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 NH₄Ac, 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.

Product Availability:
Genisphere Inc.