Detection of ras point mutations by polymerase chain reaction using mutation-specific, inosine-containing oligonucleotide primers

doi:10.1016/0006-291X(89)92452-2

Biochemical and Biophysical Research Communications

Volume 160, Issue 2, 28 April 1989, Pages 441-447

https://doi.org/10.1016/0006-291X(89)92452-2 Get rights and content

Abstract

The use of DNA primers with 3′-ends complementary to specific genetic point mutations allowed for the rapid detection of such mutations in genomic DNA by polymerase chain reaction. The sensitivity of this approach was such that mutations could be detected in DNA samples mixed with a 10⁷-fold excess of normal non-mutated DNA. To increase the practicality of this approach for the detection of point mutations affecting all 3 of the known ras oncogenes we synthesized mutation-specific primers complementary to all 3 genes by substituting inosine residues at positions corresponding to ambigous bases on the genes.

References (20)

Verlaan-de-VriesM. et al.
Gene
(1986)
AlmogueraC. et al.
Cell
(1988)
OhtsukaE. et al.
J. Biol. Chem.
(1985)
BosJ.L.
Mutation Res.
(1988)
HorowitzJ.M. et al.
Science
(1989)
SaikiR.K. et al.
Science
(1985)
SaikiR.K. et al.
Science
(1988)
LandegrenU. et al.
Science
(1988)
CottonR.G.H. et al.
MeyersR.M. et al.
Nature
(1985)

There are more references available in the full text version of this article.

Cited by (110)

Development of a sensitive double TaqMan Probe-based qPCR Angle-Degree method to detect mutation frequencies
2023, Mitochondrion
We designed a method to examine the mutation frequencies of the A3243G mutation of mitochondrial DNA (mtDNA) in patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. We performed a qPCR assay using the FAM and VIC TaqMan probes, which detect the 3243G (mutated) and 3243A (wild-type) sequences of mtDNA, respectively. The results obtained by “degree” in a series of differential mutation frequencies were used to plot a standard curve of the mutation frequency. The standard curve was then applied for qPCR assays of the desired samples. The standard deviation (%) of the samples calculated using the standard curve for the TaqMan probe was 2.4 ± 1.5%. This method could be used to examine mutation frequencies in the context of diabetes, aging, cancer, and neurodegenerative diseases.
PCR-Based Detection Methods for Single-Nucleotide Polymorphism or Mutation: Real-Time PCR and Its Substantial Contribution Toward Technological Refinement
2017, Advances in Clinical Chemistry
Citation Excerpt :
The most important step for sequence-specific amplification by PCR is the design of suitable primers or probes. Representative studies dealing with the design of allele-specific primers for nucleotide substitution were published in 1989 [25–29]. These studies basically designed specific primers with the 3′-terminal base of the primer matched to the wild-type template or mutant template, resulting in the specific amplification of the only target sequence.
Single-nucleotide polymorphisms (SNPs) and single-nucleotide mutations result from the substitution of only a single base. The SNP or mutation can be relevant to disease susceptibility, pathogenesis of disease, and efficacy of specific drugs. It is important to detect SNPs or mutations clinically. Methods to distinguish/detect SNPs or mutations should be highly specific and sensitive. In this regard, polymerase chain reaction (PCR) has provided the necessary analytical performance for many molecular analyses. PCR-based methods for SNP/mutation detection are broadly categorized into two types—(1) polymorphic or mutant allele-directed specific analysis using primers matched with substituted nucleotide or using oligonucleotides to block or clamp the nontargeted template, and (2) melting curve analysis, which is combined with the real-time PCR techniques using hydrolysis probes, hybridization probes, or double-stranded DNA-binding fluorescent dyes. Innovative and novel approaches as well as technical improvements have made SNP- or mutation-detection methods increasingly more sophisticated. These advances include DNA/RNA preparation and subsequent amplification steps, and miniaturization of PCR instruments such that testing may be performed with relative ease in clinical laboratories or as a point-of-care test in clinical settings.
PRISE (PRImer SElector): Software for designing sequence-selective PCR primers
2008, Journal of Microbiological Methods
Citation Excerpt :
Sequence-selective PCR primers allow the amplification and isolation of specific DNA fragments from complex mixtures. They are important in a variety of tasks, including single nucleotide polymorphism (SNP) analyses, where sequences differing by one nucleotide need to be distinguished (Ehlen and Dubeau, 1989; Nichols et al., 1989; Okayama et al., 1989; Wu et al., 1989). Sequence-selective primers are used in a variety of procedures for genomic walking (Arnold and Hodgson, 1991; Ayyadevara et al., 2000a,b; Nichols et al., 1989).
This report presents PRImer Selector (PRISE), a new software package that implements several features that improve and streamline the design of sequence-selective PCR primers. The PRISE design process involves two main steps. In the first step, target and non-target DNA sequences are identified. In the second step, primers are designed to amplify target (but not non-target) sequences. One important feature of PRISE is that it automates the task of placing primer–template mismatches at the 3' end of the primers — a property that is crucial for sequence selectivity. Once a list of candidate primers has been produced, sorting tools in PRISE speed up the selection process by allowing a user to sort the primers by properties such as amplicon length, GC content and sequence selectivity. PRISE can be used to design primers with a range of specificities, targeting individual sequences as well as diverse assemblages of genes. PRISE also allows user-defined primers to be analyzed, enabling their properties to be examined in relation to target and non-target sequences. The utility of PRISE was demonstrated by using it to design sequence-selective PCR primers for an rRNA gene from the fungus Pochonia chlamydosporia.
Tumor initiating activity of Helicobacter pylori water extract on mouse skin carcinogenesis
2003, Cancer Letters
Helicobacter pylori (H. pylori) infection has been associated with gastric carcinogenesis, but responsible and detail mechanisms are insufficient by the absence of adequate data. To obtain direct evidence regarding the carcinogenicity of H. pylori, we investigated the initiating and promoting activity of H. pylori water extract (HPE) in two-stage mouse skin carcinogenesis model. HPE treatment, as an initiation, significantly enhanced tumor formation compared with control group. Moreover, HPE treatment increased production of 8-hydroxydeoxyguanosine in epidermal cells and HPE-initiated/TPA-promoted papillomas demonstrated a point mutation of the Ha-ras gene. These results suggest an initiating activity of HPE on two-stage mouse skin carcinogenesis.
A novel activating mutation of the K-ras gene in human primary colon adenocarcinoma
2000, Biochemical and Biophysical Research Communications
We identified a novel type of point mutation at the 22nd codon of the K-ras gene in a primary colon cancer. The mutation was C to A transversion substituting lysine (AAG) for normal glutamine (CAG) codon. Biological activity of this mutant K-ras gene was tested by expression of full-length cDNA clones in NIH3T3 cells. Most of the K-ras Lys22-transfected cells exhibited an increased saturation density, a lower serum requirement, and transformed morphology reminiscent to the typical K-ras Val12 transformants. However, the tumorigenicity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants; only a high copy number transformant produced tumors. Even though the activation is incomplete, the finding that the majority of tumor cells in the specimen carried the K-ras Lys22 mutation suggests that this mutation might be advantageous for growth of tumor cells in vivo.
Discrimination of primer 3'-nucleotide mismatch by Taq DNA polymerase during polymerase chain reaction
2000, Analytical Biochemistry
We investigated the effect of primer–template mismatch on the efficiency of polymerase chain reaction. For primers with T, C, or G as the 3′ nucleotide, Thermus aquaticus (Taq) DNA polymerase was highly specific for template complementarity to this base, but was somewhat less constrained opposite the penultimate nucleotide. In contrast, primers with a 3′-terminal A were less efficiently amplified regardless of the corresponding nucleotide on the template strand. Thus, allele-specific PCR with Taq polymerase offers the greatest template discrimination (40- to 100-fold) against mismatch to a primer's 3′-terminal T, G, or C, but not A. Nucleotides at the penultimate position are responsible for roughly one-fifth as much mismatch discrimination (8- to 20-fold), and amplification efficiency is reduced when T and especially A occupy this primer position. We thus have defined conditions which allow robust discrimination for PCR-mediated analysis of single-nucleotide polymorphisms (SNPs), and for reduction in complexity of anchor–ligation PCR products.

View all citing articles on Scopus

View full text

Detection of ras point mutations by polymerase chain reaction using mutation-specific, inosine-containing oligonucleotide primers

Abstract

Gene

Cell

J. Biol. Chem.

Mutation Res.

Science

Science

Science

Science

Nature