Genotyping β-Globin Gene Mutations on Copolymer-Coated Glass Slides with the Ligation Detection Reaction

Abstract

Background: Methods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity. Methods: We developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA–templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides. Results: The assay was designed to detect 7 prevalent mutations in the β-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known β-thalassemia mutations. Results show good correspondence between the patients’ genotypes as assessed by DNA sequence analysis and those generated from the LDR assays. Conclusions: The developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.

A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β-Thalassemia Mutations

“…[5][6][7][8][9][10] Translation of the data into clinical practice requires a simple, reliable, cost-effective method that is able to detect all of these mutations. Among dozens of methods that have been developed for detecting ␤-thalassemia mutations, [11][12][13] reverse dot blot (RDB) analysis is the only method approved in China. 14 Despite its costeffectiveness, broad coverage of mutations, and longterm adoption in clinical settings, its use in carrier screening is limited, primarily because of low throughput and subjective results.…”

Fluorescent detection of point mutation via ligase reaction assisted by quantum dots and magnetic nanoparticle-based probes

“…The assay was designed to detect 7 prevalent mutations in the beta globin gene. 46 Although this strategy had sufficiently high sensitivity and specicity to differentiate multiple targets but the combined PCR-LDR process suffer from the complications of PCR.…”

Copy Number Variation Analysis by Ligation-Dependent PCR Based on Magnetic Nanoparticles and Chemiluminescence

“…The concentration of ligation probes should also be controlled at an extremely low level in comparison with the universal primers, as this could largely eliminate the non-specific signals. Additionally, adding certain exonucleases to digest unligated oligonucleotides before amplification was also a useful method 55 . The non-specific amplification was checked from the blank control group performed under the same reaction conditions without template.…”