scholarly journals Inclusion of double helix structural oligonucleotide (STexS) results in an enhance of SNP specificity in PCR

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jae Jong Kim ◽  
Hyoung-Min Park ◽  
A. Young Kyoung ◽  
In Kyung Park ◽  
Si-Kyu Lim ◽  
...  
Keyword(s):  
Double Helix ◽  
Genetic Disorders ◽  
Egfr Mutations ◽  
Clinical Samples ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphisms ◽  
Technical Problems ◽  
Mismatched Dna ◽  
Efficient Detection ◽  
Mutation System

AbstractGenetic mutations such as single nucleotide polymorphisms (SNP) are known as one of the most common forms which related to various genetic disorders and cancers. Among of the methods developed for efficient detection of such SNP, polymerase chain reaction (PCR) methods are widely used worldwide for its cost and viable advantages. However, the technique to discriminate small amounts of SNP mixed in abundant normal DNA is incomplete due to intrinsic technical problems of PCR such as amplification occurring even in 3’mismatched cases because of high enzyme activity of DNA polymerases. To overcome the issue, specifically designed PCR platform, STexS (SNP typing with excellent specificity) using double stranded oligonucleotides was implemented as a means to emphasize the amplification of SNP templates by decreasing unwanted amplification of 3’mismatched DNA copies. In this study, the results indicate several EGFR mutations were easily detected specifically utilizing the STexS platform. Further trials show the novel method works effectively to discriminate mutations in not only general allele specific (AS)-PCRs, but also amplification refractory mutation system (ARMS)-PCR. The STexS platform will give aid in PCRs targeting potential SNPs or genetically mutated biomarkers in human clinical samples.

scholarly journals Inclusion of Double Helix Structural Oligonucleotide (STexS) Results in an Enhance of SNP Specificity in PCR

2021 ◽  
Author(s):  
Byoung Park ◽  
Jae Jong Kim ◽  
Hyoung-Min Park ◽  
AYoung Kyoung ◽  
In Kyung Park ◽  
...  
Keyword(s):  
Double Helix ◽  
Genetic Disorders ◽  
Egfr Mutations ◽  
Clinical Samples ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphisms ◽  
Technical Problems ◽  
Mismatched Dna ◽  
Efficient Detection ◽  
Mutation System

Abstract Genetic mutations such as single nucleotide polymorphisms (SNP) are known as one of the most common forms which related to various genetic disorders and cancers. Among of the methods developed for efficient detection of such SNP, polymerase chain reaction (PCR) methods are widely used worldwide for its cost and viable advantages. However, the technique to discriminate small amounts of SNP mixed in abundant normal DNA is incomplete due to intrinsic technical problems of PCR such as amplification occurring even in 3’mismatched cases because of high enzyme activity of DNA polymerases. To overcome the issue, specifically designed PCR platform, STexS (SNP typing with excellent specificity) using double stranded oligonucleotides was implemented as a means to emphasize the amplification of mutated SNP templates by decreasing unwanted amplification of 3’mismatched DNA copies. In this study, the results indicate several EGFR mutations were easily detected specifically utilizing the STexS platform. Further trials show the novel method works effectively to discriminate mutations in not only general allele specific (AS)-PCRs, but also amplification refractory mutation system (ARMS)-PCR. The STexS platform will give aid in PCRs targeting potential SNPs or genetically mutated biomarkers in human clinical samples.

scholarly journals Association of Common Variants in HNF1A Gene with Serum AFP Level in Healthy Chinese Individuals and HCC Patients

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Xue-jun Li ◽  
Dong-hua Shao ◽  
Mei-lin He ◽  
Guo-wei Liang
Keyword(s):  
Elevated Serum ◽  
Gene Promoter ◽  
Taqman Probe ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphisms ◽  
Genetic Determinants ◽  
Mutation System ◽  
Hereditary Persistence ◽  
Hnf1a Gene ◽  
Healthy Chinese

Although alpha-fetoprotein (AFP) is a widely used tumor marker in hepatocellular carcinoma (HCC), 40% of newly diagnosed patients do not have an elevated AFP level. Research has revealed that mutations in the HNF1A binding site of the AFP gene promoter cause significantly elevated serum AFP levels in patients with hereditary persistence of AFP. This study investigated the relationship between HNF1A genetic variants and serum AFP levels. We examined the association between the HNF1A-rs1169288 (A/C), rs2464196 (G/A), and rs1169310 (C/T) polymorphisms and AFP levels in a healthy Chinese population (n=1010) and HCC patients (n=185). Single nucleotide polymorphisms were genotyped by the amplification refractory mutation system combined with TaqMan probe in real-time PCR. The serum AFP concentrations were measured using the Architect i2000 immunochemistry analyzer. In healthy individuals, serum AFP levels were significantly lower with the rs2464196-AA and rs1169310-TT genotypes. Similar significant differences were observed in HCC patients. Moreover, in HCC patients, the distribution frequencies of rs2464196-AA+AG and rs1169310-TT+TC among those with AFP≤20 ng/ml or ≤400 ng/ml were significantly lower than those in patients with AFP>20 ng/ml or >400 ng/ml. Among all subjects, those carrying the HNF1A-rs2464196-A or rs1169310-T allele tended to have low levels of AFP. However, the HNF1A-rs1169288 polymorphism showed no significant association with the serum AFP level. These findings provide new insight into the genetic determinants of serum AFP level and can aid the differential diagnosis of HCC patients with low serum AFP.

scholarly journals Heparanase (HPSE) gene polymorphism (rs12503843) contributes as a risk factor for hepatocellular carcinoma (HCC): a pilot study among Egyptian patients

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Faten Saad ◽  
Mahmoud Gadallah ◽  
Ahmed Daif ◽  
Nahed Bedair ◽  
Moustafa A. Sakr
Keyword(s):  
Hepatocellular Carcinoma ◽  
Risk Factor ◽  
Pilot Study ◽  
Human Cancer ◽  
Rflp Analysis ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphisms ◽  
Allelic Distribution ◽  
Egyptian Patients ◽  
Mutation System

Abstract Background Heparanase activity was found to be included in human cancer development and growth. Heparanase (HPSE) gene single nucleotide polymorphisms (SNPs) have been found to be correlated with different human cancers. In the current study, we investigated whether HPSE SNPs were a hepatocellular carcinoma (HCC) risk factor by carrying out a comprehensive case-control pilot study. HPSE rs12331678 and rs12503843 were genotyped in 70 HCC-diagnosed patients and 30 healthy controls by modified amplification refractory mutation system (ARMS PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results HPSE rs12331678 distributions showed that there were no statistically significant differences between both cohorts either in genotypic or allelic distribution but there was a significant correlation between the rs12503843 (T allele) and the HCC risk in the whole samples (P = 0.042). No significant association was observed between the HPSE rs12331678 and rs12503843 gene polymorphisms and all clinicopathologic markers or with SNP stratification based on HCV carrier in HCC groups. Conclusion Our findings suggest for the first time the HPSE gene SNP characterization in HCC Egyptian patients, and our findings reveal there were associations between the HPSE rs12503843 (T allele) and the susceptibility to HCC.

Association between single nucleotide polymorphisms rs12722489 and multiple sclerosis in Iranian patients with multiple sclerosis

2020 ◽  
Author(s):  
Habib Ahmadi ◽  
Vahid Reza Yassaee ◽  
Reza Mirfakhraie ◽  
Feyzollah Hashemi-Gorji
Keyword(s):  
Multiple Sclerosis ◽  
Single Nucleotide Polymorphisms ◽  
Interleukin 2 ◽  
European Population ◽  
Control Group ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Mutation System ◽  
Iranian Patients

Abstract Background: Multiple sclerosis (MS) is a complex incurable neurodegenerative disease featuring demyelination of neurons, resulting in impairment of neuron impulses. Recently, an association of two single nucleotide polymorphisms (SNP) (rs2104286 and rs12722489) in interleukin 2 receptor subunit alpha (IL2RA) gene was found to be a risk factor of MS in white European population. Therefore, we performed a study to investigate the contribution of these two intronic variations in Iranian patients with MS. Methods: We determined the genotypes of rs2104286 and rs12722489 in patients with MS (n = 100) and in the control group (n = 111). The SNPs were genotyped using tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) for both of SNPs. Statistical analysis was performed by SPSS software. Also, odds ratios (ORs) and 95% confidence interval (CI) were calculated. Results: Logistic regression revealed that various genotypes of rs12722489, regarding sex-adjusted effect, yielded meaningful association with MS risk in Iranian patients (OR = 2.67, 95% CI: 1.03-6.90). However, no association was obtained for rs2104286 and rs12722489 with MS. Conclusion: The results confirmed partially the reports in white European population performed recently. However, further investigation in larger scale is necessary to validate our study.

Plasma levels of estrone sulfate (ES) in postmenopausal women with breast cancer (BC) during letrozole (L) treatment: Association with single nucleotide polymorphisms (SNPs) of CYP19A1

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 555-555
Author(s):  
G. Lunardi ◽  
L. Del Mastro ◽  
M. Serra ◽  
P. Driol ◽  
C. Boni ◽  
...  
Keyword(s):  
Amplification Refractory Mutation System ◽  
Aromatase Activity ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Estrone Sulfate ◽  
Mutation System ◽  
Key Enzyme ◽  
The Difference ◽  
Immuno Assay ◽  
Estrogen Biosynthesis

555 Background: The CYP19A1 gene encodes aromatase, a key enzyme for estrogen biosynthesis. We investigated the association between four CYP19A1 SNPs, affecting different circulating estrogen levels (rs10046 C>T, rs4646 G>T, rs749292 C>T, rs727479 T>G), and suppression of plasma ES levels induced by 6 weeks (time needed to reach L steady-state concentrations) of L (25 mg/d). Methods: Patients were enrolled into a prospective, italian multi-centre trial (GIM5) testing the correlation of CYP19A1 SNPs with the efficacy of L adjuvant therapy, after 5 years of tamoxifen, in postmenopausal early BC patients. Blood samples for hormone measurements were obtained immediately before starting L (baseline) and following 6 wks of treatment. SNPs were identified from DNA obtained from peripheral blood cells by Hexaprimer Amplification Refractory Mutation System PCR (H-ARMS-PCR). Plasma ES levels were evaluated by Radio Immuno Assay (RIA). Results: SNPs and hormone levels were evaluated in at least 129 patients. SNPs of CYP19A1 were associated with different baseline plasma ES levels. Mean inhibition of aromatase activity induced by L ranges from 71% to 79%, as a function of the SNPs. After 6 weeks of L no difference in ES levels was observed between patients with different SNPs (Table). Conclusions: L therapy induces a higher aromatase suppression in patients with SNPs associated with higher baseline plasma ES levels. The difference in ES levels associated with genetic variation at the CYP19A1 locus disappeared after L therapy. [Table: see text] [Table: see text]

scholarly journals Multiplexed Assays for Detection of Mutations in PIK3CA

2008 ◽  
Vol 54 (4) ◽  
pp. 757-760 ◽  
Author(s):  
Ruth E Board ◽  
Nicola J Thelwell ◽  
Paul F Ravetto ◽  
Stephen Little ◽  
Malcolm Ranson ◽  
...  
Keyword(s):  
Pik3ca Mutation ◽  
Clinical Samples ◽  
Amplification Refractory Mutation System ◽  
Pik3ca Mutations ◽  
Specific Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
Phosphoinositide 3 Kinase ◽  
Low Sensitivity ◽  
Detection Of Mutations

Abstract Background: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. Methods: We combined Amplification Refractory Mutation System (ARMS™; AstraZeneca) allele-specific PCR and Scorpions™ (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). Results: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. Conclusions: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.

scholarly journals Association between risk of brucellosis and genetic variations in MicroRNA-146a

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sima Kazemi ◽  
Saeid Afshar ◽  
Manoochehr Karami ◽  
Massoud Saidijam ◽  
Fariba Keramat ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacterial Infections ◽  
Inflammatory Diseases ◽  
Genetic Variations ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphisms ◽  
Chain Reaction ◽  
Phenotypic Differences ◽  
Mutation System ◽  
Polymerase Chain

Abstract Background Single nucleotide polymorphisms (SNPs) are the most common types of DNA changes in the human genome that leading to phenotypic differences in humans. MicroRNAs (miRNAs) are usually affected by various bacterial infections, and they are involved in controlling the immune responses. MicroRNA-146a (miR-146a) plays an essential role in the development of infectious and inflammatory diseases. The aim of the present study was to investigate the association between risk of brucellosis and genetic variations in miR-146a. Methods This case–control study was conducted on 108 Brucellosis patients and 108 healthy controls. We genotyped two SNPs (rs2910164 and rs57095329) of the miR-146a using tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) methods. Results The rs2910164 SNP was significantly associated with brucellosis in co-dominant [OR = 4.27, 95% CI = (2.35–7.79, P = 0.001] and dominant [OR = 3.52, 95% CI = (1.97–6.30, P = 0.001] models. Co-dominant (P = 0.047) and recessive (P = 0.018) models were significant at position rs57095329 between the two groups of patient and healthy. The A C haplotype (rs2910164 and rs57095329) was associated with brucellosis in the assessed population [OR (95% CI) = 1.98 (1.22–3.20), P = 0.0059]. Conclusions Consequently, our study demonstrated significant differences in genotype and haplotype frequencies of miR-146a variants between brucellosis patients and controls. Further studies on the larger sample sizes are required to verify the observed associations.

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