JP2019506625A - ゲノム配列決定及び他の適用における極端紫外放射線 - Google Patents
ゲノム配列決定及び他の適用における極端紫外放射線 Download PDFInfo
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Abstract
Description
本出願は2016年2月1日に出願された米国仮特許出願第62/289,897号の恩典を主張し、該仮出願の内容はその全体が参照により本明細書に組み込まれている。
次世代配列決定法(NGS)及びSanger配列決定法などの配列決定技術は、配列遺伝子及びゲノムの塩基対を読み取り、かつ同定するために使用される。ヒトゲノム中にはおよそ32億塩基対が存在するため、大規模で同定し、配列決定するには、ハイスループット配列決定機器が必要となる。次世代配列決定法は蛍光標識ターミネータを使用する合成及び大量の並列処理によって配列決定して、遺伝子構造及び多型の正確な同定を提供するような技術を含む。CRISPRのような技術は遺伝子配列を編集するために使用される。特殊な酵素を使用して遺伝子配列セット内の所与の配列を標的化し、識別した配列を繰り返し除去する。これが最先端の技術である。
一態様において、本件開示は一般に生物学的細胞の内側又は外側のいずれかで、遺伝子又は遺伝子配列を選択的に検出し、読み取り、同定するバイオテクノロジー適用における極端紫外(EUV)放射線、軟X線放射線の使用に関する。本件開示はさらに、ゲノム配列内の所与の位置で動的に、かつ/又は無作為に、かつ/又は選択的に遺伝子を編集すること、欠失させること、変化させること、かつ/又は修復することに関する。本明細書では、その全てが配列決定法に複雑さ、不正確さ、及び時間をもたらすDNA合成、生体分子タグ付加、蛍光標識、DNA複製、酵素的開裂、並列処理、カバレッジ深度(depth of coverage)、参照ゲノムとのアラインメント、ナノポアチャネル、ショットガン配列決定法、イオン検出、又はヌクレオチド付加を必要とすることなく選択的に、かつ非順次に配列決定する方法を提供する。
本件開示は、添付の図面と併せて解釈して、以下の詳細な説明に関してより十分に認識することができる。ここで:
本明細書では、高分子を検出し、読み取り、同定し、かつ/又は編集するために有用な装置及び方法を提供する。
本明細書で提供する装置及び方法を参照する場合、別途示されない限り、以下の用語は以下の意味を有する。別途定義されない限り、本明細書で使用される全ての技術及び科学用語は、当業者によって共通に理解されるものと同じ意味を有する。本明細書の用語に対し複数の定義がある場合、別途言及のない限り本節の定義が優先する。
本明細書では、高分子を検出し、読み取り、同定し、かつ/又は編集するために有用な装置を提供する。装置は一般に、放射線源、放射線の少なくとも一部を吸収するように構成された1以上の高分子、透過放射線及び/又は吸収放射線を検出することの可能な検出器を備える。
高分子は、当業者が好適であると考える任意の高分子とすることができる。ある実施態様において、高分子はポリマーである。ある実施態様において、高分子はポリペプチドである。ある実施態様において、高分子はペプチド又はタンパク質である。ある実施態様において、高分子はポリヌクレオチドである。ある実施態様において、高分子はDNAである。ある実施態様において、高分子はRNAである。ある実施態様において、高分子はオリゴ糖である。
本明細書で提供する方法において、放射線源は放射線を生成する。放射線は、放射線の少なくとも一部を吸収する高分子と接触する。高分子によって吸収され、かつ/又は透過された放射線を検出器によって検出する。
(実施例1)
分子スペクトルの評価において、各核酸塩基を0 nm〜5.0 nmの波長のEUV放射線と接触させる。各核酸塩基の吸収スペクトルを計算して図1で提供し、透過スペクトルを図4で提供する。C、G、A、及びTはそれらの固有の分子組合わせ及び密度のために固有のスペクトルを有する。いくつかの事例において、スペクトルシグネチャーは2.2 nm(酸素)、2.8 nm(窒素)、及び4.3 nm(炭素及び水素)の波長で図4の3つのスペクトル降下(グアニン)からなり、いくつかのそのような事例においてスペクトルシグネチャーは2.8 nm及び4.3 nmの波長での2つのスペクトル降下(アデニン)からなる。さらに、各々のスペクトル降下の相対的スペクトル強度は、高分子の各核酸塩基中に存在する酸素原子(2.3 nmの波長)、窒素原子(2.8 nmの波長)、及び炭素原子(4.3 nmの波長)の数に比例する。H原子は相対的に透過性である。このように、有機分子、ペプチド、アミノ酸を同定し、それらの既知の構造と相互に関連付けることができる。さらに、既知の対合構造に関する情報及び空間情報は、高分子成分をさらに識別する一助となる。
いくつかの事例において、高分子は識別子原子、例えば塩素原子を有する。この事例において追加のスペクトル降下がもう1つの波長、例えば6.5 nmで観察される。EUV及び軟X線のプラズマ源は、そのスペクトル範囲が広いため、固有の原子を有する一部の高分子成分は容易に同定できる。
Claims (26)
- 高分子による吸収を検出するための装置であって:
a. 0.1 nm〜250 nmの波長を有する放射線を送出するように構成された放射線源;
b. 任意に、該放射線を集束させることが可能な1以上の集束構成要素;
c. 該放射線の少なくとも一部を吸収するように構成された高分子;
d. 該高分子によって吸収された放射線を検出することが可能な検出器であって、高分子の配列を検出するために使用される、前記検出器を備える、前記装置。 - 前記放射線源が極端紫外線源(EUV)である、請求項1記載の装置。
- 前記放射線源が軟X線源である、請求項1記載の装置。
- 前記放射線を集束させることが可能な前記1以上の集束構成要素が存在し、かつ前記高分子に該放射線を集束させることが可能な1以上のミラー、レンズ、又は反射鏡、及びそれらの組合わせから選択される、請求項1記載の装置。
- 前記高分子に前記放射線を集束させることが可能な1以上のミラーを備える、請求項1記載の装置。
- 前記高分子を前記放射線の範囲内で接触させるように構成されたステージを備える、請求項1記載の装置。
- 前記ステージが、前記高分子を前記放射線の範囲内で並進させるように構成されている、請求項6記載の装置。
- 前記高分子が、ゲノム配列、DNA配列、RNA配列、オリゴヌクレオチド、ヌクレオチド、塩基対、一塩基多型、突然変異、コピー数多型、リード、タンパク質配列、アミノ酸、ペプチド、塩基対配列、細菌、対立遺伝子、染色体、又は分子である、請求項1〜7のいずれか1項記載の装置。
- 前記高分子がペプチド又はタンパク質である、請求項1〜8のいずれか1項記載の装置。
- 前記高分子が核酸である、請求項1〜9のいずれか1項記載の装置。
- 前記検出器からの吸収スペクトルを前記高分子の配列へと変換するように構成された、構成要素をさらに備える、請求項8記載の装置。
- 高分子の配列を検出するための、請求項1〜11のいずれか1項記載の装置。
- 高分子の配列を読み取るための、請求項1〜12のいずれか1項記載の装置。
- 高分子の配列を編集するための、請求項1〜13のいずれか1項記載の装置。
- 1以上の遺伝子配列を検出し、読み取り、同定し、かつ編集するための装置であって、
―0.1 nm〜250 nmの範囲の波長を有する光を送出するように構成された、EUV又は軟X線光源、
―光スポットサイズを集束させるためのミラー、レンズ、又は反射鏡、
―配列決定するべき生体物質、
―該配列を同定する吸収スペクトルを備える、前記装置。 - ゲノム配列、DNA配列、RNA配列、オリゴヌクレオチド、ヌクレオチド、塩基対、一塩基多型、突然変異、 コピー数多型、リード、タンパク質配列、アミノ酸、ペプチド、塩基対配列、細菌、対立遺伝子、染色体、分子からなる請求項15記載の1以上の生体物質を検出し、読み取り、同定し、かつ編集するための装置。
- 少なくとも6つの投影ミラー及びプラズマ光源を備える投影レンズ系を使用する、請求項15記載の装置。
- 遺伝子型判定に使用する、請求項15記載の装置。
- ゲノム地図を作成するために使用する、請求項15記載の装置。
- 細胞内でゲノム配列を1以上の次元でマッピングするための、請求項15記載の装置を使用する方法。
- DNA塩基、RNA塩基、タンパク質、既知の遺伝子配列、物理的座標(これらの任意の組合わせを含む)についての特性EUV又は軟X線吸収スペクトルのデータベース又はライブラリ。
- ゲノムの3Dマップを形成する、請求項21記載のライブラリ。
- 自己学習アルゴリズム又は予測的配列決定アルゴリズムのための参照を提供する、請求項1記載のライブラリ。
- 強く集束したEUV又は軟X線放射線を標的配列に送達して、該配列中の塩基を切り離す、配列編集機構。
- 複数の固有の標的配列を編集する、請求項23記載の方法。
- 自己学習アルゴリズム又は予測的配列アルゴリズムを使用して、請求項1記載の装置を使用することから導かれる遺伝子配列の同定。
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