Does Size Matter? Comparison of Extraction Yields for Different-Sized DNA Fragments by Seven Different Routine and Four New Circulating Cell-Free Extraction Methods

Abstract

An element essential for PCR detection of microbial agents in many sample types is the extraction step, designed to purify nucleic acids. Despite the importance of this step, yields have not been extensively compared across methods to determine whether the method used contributes to quantitative differences and the lack of commutability seen with existing clinical methods. This may in part explain why plasma and blood viral load assays have proven difficult to standardize. Also, studies have identified small DNA fragments of <200 bp in plasma (cell-free DNA [cfDNA]), which may include significant quantities of viral DNA. Our study evaluated extraction yields for 11 commercially available extraction methods, including 4 new methods designed to isolate cfDNA. Solutions of DNA fragments with sizes ranging from 50 to 1,500 bp were extracted, and then the eluates were tested by droplet digital PCR to determine the DNA fragment yield for each method. The results demonstrated a wide range of extraction yields across the variety of methods/instruments used, with the 50- and 100-bp fragment sizes showing especially inconsistent quantitative results and poor yields of less than 20%. Slightly higher, more consistent yields were seen with 2 of the 4 circulating cell-free extraction kits. These results demonstrate a significant need for further evaluation of nucleic acid yields across the variety of extraction platforms and highlight the poor extraction yields of small DNA fragments by existing methods. Further work is necessary to determine the impact of this inconsistency across instruments and the relevance of the low yields for smaller DNA fragments in clinical virology testing.

Keywords: ccfDNA; extraction methods; extraction yield; viral DNA processing; viral diagnostics.

FIG 1

Quantitative yields for all routine DNA extraction methods separated by instrument and fragment concentration. Dotted horizontal lines represent the theoretical 100% yield at each concentration.

FIG 2

CCF kit extraction yields. Quantitative yields for all CCF kits are shown, with the data being separated by instrument and fragment concentration. Dotted horizontal lines represent the theoretical 100% yield at each concentration. The unextracted samples were fragments made to 4.0, 5.0, and 6.0 log₁₀ copies/ml in TE (Tris-EDTA) buffer and run by ddCPR without extraction. M16, Maxwell 16; QiaSym, QIAsymphony.

FIG 3

Quantitative yields displayed on the basis of the fragment size for the 50-, 100-, and 200-bp fragments. (A, C, E) Results obtained by routine extraction methods; (B, D, F) results obtained with the CCF kits.

FIG 4

Quantitative yields displayed on the basis of the fragment size for all fragment sizes at 5.0 log₁₀ copies/ml. (A) Fragment yields separated by the instrument/kit utilized and then the yields of each fragment size. (B) The yield for each method compared to the yield for the unextracted (TE buffer) control (extracted/unextracted control).

FIG 5

Comparison of the coefficients of variation for four selected combinations of fragment size and concentration. The percent CVs for the extraction yields for 4 selected combinations of fragment size and concentration are represented graphically. The unextracted control is represented by the black bar, and for each of the 4 sets represented, the remaining bars present in order from left to right the results for EasyMag, EZ1 DSP, MP96, MinElute, Maxwell 16, m2000sp, QIAsymphony and then a space, followed by the results for the CCF kits (EZ1 CCF, MinElute CCF, Maxwell 16 CCF, and QIAsymphony CCF).