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An Efficient Method for the Preparation of Long Heteroduplex DNA as Substrate for Mismatch Repair by the Escherichia coli MutHLS System

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Published/Copyright: June 1, 2005
Biological Chemistry
From the journal Volume 383 Issue 9

Abstract

We present a method that allows preparing long DNA containing defined mismatches without the use of gel electrophoretic or chromatographic purification steps. The preparation starts with the synthesis of two PCR products, which are identical except for those positions that will later form the mismatches. One of the PCR primers must be 5phosphorylated, such that in two separate reactions two PCR products are obtained, which are 5phosphorylated in one strand. After removal of the phosphorylated strands by λexonuclease, the resulting single strands are hybridized to form the mismatchcontaining heteroduplex. The application of this procedure is demonstrated for the analysis of the Escherichia coli MutHLS system.

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Published Online: 2005-06-01
Published in Print: 2002-09-17

Copyright © 2002 by Walter de Gruyter GmbH & Co. KG

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https://doi.org/10.1515/BC.2002.166

Articles in the same Issue

  1. Kaposis Sarcoma and Human Chorionic Gonadotropin: Mechanisms, Moieties and Mysteries
  2. Dimerization Inhibitors of HIV-1 Protease
  3. A Dimeric Mutant of the Homotetrameric Single-Stranded DNA Binding Protein from Escherichia coli
  4. The Hsp90 Co-Chaperones Cdc37 and Sti1 Interact Physically and Genetically
  5. Cloning and Sequence Analysis of Galleria mellonella Juvenile Hormone Binding Protein A Search for Ancestors and Relatives
  6. Neuronal NO Synthase and Its Inhibitor PIN Are Present and Influenced by Glucose in the Human β-Cell Line CM and in Rat INS-1 Cells
  7. Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme
  8. The in vitro Assembly of Hair Follicle Keratins: Comparison of Cortex and Companion Layer Keratins
  9. Purification, Structural and Immunological Characterization of a Timothy Grass (Phleum pratense) Pollen Allergen, Phl p 4, with Cross-Reactive Potential
  10. Muscarinic Toxin-Like Proteins from Taiwan Banded Krait (Bungarus multicinctus) Venom: Purification, Characterization and Gene Organization
  11. Degradation of Extracellular Matrix Protein Tenascin-C by Cathepsin B: An Interaction Involved in the Progression of Gliomas
  12. Flexibility Analysis and Structure Comparison of Two Crystal Forms of Calcium-Free Human m-Calpain
  13. Detection and Quantification of Insertion/Deletion Variations by Allele-Specific Real-Time PCR: Application for Genotyping and Chimerism Analysis
  14. Reconstitution of Phospholipid Flippase Activity from E. coli Inner Membrane: A Test of the Protein Translocon as a Candidate Flippase
  15. Identification of an Allergen Related to Phl p 4, a Major Timothy Grass Pollen Allergen, in Pollens, Vegetables, and Fruits by Immunogold Electron Microscopy
  16. Subunit Association and Conformational Flexibility in the Head Subdomain of Human CD81 Large Extracellular Loop
  17. Foldase Function of the Cathepsin S Proregion Is Strictly Based upon Its Domain Structure
  18. An Efficient Method for the Preparation of Long Heteroduplex DNA as Substrate for Mismatch Repair by the Escherichia coli MutHLS System
  19. A Simple Single-Step Method for the Synthesis of Recombinant Non-Homologous Competitor cDNA and Its Implications for Quantitative PCR in Biological Sciences and Molecular Diagnostics
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