Polyprobe DNA Dendrimers

Membrane:
Schleicher and Schuell, 4" x 5.25" Nytran Plus 0.45m.

Dot Blot Apparatus:
BRL Hybridot ~~ Two rows of dots per hybridization experiment.

Target Plasmid:
pBluescriptSK(-) with 200 bases of HIV-LTR position 420-620 (H9-IIIb) cloned in the BamHI-SalI Sites of the Multiple Cloning Site. The plasmid was digested with SalI to yield a single cut, linear 3.1KB molecule. The DNAs were denatured in 0.4 NaOH, neutralized with 2M NH
4Ac, loaded in 100ml as 1M NH4Ac, 0.2 NaOH and slowly drained through the membrane over 30 minutes. Serial 1:4 Dilutions used beginning with 4ng.

HIV Specific Sequences:
H9IIIb position 422-512 (HIV-422) was cloned adjacent to a(+) sequence in pBluescriptSK(-) with the SURE™ cell line, and sequenced (Lark Sequencing). Purified single stranded sequence consists of the a(-) sequence (for binding the core dendrimer) and 90 bases of HIV sequence. The same methods were used in the preparation of the HIV-520 sequence (H9IIIb position 520-610), except that the purified single stranded molecule consists of the HIV sequence adjacent to the c(-) sequence, that is, HIV-520 binds the "other" arm of an even layer dendrimer, i.e. 2,4,6,8 layer etc.

HIV Specific DNA Dendrimer:
4-layer trioxsalen crosslinked DNA dendrimers prepared under denaturing conditions were hybridized to the "HIV-422" or "HIV-520" sequences in approximately stoichiometric masses. HIV sequences were crosslinked to the 4-layer DNA dendrimer. The HIV specific DNA dendrimers were then purified in the same manner as preparation of the core 4-layer dendrimer.

Oligonucelotide Labeling:
Synthetic (~30mer) oligonucleotides were purchased from the Midland Certified Reagent Company. Standard labeling reaction consisted of 10 Units T4 polynucleotide kinase (Boehringer Mannheim), 400µCi gamma 32P-ATP, 3µg oligonucleotide in 80 µl 1X Reaction Buffer (Boehringer Mannheim). Reaction for 1 hr. at 37°C. The labeled oligonucleotide was purified from unincorporated
32P via G-25 spin column (5'--->3' Inc.). The specific activity of the labeled oligonucleotide was approximately 200,000 cpm/ng (2E8 cpm/µg), Cerenkov counts.

Hybridization Buffer:
Express-Hyb™ from Clontech.

Hybridization and Washing Conditions:
12.5ng/ml of each
32P labeled oligonucleotide, 12.5ng/ml as HIV sequence of HIV specific dendrimer, total dendrimer mass ~50ng/ml was prehybridized for 30 minutes at 80°C and hybridized overnight (~15 hrs) at 65°C, then 1.5 hrs at 45°C. All washes were done at 50°C, 3x10 minutes in 2X SSC 0.1% SDS, followed by 2x20 minutes in 0.5X SSC 0.1% SDS.

Detection:
The membranes were plastic wrapped and exposed overnight to Reflection™ film with a Reflection Intensifying Screen at -70°C (DuPont). The film was developed with Kodak developer, stop bath and fix solutions. The Molecular Dynamics Phosphorimager screen was exposed overnight at room temperature and quantitated as recommended by Molecular Dynamics.

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Polyprobe ™ DNA Dendrimers and DNA Matrix Technology are covered under U.S. Patent No's 5,175,270 || 5,484,904, || 5,487,973 and patents pending.

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