Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease

Abstract

Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.

Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: Piperidine-induced destabilization of the DNA duplex?

“…STRs are the most valuable form of sequence variation for use as genetic markers because they are highly polymorphic [5,6], occur approximately every 10 -20 kb in the human genome [7][8][9], and are relatively easy to isolate using the polymerase chain reaction (PCR) [7,8,10]. PCR amplicons generated from STR loci are typically observed as duplexes when using ESI-MS [3,4,[11][12][13][14][15][16][17][18][19][20][21][22][23]. This soft ionization process enables retention of the Watson-Crick base pairing [24] of PCR amplicons.…”

“…The difficulty of obtaining accurate mass measurements within duplex PCR amplicons arises from the fact that a base substitution, such as an A 3 T transversion, cannot be resolved by mass because of the T 3 A transversion in the complementary strand resulting in a mass difference of zero. Therefore, the production of singlestranded (ss) PCR amplicons is essential for the accurate characterization of base substitutions housed within STRs [3,16].…”

“…Strategies for the routine production of singlestranded PCR products for mass spectrometry have included nozzle skimmer dissociation [29], biotinstreptavidin chemistry [30 -36], and the DNA repair enzyme, lambda exonuclease [3,16]. It has been reported that the use of nozzle skimmer dissociation on duplex 16-mer oligonucleotides with a G-C content reaching 75% showed complete dissociation with no signs of covalent fragmentation [29].…”

“…This method of producing single strands, although successful, has been shown to exhibit significant sample loss due to difficulties in removing the unbiotinylated strands [37]. Singlestranded PCR products have been successfully generated enzymatically in our lab via the use of lambda exonuclease which selectively digests the 5' phosphorylated strand of a duplex, leaving the complementary strand available for mass analysis [3,16]. While this method has great applicability, it requires an additional enzymatic digestion step and incomplete phosphorylation of the primers renders the amplicon resistant to digestion by lambda exonuclease [3,16].…”

“…Singlestranded PCR products have been successfully generated enzymatically in our lab via the use of lambda exonuclease which selectively digests the 5' phosphorylated strand of a duplex, leaving the complementary strand available for mass analysis [3,16]. While this method has great applicability, it requires an additional enzymatic digestion step and incomplete phosphorylation of the primers renders the amplicon resistant to digestion by lambda exonuclease [3,16]. However, this method is advantageous because it allows researchers the ability to select which strand is generated following the digestion process as well as reduce the complexity of the mixture (e.g., multiplex PCR).…”