New labelling technology for molecular probes applied to the ligation detection reaction-universal array system.
The ligation detection reaction (LDR) associated with universal arrays (UA) uses a fluorescently labelled probe (DP) and a Zip Code-extended probe to detect single nucleotide polymorphisms in DNA target sequences. When used for genotyping, the LDR-UA technique uses two DPs, each specific to an allele and labelled with a different fluorophore. The fluorescent signals are processed to calculate the genotype. The uneven decay of fluorophores due to ageing and freezing/thawing cycles and the consequent unequal fluoresce level can lead to erroneous genotype calls. To circumvent this problem, an indirect labelling strategy was developed based on the substitution of the fluorophore with allele-specific 22 bp universal labelling sequences (ULS). Labelling is achieved with fluorescently labelled oligos complementary to the ULS (cULS). The strategy improved the uniformity in probe labelling, and generated results comparable to those using direct-labelled probes, as shown by genotyping 22 polymorphic sites in 70 samples with both strategies. This method can be easily implemented in the routine screening with LDR-UA or other techniques. Moreover, the approach results in a significant cost reduction over traditional direct labelling, and offers the possibility to interchange fluorophores and to increase the fluorescent signal by using multiple-labelled cULS.
Lauri
Andrea
A
Parco Tecnologico Padano, Lodi, Italy. andrea.lauri@tecnoparco.org
Chessa
Stefania
S
Raschetti
Marta
M
Castiglioni
Bianca
B
Mariani
Paola
P
Caetano
Alexandre R
AR
D000483:Alleles; D001483:Base Sequence; D005838:Genotype; D015335:Molecular Probes / Q000235:genetics*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
D015335:Molecular Probes