A Method for Clone Sequence Confirmation Using a Mismatch-Specific DNA Endonuclease

Qiu, Peter; Shandilya, Harini; Gerard, Gary F.

doi:10.1385/mb:29:1:11

Cited by 13 publications

(4 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2). Qiu et al also reported that mismatch cleavage preferences fall into four groups from the most favorite CT, AC, and CC to the least preferred AA, GG, and finally followed by the least favorable AG and GT [23]. Given that for every substitution, two types of heteroduplexes are produced; in almost all cases at least one of the two heteroduplexes formed is an ideal substrate for the endonuclease.…”

Section: Discussionmentioning

confidence: 99%

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Vogiatzakis

Kekou²,

Sophocleous³

et al. 2007

Mol Biotechnol

View full text Add to dashboard Cite

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed.

show abstract

Section: Discussionmentioning

confidence: 99%

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Vogiatzakis

Kekou²,

Sophocleous³

et al. 2007

Mol Biotechnol

View full text Add to dashboard Cite

show abstract

“…Despite this, many papers refer to SURVEYOR as a very efficient method for mutation detection in human (see below) as well as non-human 5,6 genes for screening of induced point mutations (TILLING) in several organisms, [7][8][9] for detecting heteroplasmy, 10,11 and for clone sequence validation. 12 Its application to human genetic disorders resulted in the discovery and description of many novel mutations in genes such as BRCA1, 1,2,13 EGFR, 14 JAK2, 15 hCDC4, 16 ATRX, 17 mitochondrial genes, 10,11 ABCC6, 18 p53, 19 NPHS2, 20 TP53, 21 COL4A3, and COL4A4. 22 There are advantages of SURVEYOR compared with other traditional mutation detection methods like singlestrand conformation polymorphism analysis, denaturing high-performance liquid chromatography, and heteroduplex analysis.…”

mentioning

confidence: 99%

Screening for Mutations in Kidney-Related Genes Using SURVEYOR Nuclease for Cleavage at Heteroduplex Mismatches

Voskarides

Deltas

2009

The Journal of Molecular Diagnostics

View full text Add to dashboard Cite

SURVEYOR is a new mismatch-specific plant DNA endonuclease that is very efficient for mutation scanning in heteroduplex DNA. It is much faster, cheaper, more sensitive, and easier to perform than other "traditional" mutation detection methods such as single-strand conformation polymorphism analysis, denaturing high-performance liquid chromatography, heteroduplex analysis, and phage resolvases. This is the first comprehensive report on the use of SURVEYOR for screening genes implicated in a spectrum of inherited renal diseases. Of the 48.2 kb screened, 44 variations were identified, accounting for one variation per 1.1 kb. The re-sequencing of multiple samples did not reveal any variation that had not been identified by SURVEYOR, attesting to its high fidelity. Additionally, we tested this enzyme against 15 known variants, 14 of which it identified, thus showing a sensitivity of 93%. We showed that the genetic heterogeneity of renal diseases can be easily overcome using this enzyme with a high degree of confidence and no bias for any specific variations. We also showed for the first time that SURVEYOR does not demonstrate any preference regarding mismatch cleavage at specific positions. Disadvantages of using SURVEYOR include enhanced exonucleolytic activity for some polymerase chain reaction products and less than 100% sensitivity. We report that SURVEYOR can be used as a mutation detection method with a high degree of confidence, offering an excellent alternative for low-budget laboratories and for the rapid manipulation of multiple genes.

show abstract

“…[12][13][14] It has been used to accurately detect various mutations in human, mammalian, bacterial, and plant genomes. [15][16][17][18] The study developed a new, highly sensitive, and costeffective method for identifying and enriching mutant DNA. Using this method, we genotyped two majorities of activating mutations in lung cancer that occur in EGFR and KRAS genes.…”

Section: Introductionmentioning

confidence: 99%

A Sensitive PCR-Based Method for Somatic Mutations Enrichment and Screening

Xiong¹,

Tang²

2021

CMAR

View full text Add to dashboard Cite

Background: EGFR and KRAS are the most frequently mutated genes in lung cancers, occurring in about 60% of all cases. Mutation genes assay has emerged as a promising bloodbased biomarker for monitoring cancer dynamics noninvasively. However, detection can be challenging in patients where plasma often contains low levels of tumor-derived DNA fragments. Methods: We have developed a nuclease-based enrichment assay for detecting mutant alleles. The procedure is based on Surveyor endonuclease cleaves mismatched DNA molecules, and these DNA fragments were enriched for mutation screening. We screened lung cancer specimens for mutations in exons 18 and 21 of EGFR, and the majority of activating mutations in lung cancer occur in codons 12 (G12X) and 13 (G13X) of exon 2 of the KRAS gene. The method screened all mutant genes with the same pair primers and three relevant TaqMan probes. Results:The method can effectively remove wild-type sequences and enrich mutation DNA, and the sensitivity detectable mutant allele frequencies (MAF) achieved 0.001%. The method increases the sensitivity and efficiency of mutation DNA for cancers screening. This highlights the importance of complex DNA variation like mutations in exon 21 of EGFR and exon 2 of the KRAS gene detected by the same probe. Conclusion:We developed a simple and sensitive methodology for mutation gene screening. The method is a cost-effective and sensitive method for mutation DNA enrichment and detection.

show abstract

A Method for Clone Sequence Confirmation Using a Mismatch-Specific DNA Endonuclease

Cited by 13 publications

References 12 publications

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Screening for Mutations in Kidney-Related Genes Using SURVEYOR Nuclease for Cleavage at Heteroduplex Mismatches

A Sensitive PCR-Based Method for Somatic Mutations Enrichment and Screening

Contact Info

Product

Resources

About