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Focus: Nucleic Acid Capture
Specific Capture of Biotinylated Nucleic Acids on the SAM2® Biotin
Capture Membrane
QuickView |
| The SAM2®
Biotin Capture Membrane (Cat.# V2861,
V7861) can be used to bind biotinylated nucleic acids with extremely
high affintity because of the high density of streptavidin on the filter. We demonstrate
the use of the membrane for rapid and quantitative substrate binding showing the
importance of washing conditions prior to analysis. Show Me the Data! |
By Daniel Kephart, Ph.D.
Promega Corporation
Introduction
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(a,b) (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(a,b) (Cat.# N2511), 0.75µl
of AMV Reverse Transcriptase (2 units/µl; Cat.# M5101) and 5µl
of [alpha-32P]dCTP (7 x 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
[alpha-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.
| Wash Procedure |
Conditions |
Background (%) |
| 1 |
2M NaCl with 100mM NaPO4 at room
temperature |
9.8 |
| 2 |
0.1X SSC with 0.1% SDS at room temperature |
13.7 |
| 3 |
0.1X SSC with 0.1% SDS at 60°C |
3.4 |
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 [alpha-32P]dCTP from 100µl of wash solution from
each of the seven washes of the three wash conditions showed decreasing amounts of
unincorporated [alpha-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 RiboMAXTM Large
Scale Production System(a,b,d,e) (Cat.#
P1280, P1290, P1300) was used as
template for RT-PCR(c) with either nonbiotinylated primers
or with a biotinylated downstream primer. The RNA contained the beta-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(c) (Cat.# A1250, A1280).
RT-PCR Components:
| RNA, diluted in water |
29µl |
| Access RT-PCR System 5X Buffer |
10µl |
| MgSO4 (25mM) |
2µl |
| dNTP mix (10mM) |
1µl |
| Beta-Actin Primer Pair (50pmol/µl each) |
1µl |
| [alpha-32P]dCTP |
5µl |
| AMV Reverse Transcriptase |
1µl |
| Tfl DNA Polymerase(c) (5 units/µl) |
1µl |
RT-PCR Conditions:
| First Strand cDNA Synthesis |
| 1 cycle |
48°C for 45 minutes |
| Second Strand Synthesis and Amplification |
| 35 cycles |
94°C for 30 seconds
60°C for 1 minute
68°C for 1 minute |
| 1 cycle |
68°C for 7 minutes |
| 1 cycle |
4°C soak |
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).
Figure 1. Analysis of RT-PCR products amplified from
increasing copies of synthetic RNA. Synthetic RNA was used in RT-PCR as described
in the text, using biotinylated (Panel A) and nonbiotinylated (Panel
B) oligonucleotide primers. Five microliters of amplification product were
analyzed on a 6% acrylamide gel that was dried and exposed to film. Lanes 1-9 represent
PCR products amplified from 0, 102, 103, 104, 105,
106, 107, 108 and 109 copies of beta-actin
RNA, respectively.
Figure 2. Scintillation counting
results plotted as the log of RNA copies versus counts per minute (cpm) for the
amplification products shown in Figure 1. Ten microliters of
each amplification reaction (Figure 1) were applied to duplicate SAM2®
Membranes. The membranes were washed eight times in 0.5X SSC plus 0.1% SDS at 60°C for
5-10 minutes each. Membranes were analyzed by scintillation counting and count data
plotted after subtracting background counts from a sample that did not contain template.
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.
Reference
- Kephart, D. (1998) Quantitative RT-PCR: rapid construction of templates for competitive
amplification. Promega Notes 68,
16.
(a)U.S. Pat. No. 5,552,302,
Australian Pat. No. 646803 and other patents.
(b)U.S. Pat. Nos. 4,966,964,
5,019,556 and 5,266,687, which claim vectors encoding a portion of human placental
ribonuclease inhibitor, are exclusively licensed to Promega Corporation.
(c)The PCR process is
covered by patents issued and applicable in certain countries. Promega does not encourage
or support the unauthorized or unlicensed use of the PCR process. Use of this product is
recommended for persons that either have a license to perform PCR or are not required to
obtain a license.
(d)The RiboMAXTM
Large Scale RNA Production Systems-T7 and T3 (Cat.# P1290 and P1300) are covered by U.S.
Pat. No. 5,256,555 and are sold under a license from Ambion, Inc. They are intended for
research use only. Parties wishing to use these products for other applications should
contact Ambion, Inc.
(e)The method of recombinant
expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024,
5,674,713 and 5,700,673.
PolyATtract, RiboClone, RNasin and SAM2 are trademarks of
Promega Corporation and are registered with the U.S. Patent and Trademark Office. RiboMAX
is a trademark of Promega Corporation.
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