Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: An accurate method for large DNA quantification

Absolute Quantification of RNA or DNA Using Acid Hydrolysis and Mass Spectrometry

“…Another study using dilute HCl (0.2 mol/L) observed complete purine hydrolysis from DNA but with diminutive release of pyrimidines. Quantitative analysis of purine base adducts in genomic DNA has been reported previously using mild acidic treatment for short hydrolysis times. , Most recently, the National Metrology Institute of Japan (NMIJ) demonstrated formic acid’s utility for hydrolyzing purines and pyrimidines from intermediate-sized bacterial dsDNA . Here, we expand on that effort by demonstrating hydrolysis at extreme sizes (for human genomic dsDNA) and for quantification of ribo-oligos (RNA).…”

“…Predominantly though, acid hydrolysis has been applied for atypical or modified nucleobase detection (DNA damage or carcinogenic adduction − ) rather than with the intent to quantify the intact oligonucleotide. Recently, chemical hydrolysis was demonstrated using double-stranded genomic DNA from bacteriophage lambda (∼48.5 kbp), showing that DNA can be accurately quantified using just such an approach . In this work, we demonstrate a modified chemical hydrolysis workflow for any sized DNA using formic acid hydrolysis and nucleobase calibrators applied to small dsDNA (7.9 kbp linearized plasmid containing 5.2 kbp BK virus) and, for the first time, to human genomic DNA (3.2 × 10 9 base-pairs).…”

“…Recently, chemical hydrolysis was demonstrated using double-stranded genomic DNA from bacteriophage lambda (∼48.5 kbp), showing that DNA can be accurately quantified using just such an approach. 33 In this work, we demonstrate a modified chemical hydrolysis workflow for any sized DNA using formic acid hydrolysis and nucleobase calibrators applied to small dsDNA (7.9 kbp linearized plasmid containing 5.2 kbp BK virus) and, for the first time, to human genomic DNA (3.2 × 10 9 base-pairs). Most importantly, we demonstrate for the first time that this approach can be applied to ribonucleotide oligomers (RNA).…”

“…26,36 Most recently, the National Metrology Institute of Japan (NMIJ) demonstrated formic acid's utility for hydrolyzing purines and pyrimidines from intermediate-sized bacterial dsDNA. 33 Here, we expand on that effort by demonstrating hydrolysis at extreme sizes (for human genomic dsDNA) and for quantification of ribo-oligos (RNA). Neat formic acid (∼26 mol/L) can be used to hydrolyze samples in either gas phase or liquid phase.…”

Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA

“…Currently, to determine the LOD in qualitative real-time PCR, a series of standard solutions are prepared by diluting DNA solutions of known concentrations. The concentrations of the original DNA solutions are estimated by ultraviolet (UV) absorption, fluorescence spectrometry, , or mass spectrometry. − However, the UV absorption and fluorescence spectrometric methods are strongly affected by sample conditions such as pH and salt concentration. Moreover, the accuracy of DNA quantification using these methods remains uncertain .…”