Abstract
The use of short oligonucleotide probes is finding increased application in DNA sequencing and genome characterization techniques, but a lack of knowledge of the hybridization properties of short duplexes hinders their use. Melting data were acquired on 128 DNA duplexes based on the length proposed in sequencing by hybridization procedures and formed from the general sequences 5'-XYZTGGAC-3',5'-GTCCAXYZ-3',5'-GCXYZGAC-3', and 5'-GTCXYZGC-3' where X, Y, and Z are either A, T, G, or C. These molecules were designed to elucidate the effects of location and nearest-neighbor stacking on the stability of base pairing in short DNA duplexes. The type of base pairs present had a major effect on stability, but was insufficient to predict stability without the inclusion of nearest-neighbor terms. Furthermore, the addition of information on position, or distance from the end, of the nearest-neighbor doublets led to statistically better fitting of the melting data. However, the positionally dependent stabilization differences are small compared with the contributions of base pairing and stacking.
Highlights
The use of short oligonucleotide probes is finding increased application in DNA sequencing and genome characterization techniques, but a lack of knowledge of the hybridization properties of short duplexes hinders their use
Shorter oligonucleotide probes are attractive since complete probe sets are possible to obtain and implement
Only 4096 sequences compose the set of all hexamer oligonucleotides, while > 1010 make up the complete set of 18-mers
Summary
The use of short oligonucleotide probes is finding increased application in DNA sequencing and genome characterization techniques, but a lack of knowledge of the hybridization properties of short duplexes hinders their use. Melting data were acquired on 128 DNA duplexes based on the length proposed in sequencing by hybridization procedures and formed from the general sequences 5'-XYZTGGAC-3', 5'-GTCCAXYZ-3', 5'-GCXYZGAC-3', and 5'-GTCXYZGC-3' where X, Y, and Z are either A, T, G, or C. These molecules were designed to elucidate the effects of location and nearest-neighbor stacking on the stability of base pairing in short DNA duplexes. Such a task requires the advancement of technologies, through the understanding of properties of nucleic acids and the optimization of procedures These technologies exploit the specific base pairing properties of DNA for sequence information and genome localization, with shorter probes finding increased application. This strategy is being exploited by using either enzymatic ligation [2, 3] or continuous stacking [4,5,6,7] of the shorter oligomers to produce sequencing primers that may make primer walking strategies practical
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