Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

Mitani, Yuichiro; Lezhava, Alexander; Kawai, Yosuke; Kikuchi, Takeshi; Oguchi-Katayama, Atsuko; Kogo, Yasushi; Itoh, Masayoshi; Miyagi, Tohru; Takakura, Hideki; Hoshi, Kanako; Kato, Chiaki; Arakawa, Takahiro; Shibata, Kazuhiro; Fukui, Kenji; Masui, Ryoji; Kuramitsu, Seiki; Kiyotani, Kazuma; Chalk, Alistair M.; Tsunekawa, Katsuhiko; Murakami, Masami; Kamataki, Tetsuya; Oka, Toshihiko; Shimada, Hiroshi; Cizdziel, Paul E.; Hayashizaki, Yoshihide

doi:10.1038/nmeth1007

Cited by 159 publications

(166 citation statements)

References 18 publications

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“…On the other hand, a blood sample diluted with 50 mmol/L NaOH (and heated) is used as a template for our real-time PCR for diagnosis of HP del and isothermal single nucleotide polymorphism genotyping. 11,12,18 We first prepared serial dilutions to identify the suitable dilution range, and turbidity was consistently observed in the tubes with dilutions from 1:30 to 1:1000 for both amplifications. The Tt increased roughly in proportion to the dilution ratio for HP-del, but not for HP-5= (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Soejima

Egashira

Kawano

et al. 2011

The Journal of Molecular Diagnostics

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Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin antibodies. Being homozygous for the haptoglobin gene deletion allele (HP del ) is the only known cause of congenital anhaptoglobinemia, and detection of HP del before transfusion is important to prevent anaphylactic shock. In this study, we developed a loop-mediated isothermal amplification (LAMP)-based screening for HP del . Optimal primer sets and temperature for LAMP were selected for HP del and the 5= region of the HP using genomic DNA as a template. Then, the effects of diluent and boiling on LAMP amplification were examined using whole blood as a template. Blood samples diluted 1:100 with 50 mmol/L NaOH without boiling gave optimal results as well as those diluted 1:2 with water followed by boiling. The results from 100 blood samples were fully concordant with those obtained by real-time PCR methods. Detection of the HP del allele by LAMP using alkaline-denatured blood samples is rapid, simple, accurate, and cost effective, and is readily applicable in various clinical settings because this method requires only basic instruments. In addition, the simple preparation of blood samples using NaOH saves time and effort for various genetic tests. The absence of a serum protein such as IgA or haptoglobin (Hp) is one of the factors that can lead to anaphylactic transfusion reactions due to production of serum antibodies against the absent protein after a transfusion. 1 At present, a homozygous deletion of the haptoglobin gene (HP del ) is the only known cause of anhaptoglobinemia.Human Hp has a genetic polymorphism of two codominant alleles, HP 1 and HP 2 , that give rise to the three common phenotypes, Hp1, Hp2-1, and Hp2. 2,3 The HP 2 allele appears to have occurred by a 1.7-kb intragenic duplication of exons 3 and 4 of the HP 1 allele ( Figure 1A). Anomalous inheritance of the Hp phenotypes was encountered during determinations of parentage, and HP del was identified by genetic analyses of one such family in Japan. 4 The HP del allele lacks an approximately 28-kb segment of chromosome 16 extending from the promoter region of the HP gene to intron 4 of the haptoglobinrelated gene (HPR) ( Figure 1A). 4,5 The HP del allele has been found only in East and Southeast Asian populations (Chinese, Korean, Japanese, Mongols, Thais, and Indonesians), not in African, West and South Asian, and European populations so far. [5][6][7][8][9] Detection of homozygosity for HP del before blood transfusion or blood component infusion is important to prevent severe side effects of transfusion because washed red blood cells and platelet concentrate do not cause transfusion-related anaphylactic reactions. 10 Recently, we established two real-time PCR methods for detection of HP del by use of a 5=-nuclease assay using dual-labeled (TaqMan; Applied Biosystems, Foster City, CA) probes and SYBR Green I (Invitrogen, Carlsbad, CA). 11,12 These methods are rapid, robust, and suitable for high-throughput analysis but req...

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Section: Resultsmentioning

confidence: 99%

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Soejima

Egashira

Kawano

et al. 2011

The Journal of Molecular Diagnostics

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“…DNAFORM; Kanagawa, Japan) to detect mutations in EGFR exon 19, according to the manufacturer's instructions. The principles of the SmartAmp2 have been previously described (26). We designed the PNA-clamp SmartAmp2 so it would amplify almost all types of deletions that occur in the hot spot of the mutation in EGFR exon 19.…”

Section: Methodsmentioning

confidence: 99%

“…In 2007, the rapid, simple and sensitive mutation detection assays, smart amplification process version 2 (SmartAmp2) and PNA-clamp SmartAmp2 for SNP detection, were developed (26,27). The SmartAmp2 method is a unique genotyping technology that can detect a mutation within 30 min under isothermal conditions and in a single step.…”

Section: Introductionmentioning

confidence: 99%

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma

et al. 2011

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Abstract. The presence of EGFR mutations is correlated with a positive therapeutic response to tyrosine kinase inhibitors; therefore, the accurate detection of EGFR mutations is crucial when deciding appropriate therapeutic strategies. Recently, the rapid and sensitive assay smart amplification process version 2 (SmartAmp2) was developed. However, this method can only detect one type of mutation in EGFR exon 19; therefore, we applied the PNA technology to the SmartAmp2 assay to develop PNA-clamp SmartAmp2 for the detection of many types of deletions in EGFR exon 19, in a single reaction. This new assay was evaluated using 172 clinical samples. Thirty-nine (22.7%) samples were found to have deletions by PNA-clamp SmartAmp2; whereas 30 (17.4%) and 38 (22.1%) tumors were found to have deletions by direct sequencing and PNA-enriched sequencing, respectively. Three cases, in which we detected mutations with PNA-clamp SmartAmp2, but not with direct sequencing, were treated with gefitinib, and all cases showed a partial therapeutic response. Using clinical samples, we demonstrated that PNA-clamp SmartAmp2 can detect various types of mutations in EGFR exon 19 in a relatively short time and with high sensitivity. This method detected small amounts of mutant DNA and identified patients for whom clinical information was previously unavailable from other tests. This test may contribute to the administration of efficient therapeutic strategies.

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“…( Previously we developed a rapid single nucleotide polymorphism detection system named the smart amplification process version 2 (SMAP-2). 1 The SMAP-2 can rapidly detect mutations from tissue, and has been used for rapid diagnosis of specific somatic mutations. 2 However, instead of using several specific point mutation assays in parallel to analyze clinical specimens, it was more desirable to create a SMAP-2 assay that would identify many possible mutations in a single tube.…”

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confidence: 99%

“…1 The first is a unique asymmetric design of the primers flanking the target sequence. These primers are referred to as the folding primer and turn-back primer and are engaged in amplifying the target through a selfpriming mechanism.…”

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confidence: 99%

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Takagi

Mitani

Watanabe

et al. 2008

The Journal of Molecular Diagnostics

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Previously , the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele , our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNAclamp SMAP-2) , we could detect KRAS mutations within 60 minutes , including sample preparation. We compared results from PNA-clamp SMAP-2 assay , polymerase chain reaction-restriction fragment length polymorphism , and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNAclamp SMAP-2 method is a rapid , simple , and highly sensitive detection assay for cancer mutations. (J Mol

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Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

Cited by 159 publications

References 18 publications

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

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