TB #157: Maximize DNA Probe Sensitivity

Random priming is a widely used technique for generating labeled DNA probes based on a method developed by Feinberg and Vogelstein (1983). The specific activity of DNA probes is an important parameter to control, since it determines the sensitivity of blot hybridization reactions. In many cases, maximizing specific activity is desirable, since this in turn permits detection of targets present at very low levels in a sample. Figure 1 shows the effect of probe specific activity on the limits of target detection. In this experiment five different amounts of template DNA were labeled in DECAprimeĂ II reactions and hybridized to individual nylon strips containing serial dilutions (2 fold) of target DNA. Note that probe made with the smallest amount of template DNA (6.25 ng) was able to detect target present at 1/4 to 1/8 the level as the probe made with the largest amount of template (100 ng). There is a trade-off between probe yield and probe specific activity when using the random priming method for labeling DNA. The larger the amount of DNA template used, the greater the yield of probe. However, large amounts of template result in lower specific activity since the unlabeled template competes with the labeled probe for target. This principle is illustrated in Figure 2.

Figure 1. Effect of Probe Specific Activity on the Limit of Target Detection. Serial dilutions (2 fold) of a plasmid containing a Human c-myc gene were blotted onto nylon membrane strips. Strips were hybridized with 4 x 10⁶ cpm/ml ³²P-labeled probe of decreasing specific activities synthesized by adding increasing amounts of template DNA to DECAprime™ II reactions. Strips were autoradiographed with XAR-5 film.

Figure 2. Effect of DNA Template Concentration on Probe Specific Activity in a DECAprime II Reaction. The incorporation of [alpha-³²P]dATP (50 µCi, 3000 Ci/mmol) with varying concentrations of 3 kb template DNA was used to determine the specific activity of the probes. A 20 µl reaction volume was incubated at 37°C for the indicated times.

Ambion's Strip-EZ DNA Probe Synthesis and Removal Kit combines the convenience of of the DECAprime II kit with the innovative Strip-EZ probe removal reagents. StripAble DNA probes generated with this kit incorporate modified nucleotides that are easily degraded following hybridization and detection. The degraded probe is removed in a mild wash. Unlike the harsh treatments commonly used to remove DNA probes from blots, the StripAble probe removal protocol does not cause irreversible damage to the blot that results in loss of sensitivity when the blot is re-probed. This permits the use of the same blot repeatedly, enhancing consistency of data and preserving precious nucleic acid samples.

With respect to kinetics of hybridization and effect of DNA template concentration on probe specific activity, Strip-EZ DNA probe synthesis reactions behave analogously to DECAprime II reactions.

The DECAprimeĂ II DNA labeling kit produces probes with maximum specific activity even when the DNA template is impure or the quantity unknown or very low. This is especially important when, for example, the template DNA is isolated from a gel. Impurities from gels can slow the labeling reaction, and in addition, the amount of template DNA may be low or not precisely known. Figure 3 shows that low template amounts require long incubation times to reach maximum specific activity. Under these conditions, extended reaction times will increase both the yield and the specific activity of the probe.

Figure 3. Kinetics of Polymerization Using DECAprime II DNA Labeling Kit. Incorporation of [alpha-³²P]dATP (50 µCi, 3000 Ci/mmol) in a DECAprime II reaction using the indicated amounts of the 3 kb control template DNA in a 25 µl reaction volume incubated at 37°C. Aliquots were removed at the indicated times to assess total and acid precipitable counts. Note: the plots for the Strip-EZ DNA kit look very similar to the above and are not included in this technical bulletin.

Extended reaction times present a problem with conventional random priming reactions since the DNA polymerase (Klenow fragment) commonly used exhibits exonuclease activity, resulting in a loss of synthesized probe. Klenow fragment lacks the 5' to 3' exonuclease activity of intact DNA Polymerase I, but still retains 3' to 5' exonuclease activity. Exonuclease free (Exo^-) Klenow has been genetically engineered to remove the 3' to 5' exonuclease activity, leaving only the DNA polymerase activity. The absence of exonuclease activity in DECAprimeĂ II reactions means that the reaction can be terminated at the convenience of the user, since labeled probe is stable in the optimized reaction buffers for many hours (see Figure 4). Figure 3 shows the results of a series of random priming reactions using the DECAprimeĂ II Kit in which the DNA template was varied from 6.25 ng to 100 ng. The probe resulting from 6.25 ng of DNA template had a specific activity of >10⁹ cpm/µg after 7.5 minutes, and 3 x 10⁹ cpm/µg after 6 hours incubation.

Figure 4. Specific Activity of a Conventional Reaction vs DECAprime II Probe Synthesis Reaction. The incorporation of [alpha-³²P]dATP (50 µCi, 3000 Ci/mmol) using 25 ng of a 3 kb template DNA at 37°C for the indicated times was used to determine the specific activity of the probes synthesized.

The elimination of exonuclease activity and improved reaction conditions with the DECAprimeĂ II Kit results in fast labeling kinetics. With the recommended 25 ng of template, the reaction reaches completion in 7.5 minutes, generating probe with a specific activity of about 2 x 10⁹ cpm/µg. Conventional random priming reactions require 20-30 minutes to reach completion, and must be critically timed for optimum results (see Figures 4 and 5).

Figure 5. Kinetics of Polymerization Using Conventional Reaction vs the DECAprime II DNA Labeling Kit. Time course of incorporation of [alpha-³²P]dATP (50 µCi, 3000 Ci/mmol) using either DECAprime I (old DECAprime formulation, no longer sold) or DECAprime II. The reaction contained 25 ng of a 3 kb control DNA template in a 25 µl volume incubated at 37°C for the indicated times.

Complete Kit

Each DECAprimeĂ II Kit comes complete with Exo^- Klenow, random decamer solution, two reaction buffers (minus dATP and minus dCTP) so that either radiolabeled dATP or dCTP can be used, linearized control DNA, EDTA and nuclease-free water. All reagents are subject to Ambion's stringent quality control procedures, often ten to one hundred times as stringent as those experienced during actual use. All Ambion molecular biology kits come with comprehensive instruction manuals with tips for both new and experienced users.