Optimization of Transrenal DNA Analysis: Detection of Fetal DNA in Maternal Urine

Abstract

BACKGROUND:Fragments of DNA from cells dying throughout the body are detectable in urine (transrenal DNA, or Tr-DNA). Our goal was the optimization of Tr-DNA isolation and detection techniques, using as a model the analysis of fetal DNA in maternal urine.

Cell-Free DNA: An Upcoming Biomarker in Transplantation

“…Low concentrations of urinary cfDNA (2-200 ng/mL) (32,33) DNA isolation kit: low molecular weight fragments (50-200 bp, transrenal) versus high molecular weight DNA (>1 kb, nontransrenal) (33,57,58) Use of Q-sepharose anion exchange resin instead of silica based methods improves sensitivity (58) Quantification of urinary ddcfDNA:…”

“…Use of PCR targeting ultrashort DNA targets improves sensitivity for Tr-DNA detection (58) Low fraction of total cfDNA (8.7%) (22) cfDNA, cell-free DNA; Tr-DNA, transrenal DNA; bp, base pairs; kb, kilobase; qPCR, quantitative polymerase chain reaction; ddPCR, digital droplet PCR; SNPs, single nucleotide polymorphisms; MPSS, massive parallel shotgun sequencing; EDTA, ethylenediaminetetraacetic acid.…”

Molecular Triage of Premalignant Lesions in Liquid-Based Cervical Cytology and Circulating Cell-Free DNA from Urine, Using a Panel of Methylated Human Papilloma Virus and Host Genes

“…These fragments are known as transrenal DNA (TrDNA) (47)(48)(49). Recently, a capillary electrophoresis TrDNA test that targets the E1 region of the HPV genome for the detection of hrHPV demonstrated high sensitivity and modest specificity for urine-based detection of cervical precancerous lesions (50).…”

Strategic Assay Developments for Detection of HBV 1762T/1764A Double Mutation in Urine of Patients with HBV-Associated Hepatocellular Carcinomas

“…This LNA clamp complementary to HBV wild type DNA can reduce interference from HBV wild type to allow for increased sensitivity for detecting the HBV double mutation. However, Sikora et al (Sikora, Zimmermann et al 2010) and Shekhtman et al (Shekhtman, Anne et al 2009) suggested that polymerase chain reaction (PCR) assays targeting template sequences of 50 nucleotides (nt) or less are necessary to obtain a sensitivity greater than 50% in order to detect DNA of interest in urine derived from the circulation. Thus, a second assay containing a 2-step nested PCR assay (HBVDM_40) with a target template size of 40 bp and incorporation of locked nucleic acid was also developed.…”