台灣轉錄科技有限公司     分生器材網

PCRonBlood ^TM

產地：丹麥

無需前處理萃取   採用全血直接增幅

Traditional Method

Blood sample →Lymphocyte separation→DNA extraction→PCR→Detection

Method by PCRonBlood^TM

Blood sample→PCR→Detection

PCRonBlood^TM for Human Blood is delivered with or with-out Ampliqon Taq DNA Polymerase and dNTP (5mM) for direct amplification.

PCRonBlood^TM for Mouse blood is delivered with or with-out Ampliqon Taq DNA Polymerase and dNTP (5mM) for direct amplification.

Mammalian body fluids including blood contain many substances that inhibit the activity of enzymes such as Taq DNA Polymerase. Therefore it is generally necessary to remove the inhibitory substances and purify DNA before performing DNA analysis on cells present in these samples. 

Traditional preparation of DNA samples from mammalian cells includes overnight treatment with proteinase K, followed by purification by phenol/chloroform extraction and EtOH precipitation. Recently, many kinds of DNA isolation kits have become commercially available, so DNA isolation has become simpler than before, but is still time-consuming. Finally, the DNA isolation procedure generally increases the likelihood of sample contamination with foreign DNA, including DNA from samples processed earlier. 

A novel reagent PCRonBlood^TM capable of effectively neutralizing the substances in Human or Mouse blood that inhibit DNA amplification.

• Use of PCRonBlood^TM for Human Blood enables direct amplification of DNA from Human blood.
• Use of PCRonBlood^TM for Mouse Blood enables direct amplification of DNA from Mouse blood.

Three types of anticoagulants (sodium citrate, dipotassium EDTA and sodium heparinate) are acceptable.

Features of PCRonBlood^TM

• DNA present in blood can be amplified directly.
• No DNA extraction is required
• Only small volumes of blood are necessary for the procedure.
• Risk of cross contamination and/or procedural error is dramatically reduced.
• Blood (citrated or EDTA-treated blood is recommended) stored frozen for long periods can be analyzed.
• Easy PCR method permits mass screening and routine use in a broad field of applications.

Protocol for PCRonBlood^TM of Human Blood or Mouse Blood

Prepare the PCR reaction mixture with 5x PCRonBlood^TM
   ↓
Aliquot PCR reaction mixture to each PCR tube
   ↓
Add Human Blood/Mouse Blood to each tube
   ↓
Overlay mineral oil, if needed
   ↓
PCR

Experimental Result PCRonBlood^TM for Human Blood

PCRonBlood^TM on human blood treated with three different types of anticoagulants (sodium citrate, dipotassium EDTA and sodium heparinate) was examined. Human blood (0.68µl-5.0µl) treated with each anticoagulant was added to PCR reaction mixture (final 50µl) prepared using PCRonBlood^TM for Human Blood or Standard Buffer O. The target sequence (β-globin: 408 bp) was then amplified directly. 
O 10mM tris-HCl, pH 8.3, 50mM KCl and 1.5mM MgCl₂

Lane M
DNA marker

Lanes 1-4, for each anticoagulant
Direct amplification of human blood. Respective volumes of blood within each set were 5.0µl (Lane 1), 2.5µl (Lane 2), 1.25µl (Lane 3) and 0.68µl (Lane 4)

Lane N
Negative Control

PCR was performed at 94°C for 4.5 min, followed by 40 cycles of amplification (94°C for 30 s, 55°C for 1 min, 72°C for 1 min) followed by final extension (72°C for 7 min.)

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