Design and application of noncontinuously binding probes used for haplotyping and genotyping.
BACKGROUND
Many methods for genotyping use melting temperature (Tm) of sequence-specific probes. Usually the probes hybridize to a continuous stretch of DNA that contains the variant(s). In contrast, hybridization of noncontinuous probes to a template can form bulges. This report generates guidelines for the design of noncontinuous probes.


METHODS
We used software to predict hybridization structures and Tms from 10 noncontinuous probes and 54 different templates. Predicted Tms were compared to existing experimental data. The bulging template's sequences (omitted in the probe) ranged in size from 1 to 73 nucleotides. In 36 cases, we compared observed and predicted DeltaTms between alleles complementary to the probe and mismatched alleles. In addition, using software that predicts effects of bulges, we designed a probe and then tested it experimentally.


RESULTS
The mean differences between predicted and observed Tms were 0.65 (2.51) degrees C with the Visual OMP software and 0.28 (1.67) degrees C with the MeltCalc software. DeltaTms were within a mean (SD) of 0.36 (1.23) degrees C (Visual OMP) and -0.01 (1.02) degrees C (MeltCalc) of observed values. An increase in the size of the template bulge resulted in a decrease in Tms. In 2 templates, the presence of a variant in the bulge influenced the experimental Tm of 2 noncontinuous probes, a result that was not predicted by the software programs.


CONCLUSIONS
The use of software prediction should prove useful for the design of noncontinuous probes that can be used as tools for molecular haplotyping, multiplex genotyping, or masking sequence variants.
Pont-Kingdon
Genevieve
G
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA. pontkig@aruplab.com
Margraf
Rebecca L
RL
Sumner
Kelli
K
Millson
Alison
A
Lyon
Elaine
E
Schütz
Ekkehard
E
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D014644:Genetic Variation; D005838:Genotype; D006239:Haplotypes; D006454:Hemoglobins / Q000235:genetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D020641:Polymorphism, Single Nucleotide; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics; D044102:Receptor, Melanocortin, Type 1 / Q000235:genetics; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics; D012984:Software; D013816:Thermodynamics; D044366:Transition Temperature
D006454:Hemoglobins; D015345:Oligonucleotide Probes; D044102:Receptor, Melanocortin, Type 1; D018343:Receptors, Adrenergic, beta-2; D004247:DNA; D051498:Receptor, Fibroblast Growth Factor, Type 3