Mass spectrometric based analysis, characterization and applications of circulating cell free DNA isolated from human body fluids

Abstract

In the past decade, cell free DNA, or circulating cell free DNA, or cell free circulating DNA, isolated from body fluids such as plasma/serum/urine has emerged as an important tool for clinical diagnostics. The molecular biology of circulating cell free DNA is poorly understood but there is currently an increased effort to understand the origin, mechanism of its circulation, and sensitive characterization for the development of diagnostic applications. There has been considerable progress towards these goals using real time polymerase chain reaction technique (rt-PCR). More recently, new attempts to incorporate mass spectrometric techniques to develop accurate and highly sensitive high-throughput clinical diagnostic tests have been reported. This review focuses on the methods to isolate circulating cell free DNA from body fluids, their quantitative analysis and mass spectrometry based characterization in evolving applications as prenatal and cancer diagnostic tools.

Figure 1. SOMA [Short Oligonucleotides Mass Spectrometry]

SOMA is a technique in which the circulating cell free DNA having SNP and wild CCFDNA are amplified using PCR, and the PCR products formed have a restriction enzyme recognition sites. On application of restriction enzyme the digested products are formed which are characterized by LC-ESI-MS [Ref. , Used with Permission].

Figure 2. Schematic illustration of the SABER and standard MassARRAY assays

Maternal plasma detection of the paternally inherited fetal-specific -thalassaemia mutation, IVS2654 C➝ T, is presented as an illustrative example. The standard protocol involves the base extension of both the mutant fetal allele (T allele) and the background allele (C allele), whereas the SABER method only extends the fetal-specific mutant allele. The base extension reactions are terminated by dideoxynucleotides, indicated in boxes. The extension products of the standard protocol include a predominance of the nonmutant allele (open arrows) with a small fraction of the fetal-specific mutant allele (filled arrows). The low abundance of the fetal allele (filled peak) is overshadowed by the nonmutant allele (open peak) on the mass spectrum. Because SABER only involves the extension of the mutant allele, the latter’s presence (filled peak) can be robustly identified from the mass spectrum. The striped peaks represent the unextended primer. (Ref. , Used with Permission)

Figure 3

Schematic diagram comparing the principles of the SABER and ASBER protocols. The site of the point mutation is indicated in capital letters. The boxed letter indicates the type of dideoxynucleotide terminator in the base extension reaction. (Ref. , Used with Permission)