Prime-It Random Primer Labeling Kits

Prime-It 隨機放射標定試劑

Prime-It II Random Primer Labeling Kit

Random oligonucleotides have been used as primers in a method for labeling DNA to produce high specific-activity probes. The procedure relies on the ability of random hexanucleotides to anneal to multiple sites along the length of a DNA template. The primer–template complexes formed represent a substrate for the Klenow fragment of DNA polymerase I. The enzyme synthesizes new DNA by incorporating nucleotide monophosphates at the free 3´–OH group provided by the primer. The newly synthesized DNA is made radioactive by substituting a radiolabeled nucleotide for a nonradioactive one in the reaction mixture. The resulting labeled DNA can serve as a sensitive hybridization probe for screening gene libraries, probing Southern and northern blots, and in situ hybridization techniques.

Conventional protocols for random oligo-labeling of DNA require a minimum of 30 minutes to perform, and many suggest an overnight incubation. The Prime-It II random primer labeling kit makes it possible to produce high-specific-activity DNA probes within two minutes. To achieve this rate of incorporation, we used a 3´ exonuclease-deficient mutant of the Klenow fragment of DNA polymerase I [Exo(–) Klenow] and random nonamer primers (9-mers). High-specific-activity probes can be produced from fragments purified by a variety of techniques, including low-melting-temperature (LMT) agarose gels.

Prime-It RmT Random Primer Labeling Kit

The Prime It RmT random primer labeling kit uses random oligonucleotides as primers for labeling DNA to produce high-specific-activity probes. The procedure relies on the ability of random 9-mers to anneal to multiple sites along the length of a DNA template. The primer–template complexes formed represent a substrate for the magenta DNA polymerase, a thermostable polymerase that allows the Prime-It RmT random primer labeling kit to be stored at room temperature. The enzyme synthesizes new DNA by incorporating nucleotide monophosphates at the free 3´-OH group provided by the primer. The newly synthesized DNA is made radioactive by substituting radiolabeled [a-32P]dCTP for unlabeled dCTP in the reaction mixture. The resulting labeled DNA serves as a sensitive hybridization probe for screening gene libraries, probing Southern and Northern blots, and in situ hybridization techniques.

DNA labeling reaction components (random primers, dNTPs, buffers and cofactors) are supplied as a dehydrated reaction mixture, pre-aliquoted in 24 single-use reaction tubes. The Prime-It RmT random primer labeling kit also includes magenta thermostable polymerase, and dehydrated control DNA, allowing storage of the kit at room temperature (Figure 2).Conventional protocols for random oligonucleotide labeling of DNA require a minimum of 30 minutes to perform, and many suggest an overnight incubation. The Prime-It RmT random primer labeling kit produces high-specific-activity probes (>1 × 109 dpm/µg) in just 5 minutes.