Structure of cytidine and its derivatives. The derivatives featured in this figure vary in their sugar substitutes. Note that in Cytosine Arabinose (Ara-C), the arabinose sugar contains hydroxyl groups in positions 3 and 5 in a similar orientation to native ribose, thus permitting reaction with other nucleotides in DNA synthesis. However, the hydroxyls in positions 2 and 3 are in the trans orientation. Comparing position 2 on the arabinose ring to that of 2-deoxyribose reveals that the hydrogen in 2-deoxyribose, that is in trans configuration to hydroxyl 3, is replaced by the hydroxyl group found on arabinose. It should be emphasized that there are two types of polymerization arrest: a. Chain termination- the nucleotide is incorporated into the nascent DNA strand and synthesis is stalled because no new nucleotide is added. Dideoxy derivatives stall elongation after incorporation into the nascent DNA strand because they do not have hydroxl in position 3. Arabinose nucleotides also belong to this group, but they offer only partial stalling . b. Template-dependent termination-nucleotides already incorporated in the DNA (e.g. in primers) are able to stall polymerization when the polymerase reads the template. It is believed that due to stereo restraints, the polymerase falls off the template. The frequency of this event determines the efficiency of the stalling. Arabinose derivatives belong to this group. The property of template-dependent termination of Ara-C was utilized in this study to create 5' overhangs in the first PCR. However, since the template-dependent termination by Ara-C is moderate, it was utilized for the amplification in the second PCR.