A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification

• Published in: Biosensors & bioelectronics 2017-05, Vol.91, p.122-127
• Main Authors: Park, Jung Hun, Jang, Hyowon, Jung, Yun Kyung, Jung, Ye Lim, Shin, Inkyung, Cho, Dae-Yeon, Park, Hyun Gyu
• Format: Article
• Language: English
• Subjects: Allele-specific ligation; Alleles; Biosensing Techniques - methods; BRCA gene; Breast Neoplasms - genetics; Female; Genes, BRCA1; Genotype; Genotyping Techniques - methods; Humans; Label-free biosensor; Mass spectrometry; Nucleic Acid Amplification Techniques - methods; Polymorphism, Single Nucleotide; SNP genotyping; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Strand displacement amplification
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2016.10.065

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry. •A new mass spectrometry-based method for multiplex SNP genotyping was developed in label-free manner.•Multiple SNP sites were simultaneously genotyped based on the peak positions by designing mass markers with different sizes.•The interpretation of the mass spectrum is simple to analyze the multiple polymorphic sites.•Several BRCA mutations was genotyped in a multiplex manner for the real clinical patient samples.