The SAM2® Biotin Capture Membrane (Cat.# V2861, V7861) binds biotinylated molecules based on their affinity for streptavidin. The proprietary process by which the SAM2® Membrane is produced results in a high density of streptavidin on the filter, providing rapid, quantitative substrate binding at a minimum of 1.3nmol/cm2. In addition, the membrane has been optimized for low nonspecific binding. For more information, see the SAM2® Biotin Capture Membrane Technical Bulletin, #TB547.
Three experiments were designed to investigate the use of SAM2® Biotin Capture Membrane for specific capture of biotinylated nucleic acids. Experiments included: i) determination of optimal washing conditions for the membranes after nucleic acid capture; ii) specific capture of a biotinylated cDNA as compared to nonbiotinylated cDNA and; iii) specific capture and quantitation of biotinylated PCR products.
Optimal Washing Conditions for Capture of Biotinylated and Nonbiotinylated cDNA
Two cDNA synthesis reactions were assembled, differing only in the primer used. Both reactions used the Kanamycin Positive Control RNA, included in the Universal RiboClone® cDNA Synthesis System (Cat.# C4360) as template. One cDNA synthesis reaction used the Oligo(dT)15 Primer (Cat.# C1101) included with the Universal RiboClone® System to prime first strand DNA synthesis; the second reaction used biotinylated oligo(dT) from the PolyATtract® System 1000 (Cat.# Z5400, Z5420).
cDNA Synthesis: Two microliters of Kanamycin RNA (5µg/µl) were mixed with 1µl of primer and 2.75µl of water. The samples were heated to 65°C for 5 minutes and cooled on ice. Next, the following components were added: 4µl of 25mM MgCl2, 2µl of 10X RT Buffer, 2µl of 10mM dNTP Mix, 0.5µl of Recombinant RNasin® Ribonuclease Inhibitor (Cat.# N2511), 0.75µl of AMV Reverse Transcriptase (2 units/µl; Cat.# M5101) and 5µl of [α-32P]dCTP (7 × 106 cpm/5µl). Reactions were incubated for 60 minutes at 42°C, heated to 94°C for 2 minutes and then diluted to 100µl with water. Nonbiotinylated cDNA exhibited 1.79% incorporation (24,429cpm/µl TCA-precipitable counts from 1,365,674 total cpm/µl); biotinylated cDNA exhibited 1.73% incorporation (19,743cpm/µl TCA-precipitable counts from 1,141,928 total cpm/µl).
Wash Conditions: Both biotinylated and nonbiotinylated cDNA products (10µl of each) were spotted directly onto duplicate SAM2® Membranes and were subjected to three different membrane washing conditions (see Table 1). For each wash procedure, six or seven washes (5ml each) were performed on the membranes for 5 minutes per wash and 100µl of the wash supernatant were analyzed for unincorporated [α-32P]dCTP. The final percent background (nonbiotinylated cDNA) relative to specific signal (biotinylated cDNA) after the washings was calculated.
Table 1. Three Wash Conditions Tested with Biotinylated DNA Fragments on SAM2® Membranes.
Seven washes of 5ml each were performed for 5 minutes per wash, for wash procedures 1 and 3; six washes were performed for procedure 2. Membrane cpms were determined by scintillation counting. Background percent was calculated by dividing the counts from the membrane to which nonbiotinylated DNA was applied by the counts from the membranes to which biotinylated DNA was applied and multiplying by 100. Results for unincorporated [α-32P]dCTP from 100µl of wash solution from each of the seven washes of the three wash conditions showed decreasing amounts of unincorporated [α-32P]dCTP in the wash supernatant for the first three washes, with the counts remaining about the same for the last three washes (data not shown).
Capture of Biotinylated RT-PCR Products on the SAM2® Biotin Capture Membrane
A dilution series of quantitated synthetic RNA made with RiboMAX™ Large Scale Production System (Cat.# P1280, P1300) was used as template for RT-PCR with either nonbiotinylated primers or with a biotinylated downstream primer. The RNA contained the β-actin sequence used previously for quantitative RT-PCR (1)
. The nonbiotinylated and biotinylated downstream primers used were identical in sequence. RNA was amplified using the Access RT-PCR System (Cat.# A1250, A1280).
|RNA, diluted in water
|Access RT-PCR System 5X Buffer
|dNTP mix (10mM)
|β-Actin Primer Pair (50pmol/µl each)
|AMV Reverse Transcriptase
|Tfl DNA Polymerase (5 units/µl)
||48°C for 45 minutes|
||94°C for 30 seconds; 0°C for 1 minute; 68°C for 1 minute|
||68°C for 7 minutes|
After amplification, 5µl of each product were analyzed on a 6% acrylamide gel, which was dried and exposed to film. The results demonstrate that both biotinylated and nonbiotinylated primers resulted in PCR products (Figure 1).
Results and Conclusions
RT-PCR products resulted from the use of both biotinylated and nonbiotinylated primers, as shown in Figure 1. There was no significant difference in the amount of cDNA produced from biotinylated versus nonbiotinylated primers. Experiments demonstrated that wash conditions are important for optimal SAM2® Membrane performance with bound nucleic acids. The wash conditions used in this study were chosen for their compatibility with double-stranded nucleic acids, important when using the SAM2® Membrane with PCR products. Best wash results were obtained when six to eight washes were performed. Wash procedure #3 resulted in the lowest percent of nonbiotinylated versus biotinylated (background) counts on the membranes, and thus optimal performance of the membrane with bound DNA.
The RT-PCR products made with biotinylated primers, but not those made with nonbiotinylated primers, were efficiently captured by the SAM2® Membrane as shown in Figure 2. Nonbiotinylated cDNA capture was approximately 3–4% of biotinylated cDNA capture (data not shown). Note: Background counts for the biotinylated amplification products can be markedly decreased by the use of spin columns to remove primer-dimers and other non-full-length amplification products; spin columns were not used in this procedure.