PCR hot start using primers with the structure of molecular beacons (hairpin-like structure)

Kaboev, O. K.; Luchkina, L A; Tretiakov, Alexander; Bahrmand, Ahmadreza

doi:10.1093/nar/28.21.e94

Cited by 54 publications

(26 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We found no associations between the SNPs and methamphetamine use disorder in males, either by allelic or allele positivity w 2 statistics. However, the results are quite different in females: significant differences were Sequences in parentheses are to increase PCR sensitivity according to Kaboev et al 49 We marked the relative positions of the SNPs according to the mRNA maps; for intronic SNP, we marked according to the genomic maps.…”

Section: Resultsmentioning

confidence: 99%

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

et al. 2003

View full text Add to dashboard Cite

Family and twins studies have suggested that genetic factors are involved in the development of substance use disorders. Several unrelated case/control association studies have reported associations of the GABA A subunit genes on 5q33 with the development of alcohol dependence. We hypothesized that these particular GABA A subunit genes also contribute to the development of methamphetamine use disorder. To test our hypothesis, we recruited cases using a series of questionnaires. Among the polymorphic SNPs, significant differences between cases and controls were identified in the female sample in the rs2279020 of the GABA A a1 subunit gene, and the novel SNP rs4480617 in the GABA A g2 subunit gene. No associations were found in the male sample. Further haplotype analysis identified several marker blocks significantly associated with methamphetamine use disorder in females; each block consists of the rs4480617. Our study provides preliminary evidence that the GABA A subunit genes on 5q33 may preferentially contribute to methamphetamine use disorder in females. There is increasing evidence that substance use disorders are familial and that genetic factors explain a substantial degree of the familial aggregation. 3 Grove et al 4 studied a sample of 32 pairs of monozygotic twins reared apart, and reported that inherited factors explained 45% of the variance in drug abuse and dependence. Pickens et al 5 found that the variance attributable to genetic factors in abuse of illicit drugs is 31% in males and 22% in females. Similarly, Tsuang et al 6 reported that genetic factors account for 34% of the variance in the individual's risk of developing a drug use disorder; for stimulant abuse, 44% of the variance is attributable to genetic factors. The pairwise concordance rate for stimulant abuse was 14.1% for monozygotic twins and 5.3% for dizygotic twins. 7 The supporting evidence is also derived from animal studies. For example, knockout of the dopamine D4 receptor gene in mice result in animals that are supersensitive to several substances, including methamphetamine. 8 For the GABA A a1 knockout mice, administration of amphetamine sulfate caused an

show abstract

Section: Resultsmentioning

confidence: 99%

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

et al. 2003

View full text Add to dashboard Cite

show abstract

“…It is possible that the additional nucleotides added to the 5Ј end of the ELHPs result in secondary structures that mimic HP thermodynamics and that the variability of the ⌬C t s in ELHP assays is the result of differences in these unplanned structures. Previous reports (11,16) have shown that HPs (which are not designed to detect SNPs) improve the specificity of PCR assays by decreasing mispriming and primer-dimer formation compared to linear primers. This feature may be an added benefit of the HP design for SNP assays.…”

Section: Discussionmentioning

confidence: 99%

Hairpin Primers for Simplified Single-Nucleotide Polymorphism Analysis of Mycobacterium tuberculosis and Other Organisms

Hazbón

Alland

2004

J Clin Microbiol

View full text Add to dashboard Cite

We describe a novel, simple, rapid, and highly sensitive method to detect single-nucleotide polymorphisms (SNPs) in Mycobacterium tuberculosis and other organisms. Amplification refractory mutation (ARMS) SNP assays were modified by converting the SNP-detecting linear primers in the ARMS assay to hairpin-shaped primers (HPs) through the addition of a 5 tail complementary to the 3 end of the linear primer. The improved ability of these primers to detect SNPs in M. tuberculosis was compared in a real-time PCR with SYBR-I green dye. Linear primers resulted in incorrect or indeterminate allele designation for 6 of the 13 SNP alleles tested in seven different SNP assays, while HPs determined the correct SNP in all cases. We compared the cycle threshold differences (⌬C t ) between the reactions containing primer-template matches and the reactions containing primer-template mismatches (where a larger ⌬C t indicates a more robust assay). The use of HPs dramatically improved the mean ⌬C t values for the SNP assays (7.6 for linear primers and 11.2 for HPs). We designed 98 different HP assays for SNPs previously associated with resistance to the antibiotic isoniazid to test the large-scale utility of the HP approach. Assay design was successful in 72.4%, 83.7%, 88.8%, and 92.9% of the assays after one to four rounds of assay design, respectively. HP SNP assays are simple, sensitive, robust, and inexpensive. These advantages favor the application of this technique for SNP assays of M. tuberculosis and other organisms.Single-nucleotide polymorphism (SNP) analysis is becoming increasingly important for studies of drug resistance, evolution, and molecular epidemiology in Mycobacterium tuberculosis (2,7,9,18,(20)(21)(22), human immunodeficiency virus (3, 10, 15), and other organisms (12,23,26,31,32). High-throughput SNP analysis can be particularly beneficial for confirming associations between specific SNPs and a phenotype of interest, such as drug resistance. The ideal SNP detection method should be simple to design, easy to perform under uniform assay conditions (so that multiple SNP assays can be performed simultaneously), easy to automate, and inexpensive. Most approaches, including DNA sequencing and its derivations (9, 22), microarrays (6), allele-specific amplification (33), mass spectrometrybased techniques (27), TaqMan probes (14), and molecular beacons (28), represent a tradeoff between cost, throughput, and flexibility.We developed an improved SNP detection method to enable us to analyze large numbers of M. tuberculosis isolates. Most causes of drug resistance in M. tuberculosis appear to be the result of SNPs in particular target genes. However, each SNP occurs at a relatively low frequency. Therefore, in the absence of large sequencing studies, it has been difficult to establish statistically valid associations between individual SNPs and resistance to a particular drug. In the case of resistance to the antibiotic isoniazid, only mutations in codon 315 of the katG gene occur with sufficient frequency. The danger o...

show abstract

“…The HP assay uses unlabeled hairpin-shaped primers, designed following the conditions and the parameters recommended for the design of molecular beacons (26). Kaboev et al (14) reported that HPs increase the specificity and the sensitivity of PCRs by strongly diminishing the formation of primer dimers and nonspecific products. The superiority of on May 9, 2018 by guest http://cvi.asm.org/ these primers over conventional linear primers for SNP detection has previously been demonstrated (10).…”

Section: Discussionmentioning

confidence: 99%

Novel Hairpin-Shaped Primer Assay To Study the Association of the −44 Single-Nucleotide Polymorphism of theDEFB1Gene with Early-Onset Periodontal Disease

Boniotto

Hazbón

Jordan

et al. 2004

Clin Vaccine Immunol

View full text Add to dashboard Cite

A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The ؊44 C3G transversion in the DEFB1 gene (which encodes human ␤-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the ؊44 SNP. We used an HP assay to study the distribution of the ؊44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the ؊44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.

show abstract

PCR hot start using primers with the structure of molecular beacons (hairpin-like structure)

Cited by 54 publications

References 7 publications

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

Hairpin Primers for Simplified Single-Nucleotide Polymorphism Analysis of Mycobacterium tuberculosis and Other Organisms

Novel Hairpin-Shaped Primer Assay To Study the Association of the −44 Single-Nucleotide Polymorphism of theDEFB1Gene with Early-Onset Periodontal Disease

Contact Info

Product

Resources

About