Oligodeoxynucleotides separation

The dideoxy DNA sequencing method begins with the denaturation of double-stranded DNA (dsDNA) into single-stranded DNA. The ssDNA is then annealed with a fluorescent dye-labeled primer, which is an oligodeoxynucleotide 20 bases long. The heteroduplex formed is then incubated in four separate reactions. 

Fig. 1. DNA was isolated from 11 F2 individuals segregating from a cross of two Glycine max (soybean) culdvars (A and B). Each DNA sample was amplified with a single 10-mer oligodeoxynucleotide primer (5 -AGCXntjTCTG). The amplification products were separated by electrophoresis in a 1.4% agarose gel and visualized by staining with ethidium bromide. The anows at the right of the gel point to polymorphisms between parents A and B that are segregating in the F2 generation. The mol-wt standards at the left of the gel are from a Hae ni digest of 0X 174 DNA.

Because of the pure hydrocarbon backbone, monoliths prepared from NBE and DMN-H6 are strongly hydrophobic [100, 105, 110]. The impressive separation capabilities have been demonstrated by the fast separation of biologically relevant compounds such as proteins, double-stranded (ds) DNA, oligonucleotides, and phosphorothioate oligodeoxynucleotides. As an example, the separation of 10 proteins was accomplished within less than 90 s at flow rates of 2 ml min . Such separation performance at high flow rates gives an illustration of the fast mass transfer that maybe achieved [39,68,92,93]. Furthermore, the separation of 20 bp of ds-DNA was achieved on both standard (i.e., 3 x 100 mm) and microanalytical (200 mm i.d.) monolithic columns [94,160]. 

Figure 16 IP-RP-HPLC separation of an oligodeoxynucleotide (dT)i2-is on a ROMP-derived NBE-based monolith (3 x 60 mm). Mobile phase 100 mmol f triethylammonium acetate at pH 7.0 linear gradient, 11-16% acetonitrile in 10min flow rate, 2mlmin 7"=20 C detection, UV 264 nm sample (dT)i2-i8, 0.1 pg of each oligodeoxynucleotide.

Contributing to ongoing efforts toward the miniaturization of analytical devices and to develop systems applicable to the coupling to highly sensitive quantification methods such as mass spectroscopy, Buchmeiser et al. reported on an extension of the concept of ROMP-derived monolithic supports to the synthesis of capillary columns. Transferring the synthetic concepts, methods, and procedures elaborated for semipreparative scale separations to 0.2 mm i.d. capillaries, high resolution was achieved in the separation of the oligodeoxynucleotides (dT)i2-i8 (2.27 [Pg.619]

Jones, R A., Fritz, H J., and Khorana, H G. (1978) Studies on polynucleotides 147 Use of the lipophilic rerr-butyldiphenylsilyl protecting group in synthesis and rapid separation of polynucleotides. Biochemistry 17, 1268-1278 Mishra, R. K. and Misra, K. (1988) Protecting groups as purification tool in large-scale synthesis of small oligodeoxynucleotides Indian J, Chem Sect. B, 27B, 817-820. 

Juite similar in performance as the PEI is the Nucleogen DEAE colunm packing material which is available with different pore sizes, the 6 nm material being recommended for the semi-preparative separation of oligodeoxynucleotides, while the 50 and 400 nm materials have been developed for the high-resolution separation of... 

Figure 3. Preparative anion-exchange HPLC of an oligo(rA) preparation on a PEI column. Sample, S.6 n oligo(rA) column, Baker PEI wide pore (4.6 x 250 mm) apparatus, Bruker LC 21 B with a Shimadzn-SPD-6A spectrophotometer set at 270 nm with 2.56 AUFS elution, linear gradient from 1 mM potassiuiH phosphate pH 6.3, 60% (v/v) formamide to 0.3 M potassium phos( ate, pH 6.3, 60% (v/v) formamide iA 180 min. Flow-rate, 1 ml/min. It is important to note that the Baker PEI wide-pore column used here does not have memory effects after such preparative runs this means that in subsequent analytical runs no peak% due to the previous preparative run appear. With the Nucleogen DEAE 60-7 column which we have alsdl tested for preparative separations of oligodeoxynucleotides slight memory effects are apparent. ( i...

Stationary phase. The classical example of a mixed mode column packing material is the low-pressure resin RPC-5 (13). It consists of non-porous Plaskon or Teflon particles to which aliphatic quaternary ammonium salts are adsorbed. RPC-5 has been applied successfully to the separation of tRNAs. tRNA fragments and more recently to the isolation of restriction fragments (14). RPC-5 itself is not commercially available. A RPC-5 analogue, however, has been developed, NACS, which can be purchased from BRL. NACS-12 has been used for mixed mode HPLC of DNA, DNA fragments and synthetic oligodeoxynucleotides. 

Hartwick and co-workers have bonded alkyl- and chloroalkylsilane to silica particles and subsequently substituted chlorine by a tertiary amine. This mixed mode support was used for the separation of crude oligodeoxynucleotides and restriction fragments (16). 

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