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Oligonucleotides and analogues: a practical approach fritz eckstein.pdf

The authors found that in a single ladies season 1 episode 7 gene construction protocol every construct sequenced had small point deletions when using unpurified oligos (Chalmers and Curnow, 2001 a similar experiment done using page purified oligos resulted in the desired end product (Au et al, 1998).
From the above discussion on how oligos are made, it is clear that the reaction product must include unwanted species as well as the desired sequence.
We also introduce psoralen azide to our growing selection of azido tags.However, truncation species can interfere with reactions when longer oligos are used in more complex procedures.For a 60-mer oligo, less than 50 of the final product will be the desired full-length molecules (see our Purification Technical Bulletin for a more complete description).This existence of the protonated and non-protonated forms in equilibrium on a pace oligonucleotide was demonstrated by decreasing the pH with mild acid, resulting in greatly increased retention of pace DNA on a reverse-phase hplc column.Oligonucleotide phosphoramidite synthetic chemistry was introduced nearly 20 years ago (McBride and Caruthers, 1983).The one significant difference in the physical properties of these molecules compared to normal DNA is the change in hydrogen bonding that is associated with a transformation from oxygen to sulfur.H., Biochemistry, 1996, 35, 87348741.Losses of approximately.3C per linkage for pace-DNA/RNA duplexes and approximately.3C for pace-DNA/DNA duplexes were observed.Automated synthesis is performed on a solid support matrix that serves as a scaffold for the sequential chemical reactions; a series of valves and timers to deliver the reagents to the matrix, and finally a post-synthesis processing stream that includes purification, quantification, product QC, lyophilization.A loaded column is attached to reagent delivery lines on a DNA synthesizer and the chemical reactions proceed under computer control.Matzura,., Eckstein,., Eur.These compounds were typically produced using an Arbuzov reaction, whereby a halide was reacted at elevated temperature with a trialkyl phosphite in a two-step, sequential, oxidative transformation.This behavior indicates that the crosslinking occurs via a 22 cycloaddition with a double bond on the base opposite.Most oligonucleotides are made on commercial nucleic acid synthesizers (Figure 1) using phosphoramidite chemistry.These protecting groups are stable under conditions used during synthesis but are rapidly and effectively removed by treatment with ammonia.Phosphorothioates, one of the first chemically modified internucleotide bonds utilized for these studies was phosphorothioate modified oligonucleotides, as shown in Figure 3, Page.Automated synthesis is done on solid supports, usually controlled pore glass (CPG) or polystyrene.Chemical groups used to protect these sensitive sites must remain intact during all phases of the DNA synthesis cycle yet must be readily removed after synthesis so that normal, unmodified DNA results.
The two examples that have currently been reported are the phosphonoacetic acid derivative (PAA)3, and the phosphonoformic acid (PFA) derivative4, shown in Figure 2, Page.
Synthesis starts with the first base attached to the CPG solid support and elongates in a 3' » 5' direction.