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PCR DIG Probe Synthesis Kit
For generation of highly sensitive probes labeled with DIG-dUTP (alkali-labile) in polymerase chain reaction.
Application
The kit is specially designed for the generation of highly sensitive hybridization probes that are suitable for the detection of low- (single-) copy target sequences. Thus, it should be considered as an alternative to random-primed labeling when either the amount of template DNA is limited or only a specific part of the sequence of the template is required for hybridization. The nucleotide concentration in the PCR DIG Probe Synthesis Kit ensures the identification of single-copy genes in the genomic blots following hybridization to DIG-labeled PCR products. Human single-copy genes are detectable in 10 µg of genomic DNA.
PCR products can be directly generated and labeled from small amounts of genomic DNA (100 ng–1 µg), and subsequently used as hybridization probes.
The PCR DIG Probe Synthesis Kit contains an alkali-labile DIG-11-dUTP formulation. This enables simple removal of the DIG label following chemiluminescent detection, and allows the subsequent rehybridization of blots with multiple DIG-labeled probes.
Background Information
The nonradioactive DIG system uses digoxigenin, a steroid hapten, to label DNA, RNA, or oligonucleotides for hybridization, and subsequent color- or luminescent detection. The digoxigenin is coupled to dUTP via an alkali-labile ester bond. The labeled dUTP can be easily incorporated by enzymatic nucleic-acid synthesis using DNA polymerases.
The polymerase chain reaction (PCR) allows the amplification of minute amounts of DNA to levels above 1 µg. The only prerequisite is that some sequence information of the target sequence is needed in order to synthesize the appropriate primers.
The combination of nonradioactive labeling with PCR is a powerful tool for the analysis of PCR products, and also for the preparation of labeled probes from small amounts of a respective target sequence. The detection sensitivity of these labeled probes can be adjusted by the ratio of the unlabeled nucleotides to DIG-dUTP. Whereas a low ratio of DIG-dUTP to dTTP of 1:19, as in the PCR DIG Labeling Mix, ensures a high-yield amplification reaction with good sensitivity for direct detection, the hybridization to single-copy genes in complex genomes may not be successful. The PCR DIG Probe Synthesis Kit, therefore, contains a high ratio of DIG-dUTP to unlabeled nucleotides of 1:2, ensuring reliable detection of single-copy genes. However, PCR yield may be decreased by up to 50%. Nevertheless, this ratio will yield enough product for several hybridizations.
Contents
1.Enzyme Mix, Expand High Fidelity, 30 µl (105 U) Enzyme Mix, 3.5 U/µl in storage buffer, 20 mM Tris-HCl, pH 7.5 (25oC), 100 mM KCl, 1 mM dithiothreitol (DDT), 0.1 mM EDTA, 0.5% Tween 20 (v/v), 0.5% Nonidet P40 (v/v), 50% glycerol (v/v)
2.PCR DIG Probe Synthesis Mix, 10x conc., 125 µl of a mixture containing dATP, dCTP, dGTP (2 mM each), 1.3 mM dTTP, 0.7 mM DIG-11-dUTP, alkali-labile, pH 7.0
3.PCR Buffer with MgCl2, 10x conc., 1 ml Expand High Fidelity Buffer, 10x conc. with MgCl2
4.dNTP Stock Solution, 10x conc., 125 µl of a mixture containing dATP, dCTP, dGTP, dTTP (2 mM each), pH 7.0
5.Control Template, 50 µl (1 ng) plasmid DNA (20 pg/µl) in Tris/EDTA buffer, pH 8.0. The 5-kb plasmid contains the cDNA for the human tissue-type plasminogen activator (tPA)
6.Control PCR Primer Mix, 25 µl (containing 50 pmol of each primer) control PCR primer 1 and 2 (2 mM each)
Quality
Function test: Each lot of the PCR DIG Probe Synthesis Kit is function-tested in PCR. Amplification products are assayed |