Enzymatic synthesis of structure-free DNA with pseudo-complementary properties

Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free singlestranded copies of any nucleic acid without impairing Watson–Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N 4 -alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that are <25 nt in lengthThis work was supported by grants from the National Institutes of Health (NIGMS Grant 74564 to H.G.) and Agilent Technologies. Dr Carl Fuller (GE Healthcare) kindly provided us with dNTPs of 7-alkyl-7-deazaguanines and 7-iodo-7-deazaguanine. We thank Dr Jeffrey Sampson (Agilent Technologies) for his constant encouragement and Caryn Evila (Idaho State University) for her early participation in the project. At TriLink Biotechnologies we are grateful to Dr Richard Hogrefe, Dr Gerald Zon, Dr Natasha Paul and Dr David Combs for support and helpful discussions and Dr Inna Koukhareva and Stephanie Perry for synthesis of dNTPs. Funding to pay the Open Access publication charges for this article was provided by NIH GM74564.Peer reviewe

Enzymatic synthesis of structure-free DNA with pseudo-complementary properties.

Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free single-stranded copies of any nucleic acid without impairing Watson-Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N(4)-alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that arelength

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification

Measurement of single electrons and implications for charm production in Au+Au collisions at root(NN)-N-S=130 GeV

Transverse momentum spectra of electrons from Au+Au collisions at roots(NN) = 130 GeV have been measured at midrapidity by the PHENIX experiment at the Relativistic Heavy Ion Collider. The spectra show an excess above the background from photon conversions and light hadron decays. The electron signal is consistent with that expected from semileptonic decays of charm. The yield of the electron signal dN(e)/dy for p(T) \u3e 0.8 GeV/c is 0.025 +/- 0.004(stat) +/- 0.010( syst) in central collisions, and the corresponding charm cross section is 380 +/- 60(stat) +/- 200(syst ) mu b per binary nucleon-nucleon collision

Centrality dependence of pi(+/-), K-+/-, p, and (p)over-bar production from root(NN)-N-S = 130 GeV Au+Au collisions at RHIC

Identified pi(+/-), K+/-, p, and (p) over bar transverse momentum spectra at midrapidity in root s(NN) = 130 GeV Au + Au collisions were measured by the PHENIX experiment at RHIC as a function of collision centrality. Average transverse momenta increase with the number of participating nucleons in a similar way for all particle species. Within errors, all midrapidity particle yields per participant are found to be increasing with the number of participating nucleons. There is an indication that K+/-, p, and (p) over bar yields per participant increase faster than the pi(+/-) yields. In central collisions at high transverse momenta (p(T) greater than or similar to 2 GeV/c), (p) over bar and p yields are comparable to the pi(+/-) yields

Net charge fluctuations in Au+Au interactions root s(NN)=130 GeV

Data from Au+Au interactions at s(NN)=130 GeV, obtained with the PHENIX detector at the Relativistic Heavy-Ion Collider, are used to investigate local net charge fluctuations among particles produced near midrapidity. According to recent suggestions, such fluctuations may carry information from the quark-gluon plasma. This analysis shows that the fluctuations are dominated by a stochastic distribution of particles, but are also sensitive to other effects, like global charge conservation and resonance decays

Measurement of the midrapidity transverse energy distribution from root(NN)-N-S=130 GeV Au+Au collisions at RHIC

The first measurement of energy produced transverse to the beam direction at the Relativistic Heavy-Ion Collider at Brookhaven National Laboratory is presented. The midrapidity transverse energy density per participating nucleon rises steadily with the number of participants, closely paralleling the rise in charged-particle density, such that ⟨ET⟩/⟨Nch⟩ remains relatively constant as a function of centrality. The energy density calculated via Bjorken’s prescription for the 2% most central Au+Au collisions at √sNN=130GeV is at least εBj=4.6 GeV/fm3, which is a factor of 1.6 larger than found at √sNN=17.2 GeV ( Pb+Pb at CERN)

Transverse-mass dependence of two-pion correlations in Au+Au collisions at root(NN)-N-S=130 GeV

Two-pion correlations in roots(NN) = 130 GeV Au+Au collisions at RHIC have been measured over a broad range of pair transverse momentum k(T) by the PHENIX experiment at RHIC. The k(T) dependent transverse radii are similar to results from heavy-ion collisions at roots(NN) = 4.1 , 4.9, and 17.3 GeV, whereas the longitudinal radius increases monotonically with beam energy. The ratio of the outwards to sidewards transverse radii (R-out/R-side) is consistent with unity and independent of k(T)