Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay

Rothschild, Cynthia B.; Brewer, Catherine; Loggie, Brian W.; Beard, G. Alan; Triscott, Mark

doi:10.1016/s0022-1759(97)00078-1

Cited by 23 publications

(18 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…17,18 The oligonucleotide ligation assay (OLA), even with sensitive capillary electrophoresis detection, can only detect minor species at about 0.1-1%. 19,20 Ligation chain reaction uses four oligonucleotides and ligase as an alternative to polymerase, but its limit of detection is approximately 0.1 to 1%. 21 Allele-specific PCR (AS-PCR) or the amplification refractory mutation system (ARMS) can only detect approximately 1% of mutant DNA.…”

Section: Research Papermentioning

confidence: 99%

Sensitive and quantitative detection of KRAS2 gene mutations in pancreatic duct juice differentiates patients with pancreatic cancer from chronic pancreatitis, potential for early detection

Shi

Fukushima

Abe

et al. 2008

Cancer Biology & Therapy

View full text Add to dashboard Cite

KRAS2 gene mutations are found in 75-90% of infiltrating pancreatic ductal adenocarcinomas but can also be present with other nonneoplastic pancreatic diseases. We recently developed a novel sensitive assay for point mutation detection, called "LigAmp", which can detect one mutant molecule in the presence of 10,000 wild-type molecules and can quantify mutant DNA over a wide dynamic range. We analyzed KRAS2 mutations in surgically-collected pancreatic duct juice samples from patients with pancreatic adenocarcinoma (n = 27) and chronic pancreatitis (n = 9). DNA sequencing demonstrated that 17 of the 27 pancreatic cancers harbored KRAS2 mutations at codon 12, including G12D (GGT → GAT), G12V (GTT), and G12R (CGT). We determined the relative amounts of each KRAS2 mutant by simultaneously quantifying wild-type and mutant KRAS2 DNA. For all pancreatic adenocarcinoma patients, the dominant KRAS2 mutation detected in the pancreatic juice corresponded to that found in the primary cancer. Mutation levels were substantially higher in patients with pancreatic cancer (0.05 to 82% of total KRAS2 molecules) compared to those with chronic pancreatitis (0 to 0.7%). Among patients with mutant KRAS2 positive cancers, all but one (94%) had mutant KRAS2 DNA concentrations of more than 0.5% in their pancreatic juice samples, whereas only 1 of 9 (11%) pancreatic juice samples from patients with chronic pancreatitis had more than 0.5% mutant KRAS2 DNA, corresponding to a sensitivity of 94% and a specificity of 89%. LigAmp quantification of mutant KRAS2 in pancreatic juice differentiates pancreatic adenocarcinoma from chronic pancreatitis, and may be a useful early detection tool for pancreatic cancer.

show abstract

Section: Research Papermentioning

confidence: 99%

Sensitive and quantitative detection of KRAS2 gene mutations in pancreatic duct juice differentiates patients with pancreatic cancer from chronic pancreatitis, potential for early detection

Shi

Fukushima

Abe

et al. 2008

Cancer Biology & Therapy

View full text Add to dashboard Cite

show abstract

“…The problem of using somatic mutations as markers of malignancy is minimal amounts of mutant DNA in a large excess of wild-type DNA containing in clinical samples. Variety of technologies based on allele discrimination strategies have been applied in the identifi cation of point mutations, such as oligonucleotide ligation (12,13), PCR enriched enzymatic cleavage (14,15,16), allele-specifi c oligonucleotide hybridization (17) and integration of allele-specifi c oligonucleotide ligation assay (OLA) with magnetic beads based on electrochemiluminescence (18). Pyrosequencing methods based on chemiluminescence (19,20) and primer extension (21) was also described.…”

mentioning

confidence: 99%

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Ugorcakova

Hlavatý²,

Novotna³

et al. 2012

BLL

View full text Add to dashboard Cite

Abstract:Objectives: The determination of gene mutations is important for the diagnosis and prognosis of various gastrointestinal cancers. The aim of our study was to develop a new procedure for the analysis of KRAS gene mutation by application of the real-time PCR method. Background: The detection process requires discriminate trace amount of mutant allele in a large excess of wild-type DNA in various samples. Methods:The real-time PCR based technique using hybridization probes for fi ve most frequently KRAS codon 12 mutations and WT specifi c peptide nucleic acid (PNA) was performed. Our multiplex detection system was tested in various DNA samples (tissue, bile, pancreatic juice) of patients with different diagnoses of gastrointestinal tract disease obtained by endoscopy and ERCP. Results: We designed and optimized the real-time PCR conditions and tested various amount of PNA in PCR reaction to suppress amplifi cation of the wild-type DNA. We determined the interassay variability of the melting temperatures and the results of mutation testing were confi rmed by DNA sequencing with the 100 % accuracy. Incidence of searched mutations was 67.5 % in cohort of 40 patients; for KRAS G12D it was in 44.4 %, KRAS G12V in 22.2 %, KRAS G12S in 14.8 %, KRAS G12A in 14.8 % and KRAS G12C in 3.8 %. The sensitivity of the assays is 1x10 -5 . Conclusions: Advantages of this technique are rapidity, accuracy and it is generally easy to perform. This method can be adapted for synchronic detection of multiple mutations and after readjustment by other type mutation of KRAS gene may serve as useful clinical tool for analyzing point mutations in various clinical samples (Tab. 3, Fig. 3, Ref. 42). Full Text in PDF www.elis.sk.

show abstract

“…[18][19][20][21] Thus, a convenient analysis of K-ras mutant is meaningful in clinical applications. [21][22][23] Figure 3 shows electropherograms of both sequences. In this case, anti-sence sequence of c-K-ras codon 10-13 (5′-GCCACCAGCTCC-3′) was immobilized onto a capillary inner surface.…”

Section: Resultsmentioning

confidence: 99%

An Affinity Capillary Electrophoresis for the Detection of Gene Mutation Using Immobilized Oligonucleotides-Polyacrylamide Conjugate

et al. 1999

View full text Add to dashboard Cite

It has been found that small mutations of certain genes are the definitive origin of many heritable disorders and cancers, thanks to striking developments of recent molecular biology. Such new findings have highlighted the importance of gene mutation assays based on the difference of DNA base sequences in diagnostic or medical fields. Capillary electrophoresis can be a good candidate for an ideal method on such gene analysis 1,2

show abstract

Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay

Cited by 23 publications

References 21 publications

Sensitive and quantitative detection of KRAS2 gene mutations in pancreatic duct juice differentiates patients with pancreatic cancer from chronic pancreatitis, potential for early detection

Sensitive and quantitative detection of KRAS2 gene mutations in pancreatic duct juice differentiates patients with pancreatic cancer from chronic pancreatitis, potential for early detection

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

An Affinity Capillary Electrophoresis for the Detection of Gene Mutation Using Immobilized Oligonucleotides-Polyacrylamide Conjugate

Contact Info

Product

Resources

About