トリプルヘリックス形成によって媒介される酵母ゲノムDNAの単一サイト酵素分裂
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Nature
|March 14, 1991
まとめ
この研究では,オリゴヌクレオチド誘導型トリプルヘリックス形成を用いた精密なDNA分裂のための新しい方法が紹介されています. この技術により,大型ゲノムDNAの標的切断が可能になり,染色体マッピングと遺伝子分離が容易になります.
科学分野:
- 分子生物学は分子生物学である.
- ゲノミクスゲノミクスとは
- バイオテクノロジー バイオテクノロジー
背景:
- 染色体の物理的なマッピングには,制御されたDNA断片化の方法が必要です.
- 配列特異のDNA分裂反応剤は,結合特異性,モチーフ一般化性,分裂効率に関する課題に直面しています.
- オリゴヌクレオチド指向のトリプルヘリックス形成は,12塩基対以上の配列に特定のDNA結合のための一般化可能なアプローチを提供します.
研究 の 目的:
- 大型ゲノムDNAの高度に特異的な単一サイト酵素分裂のための方法を開発し,実証.
- 複雑なDNA分子のオーケストラによる分裂のためにトリプルヘリックス形成を適応させる.
- 広範な図書館のスクリーニングなしに,特定のゲノム領域の標的型分離を可能にします.
主な方法:
- オリゴヌクレオチド指向のトリプルヘリックス形成を使用して,特定のDNA配列を結合します.
- メチラゼ保護とトリプルヘリックス破壊を使用して,酵素へのアクセスを制御します.
- 対象部位に制限性エンドヌクレアース消化剤を適用する.
- テクニックをSaccharomyces cerevisiae染色体IIIで実証する.
主要な成果:
- 特定のEcoRI標的部位で340キロベース酵母染色体IIIのほぼ定量的な単一部位酵素分裂を達成しました.
- 予想される2つの分裂産物 (110キロ塩基と230キロ塩基) を生成し,他の染色体では非標的分裂が検出されなかった.
- DNAの少なくとも14メガベースペアの1つのサイトで,ほぼ定量的な割れ目を可能にする技術の能力を実証しました.
結論:
- オリゴヌクレオチドによるトリプルヘリックス形成と酵素的消化との組み合わせは,精密なゲノムDNA分裂のための強力なツールを提供します.
- この方法は,物理的なマッピングと標的型遺伝子分離戦略を大幅に進歩させる.
- この技術の汎用性は,大規模なゲノム研究と操作のための大きな可能性を秘めています.
関連する概念動画
Fixing Double-strand Breaks
The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
Gene Conversion
Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
DNA Isolation
DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
Single-Strand DNA Binding Proteins
For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
Maxam-Gilbert Sequencing
In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...
Challenges of the Maxam-Gilbert Method
The...


