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Tandem oligonucleotide synthesis using linker phosphoramidites.

Pon RT, Yu S - Nucleic Acids Res. (2005)

Bottom Line: Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence.When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection.Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Calgary Calgary, AB, Canada T2N 4N1. rtpon@ucalgary.ca

ABSTRACT
Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623-631] is added to the 5'-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3'-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5'- and 3'-terminal OH ends and other oligonucleotides with 5'-phosphorylated and 3'-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

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Tandem synthesis of duplex DNA fragments (T is the linker phosphoramidite 4d). (A) A 44 base-long synthesis of a 24/20mer duplex. (B) A 52 base-long synthesis of a 28/24mer duplex. (C) A 64 base-long synthesis of 34/30mer duplex. (D) A 76 base-long synthesis of a 40/36mer duplex.
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fig4: Tandem synthesis of duplex DNA fragments (T is the linker phosphoramidite 4d). (A) A 44 base-long synthesis of a 24/20mer duplex. (B) A 52 base-long synthesis of a 28/24mer duplex. (C) A 64 base-long synthesis of 34/30mer duplex. (D) A 76 base-long synthesis of a 40/36mer duplex.

Mentions: The double-stranded fragments shown in Figure 4A–D were synthesized in the same way as described above for the multiple 20mer, using the appropriately derivatized low-loaded 1000 Å LCAA-CPG support (∼30–35 mg). Single-stranded controls corresponding to the top strands of each duplex (but not 5′-phosphorylated) were prepared by conventional synthesis. After synthesis and deprotection, UV quantification of the crude material showed the following A260 unit amounts: duplex A, 97; duplex B, 77; duplex C, 124; and duplex D, 124. Both PAGE (Figure 5A) and CGE (Figure 5B) under non-denaturing conditions showed that the two self-complementary sequences in each tandem pair formed a duplex structure. MALDI-TOF MS, however, showed the presence of two single-stranded oligonucleotides (M+H)+: duplex A, calc. 6026.9 and 7521.8, obs. 6023.5 and 7517.0; duplex B, calc. 7231.0 and 8787.6, obs. 7229.5 and 8785.6; duplex C, calc. 9046.0 and 10 682.8, obs. 9047.1 and 10 683.2; and duplex D, calc. 10 901 and 12 530, obs. 10 899.4 and 12 529.


Tandem oligonucleotide synthesis using linker phosphoramidites.

Pon RT, Yu S - Nucleic Acids Res. (2005)

Tandem synthesis of duplex DNA fragments (T is the linker phosphoramidite 4d). (A) A 44 base-long synthesis of a 24/20mer duplex. (B) A 52 base-long synthesis of a 28/24mer duplex. (C) A 64 base-long synthesis of 34/30mer duplex. (D) A 76 base-long synthesis of a 40/36mer duplex.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074720&req=5

fig4: Tandem synthesis of duplex DNA fragments (T is the linker phosphoramidite 4d). (A) A 44 base-long synthesis of a 24/20mer duplex. (B) A 52 base-long synthesis of a 28/24mer duplex. (C) A 64 base-long synthesis of 34/30mer duplex. (D) A 76 base-long synthesis of a 40/36mer duplex.
Mentions: The double-stranded fragments shown in Figure 4A–D were synthesized in the same way as described above for the multiple 20mer, using the appropriately derivatized low-loaded 1000 Å LCAA-CPG support (∼30–35 mg). Single-stranded controls corresponding to the top strands of each duplex (but not 5′-phosphorylated) were prepared by conventional synthesis. After synthesis and deprotection, UV quantification of the crude material showed the following A260 unit amounts: duplex A, 97; duplex B, 77; duplex C, 124; and duplex D, 124. Both PAGE (Figure 5A) and CGE (Figure 5B) under non-denaturing conditions showed that the two self-complementary sequences in each tandem pair formed a duplex structure. MALDI-TOF MS, however, showed the presence of two single-stranded oligonucleotides (M+H)+: duplex A, calc. 6026.9 and 7521.8, obs. 6023.5 and 7517.0; duplex B, calc. 7231.0 and 8787.6, obs. 7229.5 and 8785.6; duplex C, calc. 9046.0 and 10 682.8, obs. 9047.1 and 10 683.2; and duplex D, calc. 10 901 and 12 530, obs. 10 899.4 and 12 529.

Bottom Line: Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence.When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection.Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Calgary Calgary, AB, Canada T2N 4N1. rtpon@ucalgary.ca

ABSTRACT
Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623-631] is added to the 5'-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3'-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5'- and 3'-terminal OH ends and other oligonucleotides with 5'-phosphorylated and 3'-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

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