Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

Are-examination of NaCI effects on phosphoenolpyruvate carboxylase at high (physiological) enzyme concentrations

Inhibition of phosphoenolpyruvate carboxylase (EC 4.1.1 31) from the C₄-halophyte Salsola soda L. by NaCI is compeutive to phosphoenolpyruvate (PEP). Physiological (betaine, glycerol) and synthetic (polyethylene glycol) osmotica and the allosteric activator glucose-6-phosphate (G6P) increase the apparent affinity of the enzyme for PEP and also alleviate the inhibition by NaCl. Physiological osmotica that either increase the K_m(PEP) (proline) or are neutral (sorbitol), do not protect the enzyme against NaCI attack. In the absence of cosolutes and, G6P, the enzyme is self-protected when its concentration in the assay medium is increased to more physiological values. In addition, the amount of betaine needed for complete protection is inversely related to native protein concentration in the assay. Exogenous protein (bovine serum albumin or bovine skin gelatin) have no effect on either K_m(PEP), or extent of NaCl inhibition. These results can be better explained with the exclusion volume theory and the inferred assumption that both cosolutes and high protein concentration strengthen intrinsic aggregation properties of enzymes. It is suggested that the extremely high phosphoenolpyruvate carboxylase concentration in the cytoplasm and the accumulation of compatible solutes in response to water stress fully protect the enzyme in vivo against the chaotropic effects of NaCI.     

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N, N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1 M solute were heated for 10 min at temperatures between 35-75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1 M stabilizer solutions were frozen at -30°C and freeze-dried for 24 h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine

Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD⁺ and NADP⁺ as cofactors, but NAD⁺ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K _m values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD⁺, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress. Received: 2 May 1997 / Accepted: 2 July 1997     

High-throughput thermal stability assessment of DNA hairpins based on high resolution melting

On the basis of high-resolution melting, a high-throughput approach to measure melting temperatures (T_ms) of short DNA hairpins was developed. With this method, T_ms of thousands of triloop, tetraloop, and pentaloop hairpins involving various loop sequences and various closing base pairs (cbp) were obtained in hours. The stability of triloop hairpins decreased with the change of cbp (5′–3′) in the order of c-g > g-c > t-a ≥ a-t, showing that the cbp of 5′-Pyr-Pur-3′ (Pyr = pyrimidine, Pur = purine) contributed more stability than 5′-Pur-Pyr-3′. For tetraloop hairpins, GNNA, GNAB, and CNNG (N = A, G, C, or T; B = G, C, or T) were found to be highly stable irrespective of the cbp type. TNNA was also stable in both g-c and a-t families, while CGNA only in the c-g family. Pentaloop hairpins of cTGNAGg, cGNYNAg (Y = T or C) and cCGNNAg were exceptionally stable motifs. In most cases, pyrimidine-rich loops were more favorable to stabilize the whole structure than purine-rich ones. The present approach showed a good performance in assessing the thermal stability of large amounts of DNA hairpins comprehensively. These data are useful to understand the sequence dependence of the stability of DNA secondary structures and promising to improve the structure simulation by consummating basic databases.     

Compatible solutes and their effects on phosphoenolpyruvate carboxylase of C4-halophytes

Abstract Phosphoenolpyruvate carboxylase (PEPCase), extracted from two Poaceae (Cynodon dactylon and Sporobolus pungens) grown on saline soil, was affected physiologically by betaine and proline. Its affinity for phosphoenolpyruvate (PEP) was increased and full protection against NaCl inhibition was observed; enzymic activity was also stabilized when assayed at low PEP levels. Betaine has the same effects on PEPCase extracted from a Chenopodiaceae (Salsola soda), whereas proline behaves as a competitive inhibitor, i.e. it does not protect the enzyme against NaCl and it accelerates inactivation at low PEP levels. Betaine was only compatible with PEPCase extracted from Saisola kali, without any effect on activity, protection or stabilization, but proline was again inhibitory. The levels of free proline in the two salt-stressed Poaceae were high, whereas in the Chenopodiaceae the free proline was low, as in non-stressed plants. The above data indicate that osmoregulators could not only be compatible with cytoplasmic enzymes, but they could either promote or inhibit enzyme activity, depending on the source of enzyme. Coevolution of PEPCase with the osmolyte selected for, could also be inferred.     

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

Fate of compatible solutes during dilution stress in Ectothiorhodospira halochloris

Abstract Ectothiorhodospira halochloris reacts upon enhancement of the water activity in the environment by excreting its major compatible solute, glycine betaine, thus decreasing the osmotic pressure inside the cell. A suddenly induced dilution stress leads to an overshoot of this reaction, so that more glycine betaine than necessary to compensate the external osmotic change is released. Subsequently the cells take up glycine betaine until they reach osmotic balance with the medium. E. halochloris possesses an active transport system that allows an uptake of glycine betaine against a concentration gradient. Glycine betaine is not metabolized in E. halochloris . Ectoine, a minor compatible solute of E. halochloris , is excreted in a similar manner to that of glycine betaine during dilution stress, whereas no excretion of the third compatible solute, trehalose, was detected.     

Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability

Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8kcal/mol and increased T(m) up to 4.3 degrees C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ approximately Black Hole 2>Cy5 approximately Cy3>Black Hole 1>QSY7 approximately Iowa Black FQ>Texas Red approximately TAMRA>FAM approximately HEX approximately Dabcyl>TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.     

Glycine betaine improves Listeria monocytogenes tolerance to desiccation on parsley leaves independent of the osmolyte transporters BetL, Gbu and OpuC

Aims: To investigate the effect of glycine betaine (GB) on the survival of Listeria monocytogenes on leaf surfaces under low relative humidity (RH). Methods and Results: The addition of GB (≥25 mmol l^?1) improved the survival of L. monocytogenes under low RH on parsley leaves, thus suggesting that GB can improve the tolerance of L. monocytogenes to desiccation. Ten times less GB was needed to improve L. monocytogenes survival under low RH on nonbiological surfaces compared with parsley leaves, suggesting that, on the leaf surface, L. monocytogenes may have to compete for the available GB with autochthonous bacteria and/or the plant itself. Wild type and mutants carrying deletions in the three GB uptake systems, BetL, Gbu and OpuC, behaved similarly with and without added GB on parsley leaves (P > 0·05). In addition, preaccumulation of GB, triggered by osmotic stress prior to inoculation, failed to improve survival under low RH compared with osmotic stress without GB accumulation. Conclusions: Exogenous GB had a protective effect on L. monocytogenes cells from desiccation during survival on parsley leaves. This effect was independent of intracellular GB accumulation by the known uptake systems. Significance and Impact of the Study: Presence of GB could improve the survival of L. monocytogenes to desiccation on leaf surfaces and nonbiological surfaces.     

Exogenously supplied glycine betaine in spinach and rapeseed leaf discs: compatibility or non-compatibility?

CMS, cell membrane stability
GB, glycine betaine
PEG, polyethylene glycol
TTC, 2,3,5-triphenyltetrazolium chloride

When leaf discs of spinach ( Spinacia oleracea cv. Junius) and rapeseed ( Brassica napus var. oleifera cv. Samourai) were incubated in the light in the presence of glycine betaine (GB), they accumulated GB at a very high level. In comparison with the spinach leaf explants, the uptake of GB by rapeseed tissues was restricted, probably by the destabilizing effects exerted by GB in this plant material. In contrast, the viability of spinach leaf discs, as assessed by their capacity to reduce 2,3,5-triphenyltetrazolium chloride (TTC), was not affected, suggesting that the GB taken up was compatible in the leaf tissues of the GB accumulator. In rapeseed leaf discs treated with GB, chlorophyll loss as well as significant changes in polyamine content were induced, leading to a dramatic increase of the putrescine/(spermidine + spermine) ratio. In contrast, this ratio remained constant in the GB treated spinach explants, suggesting that spinach has the capacity to stabilize polyamine metabolism in the presence of high amounts of GB. The treatment of spinach leaf discs with GB prior to application of osmotic or salt shocks provided protection from stress. A weak capacity to accumulate proline under stress conditions was partially suppressed. The protein content decreased while the free amino acid level increased independently of the presence of GB. It is concluded that GB behaves as a true compatible solute in spinach, which is a typical GB accumulator, and that GB is damaging when loaded into the leaf tissues of rapeseed, which do not normally accumulate GB.     

一步PCR快速扩增辽宁碱蓬甜菜碱醛脱氢酶cDNA 3'末端序列

根据已获得的辽宁碱蓬甜菜碱醛脱氢酶cDNA的部分序列,设计一条基因特异性引物,与通用引物并用,一步PCR成功地克隆了辽宁碱蓬甜菜碱醛脱氢酶cDNA 3′末端。与常规的3′RACE法相比,一步PCR法具有快速、简便、经济等优点,是一种非常快捷的扩增cDNA 3′末端序列的方法。Abstract:Based on part of a known cDNA sequence of Suaeda liaotungensis betaine aldehyde dehydrogenase,we successfully cloned the 3′cDNA end of S.lianotungensis betaine aldehyde dehydrogenase using one step PCR with a gene specific primer and universal primer.Compared with the typical 3′ RACE,one step PCR is rapid,simple and inexpensive.It is very rapid to amplify an unknown cDNA 3′end using this method.     

Predicting sequence-dependent melting stability of short duplex DNA oligomers

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or t_m, of short duplex DNA oligomers (∼ 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting t_m from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental t_m's of short duplex DNA oligomers is assessed. © 1998 John Wiley & Sons, Inc. Biopoly 44: 217–239, 1997