Restriction Digest PCR (RD-PCR) for the Analysis of Gene Mutations. Application to Ki-ras
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Klaus Roland Huber
Abstract
The Kirsten-ras (onco)gene codes for a GTP-binding membrane protein that is involved in signal transduction. Activated ras triggers a cascade of protein-phosphorylations that ultimately lead to cell proliferation. Ras-mutations are the main cause for adenocarcinomas of the pancreas besides some mutations in the tumor suppressor gene p53 and the c-erbB-2 oncogene. The site of ras mutations in pancreatic cancer is restricted to codon 12 that normally encodes a glycine. For analysis of codon-12 mutations, DNA is extracted from cells in pancreatic fluid and amplified by PCR. Because most of these cells originate from normal tissue with only a few tumor cells in the fluid, “enrichment PCR” must be utilized: In a first round of the PCR, ras sequences from all cells are amplified. By utilizing an appropriate restriction enzyme, wild-type sequences can be digested and the remaining fragments containing mutated sequences be amplified again. An artificial restriction site must be introduced by the 5′ primer (…GGA C̱CT GGT…) for an enzyme (BstNI) (5′CC!WGG 3′) to differentiate between wild-type sequence (…GGA G̱CT GGT…) (during amplification, the G̱ is replaced by a C̱) and mutated sequences (̱…GGA GCT (GTT), (CGT), (CCT), etc.). The necessary manipulations pose a considerable risk for contamination for the second round of the PCR procedure. Therefore, we considered whether it would be feasible to perform the restriction digest simultaneously with the first PCR reaction, and avoiding the secound round altogether.
The results of our experiments demonstrate that one tumor cell in 1000 normal cells can be determined readily, paralleling the results with the original two step-assay. The restriction enzyme used to enrich mutated sequences is stable long enough to be included into the PCR procedure. By this, wild-type sequence amplicons are digested while they are formed and mutated sequences can be enriched selectively.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
Articles in the same Issue
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- Does Clinical Chemistry Have a Future?
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- The Human Genome Project and the Role of Genetics in Health Care
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- Population Genomics: Laying the Groundwork for Genetic Disease Modeling and Targeting
- Oligonucleotide-Based Gene Therapy for Cardiovascular Disease
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Articles in the same Issue
- Human Genomics: The Basis of the Medicine of Tomorrow. Recent Progress in Molecular Biology Technology. First IFCC-Roche Conference – Singapore, 15–18 March 1998
- Does Clinical Chemistry Have a Future?
- The Human Genome Project: From Mapping to Sequencing
- The Human Genome Project and the Role of Genetics in Health Care
- Mutation Detection and Mutation Databases
- Population Genomics: Laying the Groundwork for Genetic Disease Modeling and Targeting
- Oligonucleotide-Based Gene Therapy for Cardiovascular Disease
- Signal Transduction Pathways and Modulation of Gene Activity
- Do Overexpressed Oncoproteins Cause Malignant Growth of Cancer Cells? – Studied by Antisense Oligonucleotides
- Ethical and Legal Issues in the Procurement, Storage and Use of DNA
- DNA Extraction and Stability for Epidemiological Studies
- Simple and Broadly Applicable Sample Preparation by Use of Magnetic Glass Particles
- A Multilocus Genotyping Assay for Cardiovascular Disease
- Optimized Non-Radioactive Protein Truncation Test for Mutation Analysis of the Adenomatous Polyposis Coli (APC) Gene
- Monitoring of Inhibitors of Enzymatic Amplification in Polymerase Chain Reaction and Evaluation of Efficacy of RNA Extraction for the Detection of Hepatitis C Virus Using the Internal Control
- Quantification of Relative Expression of Genes with Homologous Sequences Using Fluorescence-Based Single-Strand Conformation Polymorphism Analysis – Application to Lactate Dehydrogenase and Cyclooxygenase Isozymes
- Automation of Polymerase Chain Reaction-Based Systems for Detection of Hepatitis C Virus RNA
- A Simple PCR with Different 3′ Ends of the Third Primer for Detection of Defined Point Mutations: HCV Genotyping as an Example
- Two Quantitative Methods for Imaging Samples in Molecular Biology
- Restriction Digest PCR (RD-PCR) for the Analysis of Gene Mutations. Application to Ki-ras
- Direct Detection of Mycobacterium tuberculosis in Respiratory Specimens Using an Automated DNA Amplification Assay and a Single Tube Nested Polymerase Chain Reaction (PCR)
- Quantitative Detection of Hepatitis B Virus DNA with a New PCR Assay
- Molecular Diagnostics for Cardiovascular Disease
- Association of Apolipoprotein E Polymorphism and Concentration with Serum Lipids and Apo-lipoprotein Level in the Chinese fromShanghai
- Angiotensin Converting Enzyme Gene Polymorphism in Coronary Artery Disease: The Indian Scenario
- Mutation C677T of Methylenetetrahydrofolate Reductase Gene Is Not Associated with Coronary Artery Disease, but Possibly with Albuminuria, in Type 2 Diabetic Patients
- Cholesteryl Ester Transfer Protein Mutations, Protein Activity and HDL-Cholesterol Concentration
- Molecular Biology of Alcohol Dependence, a Complex Polygenic Disorder
- Analysis of Clonality in T-Lymphoproliferative Diseases by Multiplex PCR
- The Impact of Molecular Diagnosis on Familial Colorectal Cancer
- Screening for Tumour Suppressor p16(CDKN2A) Germline Mutations in Israeli Melanoma Families
- CGG Repeat Interruptions in the FMR1 Gene in Patients with Infantile Autism
- Detection of CYP2D6*3 and 2D6*4 Allelic Variants by PCR-Restriction Fragment Length Polymorphism
- Genetic Basis of Congenital Hypothyroidism: Abnormalities in the TSHβ Gene, the PIT1 Gene, and the NIS Gene
- Follitropin (FSH) Deficiency in an Infertile Male due to FSHβ Gene Mutation. A Syndrome of Normal Puberty and Virilization but Under-developed Testicles with Azoospermia, Low FSH but High Lutropin and Normal Serum Testosterone Concentrations
- Molecular Analysis and Electromyoneurographic Abnormalities in Croatian Children with Proximal Spinal Muscular Atrophies
