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DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments
Published on: January 27, 2016
One-base excess adaptor ligation method for walking uncloned genomic DNA
Yuki Tonooka1, Yoichi Mizukami, Masahiro Fujishima
1Department of Biology and Chemistry, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8512, Japan. tonooka@crc.yamaguchi-u.ac.jp
Applied Microbiology and Biotechnology
|December 12, 2007
Summary
This study introduces a new method using oligo cassette-mediated PCR to sequence unknown genomic DNA regions. The technique utilizes T4 DNA ligase
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Genomic DNA sequencing is crucial for understanding gene function.
- Existing methods for sequencing unknown flanking regions can be inefficient.
Purpose of the Study:
- To develop a novel and efficient method for genomic DNA sequencing.
- To determine flanking sequences of the catalase gene in Paramecium bursaria.
Main Methods:
- Oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction (PCR).
- Genomic DNA digestion with restriction enzymes to create sticky ends.
- Ligation of a one-base excess oligo-adaptor using T4 DNA ligase.
- Exploitation of T4 DNA ligase's exonuclease activity for specific amplification.
Main Results:
- A novel method for walking genomic DNA sequences was established.
- The technique demonstrated efficient amplification of target sequences.
- The 3'- and 5'-flanking sequences of the Paramecium bursaria catalase gene were successfully determined.
Conclusions:
- The developed oligo cassette-mediated PCR technique is an efficient method for genomic DNA sequencing.
- This method allows for the determination of unknown flanking DNA sequences.
- The study successfully mapped flanking regions of a specific gene in a ciliate model organism.
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