An efficient method for the preparation of long heteroduplex DNA as substrate for mismatch repair by the Escherichia coli MutHLS system.
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 5'-phosphorylated, such that in two separate reactions two PCR products are obtained, which are 5'-phosphorylated in one strand. After removal of the phosphorylated strands by lambda-exonuclease, the resulting single strands are hybridized to form the mismatch-containing heteroduplex. The application of this procedure is demonstrated for the analysis of the Escherichia coli MutHLS system.
Thomas
Evangelos
E
Institut für Biochemie, Justus-Liebig-Universität, Giessen, Germany.
Pingoud
Alfred
A
Friedhoff
Peter
P
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D019175:DNA Methylation; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005092:Exonucleases / Q000737:chemistry; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D009692:Nucleic Acid Heteroduplexes / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D009692:Nucleic Acid Heteroduplexes; D004706:Endodeoxyribonucleases; D005092:Exonucleases; C059682:methyl-directed mismatch repair protein, E coli; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D045643:DNA Repair Enzymes