 (Fig.
was shown little bit trailing.
A cluster plot from rs4345600 (Fig.
are not well cluster and major cluster.
Those cluster also shown a trailing cluster as well as The undetermined sample also shown in rs4646243 cluster (Fig.
Figure 1.
TaqMan assay genotype result for SNP rs4646243 JURNAL KEDOKTERAN YARSI 30.
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TaqMan assay genotype result for SNP rs4646244 Figure 3.
TaqMan assay genotype result for SNP rs4646267 JURNAL KEDOKTERAN YARSI 30.
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p-ISSN : 0854-1159.
e-ISSN: 2460-9382 Figure 4.
TaqMan assay genotype result for SNP rs4345600 Figure 5.
TaqMan assay genotype result for SNP rs4646246 JURNAL KEDOKTERAN YARSI 30.
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p-ISSN : 0854-1159.
e-ISSN: 2460-9382
DISCUSSION
Study of NAT2 polymorphism in promoter region is still rarely done.
Most of study are still focusing in coding region polymorphism.
Whereas, promoter region has an important role as a regulatory region for gene expression and initiate transcription of a NAT2 gene.
Previous reported study regarding promoter region has been conducted by Yuliwulandari et al (Yuliwulandari et al.
and Kang et al (Kang et al.
in normal population with PCR-direct sequencing Therefore in this study we used TaqMan assay genotyping approach in AT-DILI cases and control to genotyped the polymorphism.
To the best of our knowledge, it is the first study in Indonesia.
Among the most robust assays for SNP genotyping.
TaqMan SNP Genotyping Assays are provides a more economic and easy to handle procedure to efficiently generate thousands of genotypes for genetic association studies (Schleinitz et al.
Each assay allows genotyping of individuals for a single nucleotide polymorphism (SNP) (Applied Biosystem 2.
The TaqMan SNP Genotyping Assay involves the use of forward and reverse primers to amplify the polymorphic sequence of interest and two dyelabeled probes for allele-specific At the 5Ao site, one probe is labeled with VIC dye, which detects the AuAllele 1Ay sequence, while the other one is labeled with 6FAM dye, which detects the AuAllele 2Ay sequence.
VIC
and 6FAM are also referred to as reporter dyes.
Based on the principle of fluorescence resonance energy transfer, at the 3Ao site the probes are labeled with a nonfluorescent quencher (NFQ), which absorbs the energy of the reporter dyes as long as both are bound to the probe and therefore are in spatial Further.
TaqMan probes incorporate a minor groove binder (MGB) at the 3Ao end.
The MGB molecule .
tripeptide [DPI.
) binds to the minor groove of the DNA helix and improves hybridization-based stabilizing the probe/ template complex (Schleinitz et al.
In general, our result showed a good separation cluster between allele A homozygous, allele B homozygoes and allele A/B heterozygous for rs4646243 (Fig.
, rs4646244 (Fig.
rs4646267 (Fig.
, and rs4646246 (Fig.
except for rs4345600 (Fig.
However, some of good separation cluster shown a trailing clusters (Fig.
1 & Fig .
Trailing cluster describes the variation of fluorescence signals among a given genotype that has the appearance of being spread across an imaginary line that extends from the no template control (NTC).
This phenomenon is often caused by variability in concentration of individual genomic DNA (Malkki & Petersdorf 2012.
Applied Biosystem 2.
The quality of genomic DNA concentration is critical TaqMan Assay Furthermore, quantitative nature of the assay, every effort should be made to ensure a uniform concentration of DNA across DNA concentration should be determined using a fluorescent method as this is more accurate than that determined by measuring absorbance at 260 nm (Hui et al.
Fluorescence assays are less prone to interference JURNAL KEDOKTERAN YARSI 30.
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Fluorophore excitation and subsequent emission produce higher sensitivity, and many fluorophores provide specificity by preferentially binding to double stranded DNA (Gallagher & Desjardins An undetermined samples as shown in rs4646243 (Fig.
and rs4345600 (Fig.
are considers as Generally, it caused by a variable DNA quality or pipetting issues (Applied Biosystem 2.
Because of low confidence in assigning the correct genotype, this small number of samples should not be used in data undetermined (Hui et al.
PCR
temperature is one of best approach to prevent an outliers samples and make a better separation clusters (Malkki & Petersdorf 2.
Besides samples, there are no-amplification sample showed in rs4345600 clusters (Fig.
There are some possibilities that cause this problem.
The presence of polymerase inhibitors can decrease PCR efficiency.
Potential PCR inhibitors can originate from the tissue source of the DNA sample or from the purification method.
Degraded DNA also can affect PCR efficiency due to the presence of fewer template copies, which will affect the success of TaqMan SNP Genotyping Assay.
Degradation can result from using very old DNA samples freezing and thawing DNA samples repeatedly and leaving DNA samples at room temperature (Applied Biosystem 2.
CONCLUSION
TaqMan genotyping assay method has shown a sensitivity and specificity to detect polymorphism in NAT2 promoter region.
It is still techniques used to genotype single SNPs or small numbers of SNPs.
It is also economical and time-saving.
An equal concentration of genomic DNA, modification of PCR annealing temperature, and prevent a PCR amplification has to be tried in further research to prevent a trailing cluster, an outliers cluster and no amplification ACKNOWLEDGEMENT This project was funded by a grant from the Indonesian Directorate General of Higher Education (DIKTI) of the Ministry of Higher Education.
Research and Technology of the Republic of Indonesia and Internal Grant of YARSI University.
We are also thankful to YARSI Foundation and Pasar Rebo General Hospital for their support in this project.
REFERENCES