| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Molecular biology relies heavily on how well and quickly nucleic acids can be separated and analyzed.One of the most commonly used techniques of nucleic acid separation is gel electophoresis. If gel electrophoresis is replaced by high performance liquid chromatography (HPLC) in molecular biology, it will enable recovery of nucleic acids in a shorter period of time. Separation by gel permeation HPLC was limited to natural DNA fragments (double stranded DNA) , whereas separation by reversed phase HPLC was widely used for synthetic oligonucleotides (single stranded DNA) . DNA fragments with a difference in hundreds in base pairs have been separated in gel permeation HPLC on appropriate columns connected in tandem, but this method is too time-consuming.
To solve this problem, we developed a novel method for the separation of double stranded DNA fragments by bis-intercalators. In order for bis-intercalators to bis-intercalate into DNA,a minimum length of double stranded DNA is required. If D
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NA is shorter than this length the bisacridine-intercalator binds to DNA by the mono-intercalation mode. In other words, the binding mode and affinity of the bis-intercalator for DNA varies depending on the length of DNA.We attempted to exploit this difference in the affinity of the bis-intercalator for the length of DNA.We synthesized a bis-intercalator carrying a connector which separates the two intercalator parts by 26A,equivalent to 8 bp of DNA.We also synthesized four self-complementary 6-, 8-, 12-, and 16-meric oligonucleotides.
The bis-acridinyl derivative could bind to calf thymus DNA with K= (1.7<plus-minus>0.3) x10^5M^<-1> even at high salt concentration (0.1M NaCl) and cover the area of nearly eight base pairs. The effect of the bis-acridine derivative on the thermal denaturation of double stranded oligonucleotides was then studied. The bis-acridinyl derivative shifted the T_m of DNA to higher temperature, but its effect, as measured by DELTAT_m, was much more pronounced for the 8-mer or longer double stranded DNA than for the shorter counterparts. In other words, the intercalator bound the 6-mer by the mono-intercalation mode, while it bound the 8-mer or longer oligonucleotides by the bis-intercalation mode.
These idea suggest that this bis-acridine derivative can discriminate double stranded DNA according to its length. By taking advantage of this characteristic precise separation of double stranded DNA of various lengths will be achieved chromatographically, once bis-intercalator is immobilized on an inert gel. Such an undertaking is now under way in our laboratory. Less
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