Reagents for Eprobe® PCR : Technology Overview

□Technical Information

Eprobe®, which was jointly developed by DNAFORM and RIKEN, is a synthetic oligonucleotide-based fluorescent probe that has an excitonic interaction. Performing polymerase chain reaction (PCR) with Eprobe® allows for specific quantification of target DNA sequences and also enables base mutation detection.

□Function of Eprobe® in real-time PCR

Eprobe® is a fluorescence-labeled oligonucleotide with one modified thymine carrying two dye moieties. When Eprobe® forms a double-stranded structure with target DNA sequences, the two dyes in Eprobe® are separated and emit a strong fluorescence caused by disruption of the excitonic interaction between dyes. Eprobe®, when added in sufficient amounts to the reaction mixture for real-time PCR, also binds to its target DNA sequence during the primer annealing step. By measuring the fluorescence generated upon binding of Eprobe®, only the target DNA sequence is quantified.

This Eprobe®-mediated real-time PCR can also be applied to the detection of base mutations. In post-PCR melting curve analysis, differences in melting temperature (Tm) between when Eprobe® binds to a perfectly matched wild-type base sequence and when the same Eprobe® binds to mismatched mutated base sequence(s) are expected. By analyzing the differences in Tm, the presence or absence of mutated DNA sequence(s) can be assessed.

□Features of Eprobe® PCR

□Eprobe® generates a fluorescence signal upon hybridization to its target nucleotide sequence.
□The use of a single Eprobe® allows for both real-time PCR and melting curve analysis.
□Higher melting temperature (Tm values) compared to normal DNA oligonucleotides are observed.

□Applications

□細菌やウイルスなど感染症の高感度検出。
□遺伝子発現の定量。
□SNP検出。
□がんなどの疾患原因遺伝子の微量変異検出。

Eprobe® PCR法については、Eprobe®/Eprimer™の技術情報もご参照ください。

→Custom Eprobe®/Eprimer™ Synthesis : Technology Overview

□Typical conditions

□E-Taq 2xPCR Mixの使用方法

□ホットスタートPCR用酵素なので、Taqポリメラーゼの活性化のためにサイクルの前に98℃で5?10秒、もしくは94℃で20〜30秒の活性化ステップを行ってください。
□長時間の活性化は酵素にダメージが加わりますので、上記条件にて活性化してください。
□72℃での伸長速度：1 min/Kb。
□使用するテンプレートは精製したものを推奨します。
□テンプレート使用量は500 ng/PCR以下を推奨します。

□典型的な反応組成

	- E-Taq 2xPCR Mix                   10μL
	- Template                       <500 ng
	- Primer1                      0.2-1.0μM
	- Primer2                      0.2-1.0μM
	- Sterilized distilled water     to 20μL

□E-Taq DNA polymeraseの使用方法

□ホットスタートPCR用酵素なので、Taqポリメラーゼの活性化のためにサイクルの前に98℃で5?10秒、もしくは94℃で20〜30秒の活性化ステップを行ってください。
□長時間の活性化は酵素にダメージが加わりますので、上記条件にて活性化してください。
□72℃での伸長速度：1 min/Kb。
□使用するテンプレートは精製したものを推奨します。
□テンプレート使用量は500 ng/PCR以下を推奨します。

□典型的な反応組成

	- 5U/μL E-Taq DNA polymerase       0.1 μL
	- E-Taq buffer (x10)                 2 μL
	- 10mM dNTP                        0.4 μL
	- Template                       <500 ng
	- Primer1                      0.2-1.0 μM
	- Primer2                      0.2-1.0 μM
	- Sterilized distilled water     to 20 μL

□Calculation of Tm in Eprobe®-mediated PCR

When introduced into Eprobe® (or Eprimer™), thiazole orange serves to increase the Tm of the hybridization product ? i.e., Eprobe®/DNA (or Eprimer™/DNA) duplex. The predicted Tm for Eprobe® (or Eprimer™) can be calculated using an online tool (available on the following website) by placing “Z” in the position of thiazole orange-labeled thymidine in the oligonucleotide.

→RIKEN > Hybridization thermodynamics of ECHO/DNA duplexes

*The data available on the website is based on publications by RIKEN.

K.K. DNAFORM