Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

Abstract

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10−13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Ethnic derivation of the Ainu inferred from ancient mitochondrial DNA data

“…This analysis was performed by using a multiplex system for analyzing mitochondrial single‐nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product‐length polymorphism (APLP) method that uses inosine‐flapped primers and is specifically designed for the detailed haplogrouping of degraded mtDNA (Kakuda et al, 2016). This analysis was performed only for the samples for which the sequence analysis was successful.…”

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

“…The thermodynamics of the primers with inosine flaps has been proven to be less influenced by the sequence of PCR templates than the thermodynamics of the primers with 5′-flaps containing ordinary bases [33]. Furthermore, we reported that our inosine-flapped APLP method in the multiplex SNP analysis showed markedly higher sensitivity than that reported in various studies [61]. These features of 5′-flap primers are likely to have contributed to the high sensitivity observed in our sex determination method.…”

Genetic Polymorphism Analysis of&nbsp;Mitochondrial DNA from&nbsp;Chinese Guangdong&nbsp;Liannan Yao ethnic group

“…Thanks to the rapid development of sequencing technology, the genetics community has conducted a large number of assessments and analysis of mtDNA haplogroup diversity in the past few decades in different regions and ethnic groups in China. [18][19][20][21][22][23][24][25][26] MtDNA plays an important role in studying the matrilineal origins of various ethnic groups in the contemporary East Asian population. Although many problems remain to be solved and the related database is not perfect, the historical migration route of the population is still relatively clear.…”