A new approach to detect a set of SNP‐SNP markers: Combining ARMS‐PCR with SNaPshot technology

Abstract

Microhaplotypes are a new promising type of forensic genetic marker. Without the interference of stutter and high mutation rates as for STRs, and with short amplification lengths and a higher degree of polymorphism than single SNP, microhaplotypes composed of two SNPs, SNP-SNP, have a strong application potential. Currently, the most common method to detect microhaplotypes is massive parallel sequencing. However, the cost and extensive use of instruments limit its wide application in forensic laboratories. In this study, we screened 23 new SNP-SNP loci and established a new detection method by combining a multiplex amplification refractory mutation system-based PCR (ARMS-PCR) and SNaPshot technology based on CE. First, we introduced an additional deliberate mismatch at the antepenultimate base from the 3 end of primers when designing ARMS-PCR for SNP 1 (the first SNP of the SNP-SNP). Then, single base extension primers for SNaPshot assay were designed next to the position of SNP 2 (the second SNP). Finally, 15 loci were successfully built into four panels and these loci showed a relatively high level of polymorphism in the Southwest Chinese Han population. All the loci had an average probability of informative genotypes (I value) of 0.319 and a combined discrimination power of 0.999999999. Therefore, this new detection system will provide a valuable supplement to current methods.

Set of 15 SNP-SNP Markers for Detection of Unbalanced Degraded DNA Mixtures and Noninvasive Prenatal Paternity Testing

“…Table 2 showed the information of all primers used to detect cffDNA in this study. The ARMS-PCR amplicons of these primers observed in previous reports were 60-150 bp, and the SBE amplicons were 32-79 bp (Zhang et al, 2020). The size of all amplicons makes the detection of cffDNA highly feasible because the median length of cffDNA is only 143 bp.…”

“…Therefore, based on the theoretical ratio of 5-20%, the concentration of cffDNA was distributed approximately between 0.0078 and 0.286 ng/μL. In previous sensitivity determinations, the detection limit of most markers was 0.025 ng (Zhang et al, 2020). Therefore, to improve the detection rate, the number of cycles in the ARMS-PCR and SBE steps was increased to 32.…”

Multi-Indel: A Microhaplotype Marker Can Be Typed Using Capillary Electrophoresis Platforms

“…We believe that a set of all variants including SNP, Indel and STR within a specifically short length can be considered as generalized microhaplotypes. Only microhaplotypes containing two SNP can presently be genotyped on the CE platform due to limitations of the system (Zhang et al, 2020). Therefore, we selected Indels from the 1000 Genomes FIGURE 5 | Effects of ultrasound on DNA extracted from fresh EDTA blood.…”

“…Thereafter, two allele-specific sequences are obtained using PCR. The genotype of haplotype markers from an individual can be determined using a capillary electrophoresis (CE) platform and a two-step detection method, but the phase of a haplotype can be unambiguously determined only when the microhaplotypes include two variants (Castella et al, 2013;Cereda et al, 2014;Oldoni et al, 2015;Tan et al, 2017;Liu et al, 2018Liu et al, , 2019Moriot and Hall, 2019;Oldoni and Podini, 2019;Zhang et al, 2020). Additionally, the main limitation of microhaplotype markers comprising only two variants is the difficulty with increasing polymorphism.…”

Análisis de la mutación c.187 C>T en el gen ATP6V0A2 mediante PCR-ARMS

“…Los resultados permitieron confirmar que la metodología establecida es capaz de identificar y discernir entre individuos sanos, heterocigotos y homocigotos, para la mutación c.187 C>T en el exón 2 del gen ATP6V0A2, comprobando que la PCR-ARMS es una técnica que permite la diferenciación para todo tipo de mutaciones puntuales Zhang-R et al, 2020;Doulabi, Moghaddam & Salehzadeh, 2020;Li, He & Liang, 2020;Medrano & De Oliveira, 2014) Identificación de individuos portadores de la mutación c.187 C>T por PCR-ARMS Un lote de diez muestras de ADNg de individuos de las poblaciones de Túnel de Potrerillo, Boca Túnel y Túneles en Rincón de Romos, Aguascalientes, localidades donde se reportó por primera vez un paciente con Cutis laxa portador de la mutación c.187 C>T en el exón del gen ATP6V0A2 (paciente 1) (Bahena-Bahena et al, 2014) se analizaron utilizando la metodología previamente descrita y utilizando como controles de comparación la amplificación de ADNg del paciente 1 y de su progenitor. Los resultados de las amplificaciones se muestran en la Figura 4.…”