scholarly journals Three-Dimensional Microarray Compared with PCR–Single-Strand Conformation Polymorphism Analysis/DNA Sequencing for Mutation Analysis of K-ras Codons 12 and 13

2004 ◽  
Vol 50 (8) ◽  
pp. 1322-1327 ◽  
Author(s):  
Masato Maekawa ◽  
Tomonori Nagaoka ◽  
Terumi Taniguchi ◽  
Hitomi Higashi ◽  
Haruhiko Sugimura ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Cell Lines ◽  
Three Dimensional ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Sscp Analysis ◽  
Polymorphism Analysis ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Conformation Polymorphism

Abstract Background: We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations. Methods: We used the PamChip® microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR–single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Results: We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4–100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was ∼16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16–25% of the total DNA. Conclusions: The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.

scholarly journals Optimization of single-strand conformation polymorphism analysis in the presence of polyethylene glycol

1997 ◽  
Vol 43 (1) ◽  
pp. 30-33 ◽  
Author(s):  
Arseni Markoff ◽  
Alex Savov ◽  
Vladimir Vladimirov ◽  
Nadia Bogdanova ◽  
Ivo Kremensky ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gc Content ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Sscp Analysis ◽  
Polymorphism Analysis ◽  
Conformation Polymorphism Analysis ◽  
Detection Of Mutations ◽  
Conformation Polymorphism

Abstract We report optimization of single-strand conformation polymorphism (SSCP) analysis in the presence of polyethylene glycol. The protocol developed separates single-strand conformers in a much shorter time (1–3 h) than conventional SSCP protocols and broadens the applicability of SSCP analysis from 150 to as much as 500 bp of DNA by different percentages of GC content present. We conclude that addition of polyethylene glycol helps improve the differential separation of conformers and, in combination with high-resolution polyacrylamide gel electrophoresis, offers an alternative to previous SSCP analysis protocols. This protocol should be very useful for clinical applications in routine detection of mutations as well as for research purposes.

Multiplex Single Strand Conformation Polymorphism Analysis by Capillary Electrophoresis with On-the-Fly Fluorescence Lifetime Detection

2005 ◽  
Vol 59 (3) ◽  
pp. 335-339 ◽  
Author(s):  
Tara Morcone Snyder ◽  
Linda B. McGown
Keyword(s):  
Capillary Electrophoresis ◽  
Fluorescence Lifetime ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
G20210a Mutation ◽  
Prothrombin Gene ◽  
Fluorescence Lifetime Detection ◽  
Conformation Polymorphism

This paper describes the use of on-the-fly fluorescence lifetime detection (OFLD) for multiplex single strand conformation polymorphism (SSCP) analysis by capillary electrophoresis (CE). The dye labels studied for multiplex SSCP-OFLD-CE analyses included RG, NBD, and BODIPY-FL. The dyes were first investigated for a model system of “Wild Type” and “Mutant” 43-base fragments designed to vary by a single A/T substitution. Two dye pairs, BODIPY-FL/RG and BODIPY-FL/NBD, were then used to detect the G20210A mutation in the human prothrombin gene. Mobility correction was required for the BODIPY-FL/RG system. Three “blind” analyses were performed of three mixtures that combined a control fragment (wild type-BODIPY-FL) with two “unknown” fragments selected among four possibilities (wild type or mutant labeled with NBD or RG). In each multiplex analysis, the “origin” of the unknown fragments was correctly identified on the basis of fluorescence lifetime of the dye label and the presence or absence of the mutation was correctly determined on the basis of conformation-induced differences in migration time.

scholarly journals Absence of WAF1 mutations in a variety of human malignancies

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3781-3784 ◽  
Author(s):  
M Shiohara ◽  
WS el-Deiry ◽  
M Wada ◽  
T Nakamaki ◽  
S Takeuchi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Polymerase Chain Reaction Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Dna Polymorphisms ◽  
Polymorphism Analysis ◽  
P53 Mutations ◽  
Cloned Gene ◽  
Transformed Cell Lines ◽  
Conformation Polymorphism

A newly cloned gene named wild-type p53-activated fragment 1 (WAF1; also known as p21, Pic-1, Cip-1, or SDI1) is directly regulated by p53 and can itself suppress tumor cell growth in culture. Induction of expression of WAF1 may be an important means by which cells with DNA injury arrest their growth to repair DNA or undergo apoptosis. Based on the hypothesis that mutations of this gene may play a role in carcinogenesis, we have studied 351 DNAs from 14 kinds of malignancies, as well as 36 human transformed cell lines, for alterations of WAF1 gene by single-strand conformation polymorphism analysis of polymerase chain reaction amplification of the DNA coding region of the WAF1 gene. No abnormal band shifts of WAF1 were noted in any of the samples or cell lines, but three major variants in exons 2 and 3 of the gene were found that are consistent with the existence of two different DNA polymorphisms. Sequence analysis of the amplified products producing these three variants in each exon from normal DNAs confirmed the presence of the polymorphisms in the WAF1 gene. Of 290 selected tumor samples previously evaluated for p53 mutations by single-strand conformation polymorphism, 90% had no detectable p53 alterations. In summary, mutations within the coding portion of the WAF1 gene were undetectable in a large series of human tumors, many of which had a normal p53 gene. This suggests that WAF1 alterations are generally caused indirectly, through p53 mutations rather than through intragenic mutation of the WAF1 itself.

scholarly journals Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation polymorphism analysis [see comments]

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3363-3368 ◽  
Author(s):  
A Hirono ◽  
S Miwa ◽  
H Fujii ◽  
F Ishida ◽  
K Yamada ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Japanese Patients ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Sscp Analysis ◽  
Polymorphism Analysis ◽  
Missense Mutations ◽  
G6pd Gene ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Conformation Polymorphism

Abstract Using a newly developed nonradioisotopic method of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis combined with the direct sequencing using the fluorescence-labeled terminator, we identified seven missense mutations, 527 A-->G, 1003 G-- >A, 1159 C--eT, 1160 G-->A, 1229 G-->A, 1246 G-->A, and 1361 G-->A, in eight Japanese patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Except for the 527 A-->G, each mutation has been reported to cause variants G6PD Chatham, G6PD Guadalajara, G6PD Beverly Hills, G6PD “Japan”, G6PD Tokyo, and G6PD Andalus, respectively. In addition, a single base deletion in intron 5 was found in the patients with G6PD Guadalajara or G6PD Andalus. The variant with unique 527 A-->G was characterized and designated as G6PD Shinshu. We also characterized G6PD “Japan” and found that the variant had the striking resemblance with G6PD Riverside, bearing a missense mutation in the same codon, but causing a different amino acid substitution. Our modified PCR-SSCP analysis using minigel and ethidium bromide staining could detect six of the eight diverse mutations in the G6PD gene. Because it is easy and requires no special apparatus, this modified method will be useful for screening mutations in the G6PD gene.

scholarly journals Molecular screening of patients with long standing extensive ulcerative colitis: detection of p53 and Ki-ras mutations by single strand conformation polymorphism analysis and differential hybridisation in colonic lavage fluid

Gut ◽  
1999 ◽  
Vol 44 (6) ◽  
pp. 822-825 ◽  
Author(s):  
S M Lang ◽  
D F Stratakis ◽  
M Heinzlmann ◽  
W Heldwein ◽  
B Wiebecke ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Point Mutations ◽  
P53 Gene ◽  
Lavage Fluid ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Conformation Polymorphism Analysis ◽  
Conformation Polymorphism

BACKGROUNDIn patients with long standing ulcerative colitis at risk of developing malignancy, mutations of the p53 and Ki-ras gene were investigated in lavage solution obtained at surveillance colonoscopy.METHODSDNA was isolated from 31 consecutive patients with total or subtotal ulcerative colitis and a disease duration of between seven and 26 years. Twenty seven control patients showed no macroscopic or microscopic inflammation on colonoscopy. Exons 5–8 of the p53 gene and exon 1 of the Ki-ras gene were amplified by polymerase chain reaction. Mutations of the p53 gene were detected by single strand conformation polymorphism analysis. Point mutations of the Ki-ras gene were hybridised on dot blots with oligonucleotides marked with digoxigenin.RESULTSIn all cases of ulcerative colitis and in all of the 27 control patients, wild type p53 and wild type Ki-ras could be detected. In four patients with ulcerative colitis, a mutation in exon 5 to 7 of the p53 gene was found, and two patients had a mutation of the Ki-ras gene (Gly to Asp-12, Gly to Val-12). None of these patients had dysplasia in serial biopsy specimens, and all but one had had the disease for more than 10 years. One control patient had a mutation.CONCLUSIONSMutations were more frequent in patients with long standing ulcerative colitis (19%) than in control patients (3%, p = 0.07). The technique may be useful for screening for early malignancy in ulcerative colitis.

scholarly journals High sensitivity of the single-strand conformation polymorphism method for detecting sequence variations in the low-density lipoprotein receptor gene validated by DNA sequencing

1996 ◽  
Vol 42 (8) ◽  
pp. 1140-1146 ◽  
Author(s):  
H K Jensen ◽  
L G Jensen ◽  
P S Hansen ◽  
O Faergeman ◽  
N Gregersen
Keyword(s):  
Dna Sequencing ◽  
Receptor Gene ◽  
Ldl Receptor ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Sscp Analysis ◽  
Oligonucleotide Primer ◽  
In Vitro Mutagenesis ◽  
Sequence Variations ◽  
Conformation Polymorphism

Abstract We designed oligonucleotide primer pairs to amplify the promoter region, the translated exon sequences, and the flanking intron sequences of all 18 exons of the LDL receptor gene to compare the ability of the PCR single-strand conformation polymorphism (PCR-SSCP) method with semiautomated solid-phase genomic DNA sequencing to detect sequence variations. In 20 apparently unrelated Danish patients with a clinical diagnosis of heterozygous familial hypercholesterolemia (FH), we identified 13 different mutations in the LDL receptor gene: two silent (C331C, N494 N); five missense (W66G, E119K, T383P, W556S, T7051); one nonsense (W23X); three splice-site (313 + 1G-->A, 1061-8T-->C, 1846-1G-->A); and two frameshift (335del10, 1650delG) mutations. Four of these mutations, N494 N, T383P, 1061-8T-->C, and W556S, have not been reported earlier. The pathogenicity of the T383P, 1061-8T-->C, and W556S mutations remains to be established by in vitro mutagenesis and transfection studies. One patient had three mutations (335del10, 1061-8T-->C, and T705I) on the same allele. Further, nine well-known polymorphisms were detectable with this methodological setup. Direct DNA sequencing of the PCR products used for the SSCP analysis did not reveal any sequence variations not detected by the PCR-SSCP method. In two patients we did not detect any mutation by either method. We conclude that the PCR-SSCP analysis, performed as described here, is as sensitive and efficient as DNA sequencing in the ability to identify the sequence variations in the LDL receptor gene of the patients with heterozygous FH of this study.

Evaluation of PCR–Single-Strand Conformational Polymorphism Analysis for Identification of Gram-Negative Histamine-Producing Bacteria Isolated from Fish

2007 ◽  
Vol 70 (5) ◽  
pp. 1200-1205 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
MIKI SATO ◽  
BON KIMURA ◽  
TATSUYA ISHIKAWA ◽  
TATEO FUJII
Keyword(s):  
Histidine Decarboxylase ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Sscp Analysis ◽  
Polymorphism Analysis ◽  
Single Strand Conformational Polymorphism ◽  
Banding Patterns ◽  
Gram Negative ◽  
Fish Samples ◽  
Conformation Polymorphism

In this study, the previously established PCR–single-strand conformation polymorphism (SSCP) system for detecting and identifying gram-negative histamine-producing bacteria was evaluated. This system can detect and identify histamine-producing bacteria directly from seafood by the use of sequence polymorphisms of the histidine decarboxylase gene (hdc). First, we isolated 81 histamine-producing strains of bacteria from fish samples and analyzed the hdc gene by the PCR-SSCP system. The 22 newly obtained SSCP banding patterns were added to our database, and the utility of our modified database was tested in a second experiment consisting of 18 strains of histamine-producing bacteria isolated from 25 fish samples. Approximately 80% of the histamine-producing strains corresponded to those in the new database. Use of the database for PCR-SSCP analysis, including the band patterns newly added in this study, for the hdc gene makes it possible to more accurately identify histamine producers.

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