Expansion of a SNaPshot assay to a 55‐SNP multiplex: Assay enhancements, validation, and power in forensic science

Abstract

A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty-four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single-base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father-child-mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10(-22) and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.

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

“…In recent years, the SNaPshot technology has been used to analyze SNPs of forensic DNA with CE as its detection system [10,11]. The advantage of this multiplexing technology is that it can be easily integrated into an operational forensic laboratory with less time requirements and greater cost effectiveness, without any additional equipment [12].…”

Targeted Detection of Single-Nucleotide Variations: Progress and Promise

“…84 Notably, such SBE assays possess excellent capabilities for the highly multiplexed detection of various SNVs; a 55plex SBE assay was recently reported by Wang and co-workers. 85 Allele-specific PCR (ASPCR), 86 also known as amplification refractory mutation system (ARMS), 87 is a modification of PCR that allows preferential amplification and detection of sequences differing by as little as a single nucleotide. The method relies on the higher efficiency of the primer extension process by DNA polymerase when the primer is perfectly complementary to a given DNA template than when there is a 3′-terminal mismatch in the primer−template duplex.…”

“…The fluorescence color readout reports the incorporated base whereby the SNV can be determined . Notably, such SBE assays possess excellent capabilities for the highly multiplexed detection of various SNVs; a 55plex SBE assay was recently reported by Wang and co-workers …”

Large-Scale Heat-Tolerance Screening and Genetic Diversity of Pea (Pisum sativum L.) Germplasms

“…Numerous approaches are available for performing SNP typing, including polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP), allele-specific PCR (AS-PCR) markers combined with polymerase chain reaction (PCR) technology and cleaved amplified polymorphic sequences (CAPS), next-generation sequencing (NGS), competitive allele-specific PCR (KASP), and mini-sequencing technology [ 41 ]. SNaPshot technology, also known as micro-sequencing technology, achieves mid-throughput SNP typing [ 47 , 48 ] and has been widely used in forensic medical identification [ 49 ] and in SNP detection of human genes [ 50 ] due to its high sensitivity, good repeatability, and lack of need for additional equipment. SNaPshot shows broad application prospects in the field of plant genetics research, such as SNP typing and marker development [ 51 ], molecular-marker-assisted breeding [ 52 ], and genetic diversity analysis [ 53 , 54 , 55 , 56 ].…”