ラーニングコーナー

2017/10/26

Dynabeadsアプリケーション紹介 ゲノム解析、遺伝子発現解析

  • タンパク・核酸発現解析

ここで紹介するのはDynabeads™磁気ビーズを用いたDifferential display(ディファレンシャルディスプレイ)法、5'RACE法、ヌクレアーゼS1 (S1ヌクレアーゼ・マッピング)法、DNase Iフットプリント法、RNA/DNA結合性タンパクの精製と腫瘍特異性の変更の隔離と病原体中の毒素遺伝子の識別のためのSubtractive hybridizationなどゲノム解析、遺伝子発現解析に関わるアプリケーションです。

例えばライブラリからcDNAのクローニングとシーケンシングに代表される多くの遺伝子発現プロファイリング法において「Dynabeads™ Streptavidinシリーズ」あるいは「Dynabeads™ Oligo(dT)25」(mRNA purificationシリーズ)が多数利用されています。

2kbより長い二重鎖DNAは「Dynabeads™ kilobase BINDERキット」の利用が効果的です。

Dynabeads Streptavidinの特長

なぜゲノム分析、遺伝子発現解析にDynabeads Streptavidinが選ばれるのか?

  • 簡便なハンドリング
  • 高い反応速度
  • 低い非特異結合
  • 純粋で均一な転写制御因子を30分で単離
  • タンパク質の単離に最適
  • DNA/RNA結合タンパク質を最大20,000倍に濃縮
  • Dynabeads streptavidinに結合したDNAは、少なくとも10回再利用可

アプリケーション例

Reverse transcription PCR

  • Jost R et al, (2007) Biotechniques 43: 206-211. Magnetic quantitative reverse transcription PCR: A high-throughput method for mRNA extraction and quantitative reverse transcription PCR.

 

DNA/RNA binding protein isolation

  • Mehta A et al. (1998). A sequence-specific RNA binding protein complements Apobec-1 to edit apolipo protein B mRNA. Mol. Cel. Biol. 18(8):4426-4432.
  • Biroccio A. et al. (2002). Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase. J. Virol. 76(8):3688-3696.
  • Nordhoff E. et al. (1999). Rapid identification of DNA-binding proteins by mass spectrometry. Nat. Biotechnol. 17: 884-888.
  • Brodsky AS. and Silver A. (2002). A microbead based system for identifying and characterizing RNA-protein interactions by flow cytometry. Mol. Cel. Proteomics 1(12):922-929.

Solid-phase DNase footprinting

  • Fletcher TM. et al. (2002). Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition. Mol. Cel. Biol. 20(17): 6466-6475.

 

Solid-phase S1 nuclease mapping

  • Dziembowski A. et al. (2001). Analysis of 3’ and 5’ ends of RNA by solid-phase S1 nuclease mapping. Anal. Biochem. 294:87-89.

 

Subtractive hybridization

  • Hansen-Hagge TE. et al. (2001). Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation mediated subtraction (Limes). Nucl. Acids Res. 29(4):e20.
  • Pradel N. et al. (2002). Genomic subtraction to identify and characterize sequences of Shiga toxin-producing Escherichia coli O91:H21. Appl. Env. Microbiol. 68(5):2316-2325.
  • Laveder P. et al. (2002). A two-step strategy for constructing specifically self-subtracted cDNA libraries. Nucleic Acids Res. 30(9): e38

 

Differential display

  • Kornmann B. et al. (2001). Analysis of circadian liver gene expression by ADDER, a highly sensitive method for the display of differentially expressed mRNAs. Nucleic Acids Res. 29 (11). e51
  • Brenner S. et al. (2000). In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs. PNAS. 97(4): 1665-1670.

 

Limes

  • Hansen-Hagge TE. et al. (2001). Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation mediated subtraction (Limes). Nucl. Acids Res. 29(4):e20.

 

5’ RACE

  • Schramm G. et al. (2000).A simple and reliable 5’-RACE approach. Nucl. Acids Res. 28(22):e96

 

SAGE®

  • Velculescu VE. et al. (1995). Serial analysis of gene expression. Science. 270(5235): 484-487.

 

TOGA

  • Sutcliffe JG. et al. (2000). TOGA: An automated parsing technology for analyzing expression of nearly all genes. PNAS. 97(5): 1976-1981.

 

RAGE

  • Wang A. et al. (1999). Rapid analysis of gene expression (RAGE) facilitates universal expression profiling. Nucleic Acids Res. 27(23): 4609-4618.

 

Double stranded DNA fragments > 2 kB

  • Fletcher TM. et al. (2002). Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition. Mol. Cel. Biol. 20(17): 6466-6475.
  • Heald R. et al. (1996) Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382:420-425.

 

S1 nuclease mapping

  • Lindblad-Toh K. et al. (2000). Large-scale discovery and genotyping of single nucleotide polymorphisms in the mouse. Nature Genetics. 24:381-386.

 

RTPCR/ECL

  • Miyashiro I. et al. (2001). Molecular strategy for detecting metastatic cancers with use of multiple tumor-specific MAGE-A genes. Clin. Chem. 47(3):505-512.

 

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