Sequencing in the presence of betaine: Improvement in sequencing of the localized repeat sequence regions.
The presence of several copies of the same class of repetitive element in DNA templates increases the probability of ambiguous base calling caused by band compression artifacts in the BigDye (Applied Biosystems, Foster City,CA) terminator cycle sequencing method. The presence of di-, tri-, and tetranucleotide repeats and short tandem repeats, which is widespread in the genome, poses a daunting task in sequencing laboratories, where a variety of DNA templates are submitted for sequencing.These base anomalies arise mainly as a result of the formation of secondary structures, including hairpins, and intramolecular base pairing between guanine and cytosine bases on the template strand. A common approach to the optimization of such sequencing reactions is either to replace the guanine with a base analog (such as deoxyinosine 5'-triphosphate [dITP] or 7-deaza-deoxyguanosine 5'-triphosphate [dGTP]) or to add a denaturant (such as dimethylsulfoxide [DMSO]) to the reaction mixture to overcome the undesired sequencing artifacts. Additives sometimes are ineffective for sequencing templates with GC-rich regions and repeat sequences. Herein we describe the effectiveness of (carboxymethyl)trimethylammonium (betaine) inner salt as an additive in the reaction mixture for reducing band compressions. The results presented show that betaine outperformed DMSO in sequencing through the localized regions containing GC-rich base pairs, guanine stretches, or TGC-type repeats in several DNA templates.
Haqqi
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Molecular Biotechnology Core, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 4195, USA.
Zhao
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Panciu
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Yadav
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