JP5188228B2 - 蛍光標識された生体関連分子の検出方法 - Google Patents
蛍光標識された生体関連分子の検出方法 Download PDFInfo
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- JP5188228B2 JP5188228B2 JP2008078128A JP2008078128A JP5188228B2 JP 5188228 B2 JP5188228 B2 JP 5188228B2 JP 2008078128 A JP2008078128 A JP 2008078128A JP 2008078128 A JP2008078128 A JP 2008078128A JP 5188228 B2 JP5188228 B2 JP 5188228B2
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Description
(1)生体関連分子が固定化された担体を用いて、蛍光標識された生体関連分子を含む試料を分析する方法であって、担体に試料を接触させることにより、蛍光標識された生体関連分子と担体に固定化された生体関連分子とを相互作用させる相互作用工程、担体を洗浄することにより、担体に固定化された生体関連分子と相互作用しなかった生体関連分子を除去する洗浄工程、および担体を乾燥させることなく、担体に励起光を照射し、結像光学系の検出器で蛍光を検出する検出工程を含む前記方法。
(2) 前記検出器が結像光学系の検出器である、(1)の方法。
(3)洗浄工程において、潮解性物質を含む洗浄液を用いて担体を洗浄する、(1)又は(2)記載の方法。
(4)相互作用工程において、試料として、蛍光標識された生体関連分子とともに潮解性物質を含む試料を用いる、(3)記載の方法。
(5)潮解性物質が、塩化マグネシウム、水酸化マグネシウム、炭酸カリウム、臭化ナトリウム、及び塩化カルシウムからなる群から選択される、(3)又は(4)記載の方法。
(6)洗浄工程の後、励起光及び蛍光を透過するカバーで担体を覆うことにより担体の乾燥を防止する、(1)〜(5)のいずれかに記載の方法。
3mm角のシリコン基板にイオン化蒸着法を用いて、下記の条件で2層のDLC層の製膜を行った。
以下の5種のDNAプローブをSol.6で10μMに溶解し、前記で得られた担体上に横一行に4スポットずつスポットした。
TTATGACTCTGCCGCCGTCA(配列番号1)
CTGAAATTGAGAACGAAAAG(配列番号2)
ACGCACAGGAACTGAAGAAT(配列番号3)
CTGAGTGAATATATCGAACA(配列番号4)
TTGTCCGCGCCGGGCTTCGCTC(配列番号5)
80℃で1時間、ベーキングを行い、2×SSC/0.2%SDSで洗浄した後、超純水で洗浄し、遠心乾燥を行うことにより、担体にDNAプローブを固定化した。
ラムダファージのゲノムDNAを鋳型とし、上記5種のプローブとハイブリダイズする領域を以下のプライマーセットを用いてPCRで増幅した。
プライマー1:ACAGGGAATGCCCGTTCTGC(配列番号6)
プライマー2:AATAACCGACACGGGCAGAC(配列番号7)
標識は、CyDyeを用いて行った。PCR溶液の組成は以下のとおりとした。
実施例3で洗浄を行った後、乾かさない状態でカバーガラス(18mm角、0.15mm厚)を上から被せた。そして、図1に示される結像光学系の検出器を用いて、蛍光を検出した。
実施例3において、得られたPCR産物30μlを、ハイブリダイズ溶液(2×SSC/0.2%SDS)30μlに溶かして試料を調製した。試料を、DNAプローブを固定化した担体に滴下し、50℃で30分間静置し、湿箱に入れて反応を行った。続いて、以下の条件で洗浄を行った。
洗浄液2(1N MgCl2/0.5% Tween20)280μl×1分×1回、揺動
12 レーザー
13 レンズ
14 担体
15 吸収ホーン
16 スリット
17 蛍光
18 蛍光フィルター
19 CCD素子
Claims (5)
- 生体関連分子が固定化された担体を用いて、蛍光標識された生体関連分子を含む試料を分析する方法であって、
担体に試料を接触させることにより、蛍光標識された生体関連分子と担体に固定化された生体関連分子とを相互作用させる相互作用工程、
担体を、0.05〜1.0mol/lの濃度で潮解性物質を含む洗浄液を用いて洗浄することにより、担体に固定化された生体関連分子と相互作用しなかった生体関連分子を除去する洗浄工程、および
担体を乾燥させることなく、担体に励起光を照射し、結像光学系の検出器で蛍光を検出する検出工程
を含む前記方法。 - 検出工程において、担体表面とレーザー光のなす角度が30〜70°となるように、レーザーで一度に担体全体に励起光を照射し、担体の正面から結像光学系の検出器で蛍光を検出する、請求項1記載の方法。
- 相互作用工程において、試料として、蛍光標識された生体関連分子とともに潮解性物質を含む試料を用いる、請求項1又は2記載の方法。
- 潮解性物質が、塩化マグネシウム、水酸化マグネシウム、炭酸カリウム、臭化ナトリウム、及び塩化カルシウムからなる群から選択される、請求項1〜3のいずれか1項記載の方法。
- 洗浄工程の後、カバーガラスで担体を覆うことなく励起光を照射し、結像光学系の検出器で蛍光を検出する、請求項1〜4のいずれか1項記載の方法。
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