Subfamily-specific Quantification of Endogenous Mouse L1 Retrotransposons by Droplet Digital PCR

Overview

Journal Anal Biochem

Publisher Elsevier

Specialty Biochemistry

Date 2020 May 23

PMID 32442414

Citations 2

Authors

Simon J Newkirk

Lingqi Kong

Mason M Jones

Chase E Habben

Victoria L Dilts

Ping Ye

Wenfeng An

Affiliations

Soon will be listed here.

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.

Citing Articles

Early maternal care restores LINE-1 methylation and enhances neurodevelopment in preterm infants.

Fontana C, Marasca F, Provitera L, Mancinelli S, Pesenti N, Sinha S BMC Med. 2021; 19(1):42.

PMID: 33541338 PMC: 7863536. DOI: 10.1186/s12916-020-01896-0.

Establishment of Quantitative PCR Assays for Active Long Interspersed Nuclear Element-1 Subfamilies in Mice and Applications to the Analysis of Aging-Associated Retrotransposition.

Kuroki R, Murata Y, Fuke S, Nakachi Y, Nakashima J, Kujoth G Front Genet. 2020; 11:519206.

PMID: 33193604 PMC: 7525186. DOI: 10.3389/fgene.2020.519206.

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