Subfamily-specific quantification of endogenous mouse L1 retrotransposons by droplet digital PCR.
Anal Biochem
; 601: 113779, 2020 07 15.
Article
em En
| MEDLINE
| ID: mdl-32442414
ABSTRACT
Long interspersed element type 1 (LINE-1; L1) mobilizes during early embryogenesis, neurogenesis, and germ cell development, accounting for 25% of disease-causing heritable insertions and 98% of somatic insertions in cancer. To better understand the regulation and impact of L1 mobilization in the genome, reliable methods for measuring L1 copy number variation (CNV) are needed. Here we present a comprehensive analysis of a droplet digital PCR (ddPCR) based method for quantifying endogenous mouse L1. We provide experimental evidence that ddPCR assays can be designed to target specific L1 subfamilies using diagnostic single nucleotide polymorphisms (SNPs). The target and off-target L1 subfamilies form distinct droplet clusters, which were experimentally verified using both synthetic gene fragments and endogenous L1 derived plasmid clones. We further provide a roadmap for in silico assay design and evaluation of target specificity, ddPCR testing, and optimization for L1 CNV quantification. The assay can achieve a sensitivity of 5% CNV with 8 technical replicates. With 24 technical replicates, it can detect 2% CNV because of the increased precision. The same approach will serve as a guide for the development of ddPCR based assays for quantifying human L1 copy number and any other high copy genomic target sequences.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Reação em Cadeia da Polimerase
/
Elementos Nucleotídeos Longos e Dispersos
Limite:
Animals
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2020
Tipo de documento:
Article