Even modest climate change may lead to major transitions in boreal forests.

The sensitivity of forests to near-term warming and associated precipitation shifts remains uncertain1-9. Herein, using a 5-year open-air experiment in southern boreal forest, we show divergent responses to modest climate alteration among juveniles of nine co-occurring North American tree species. Warming alone (+1.6 °C or +3.1 °C above ambient temperature) or combined with reduced rainfall increased the juvenile mortality of all species, especially boreal conifers. Species differed in growth responses to warming, ranging from enhanced growth in Acer rubrum and Acer saccharum to severe growth reductions in Abies balsamea, Picea glauca and Pinus strobus. Moreover, treatment-induced changes in both photosynthesis and growth help explain treatment-driven changes in survival. Treatments in which species experienced conditions warmer or drier than at their range margins resulted in the most adverse impacts on growth and survival. Species abundant in southern boreal forests had the largest reductions in growth and survival due to climate manipulations. By contrast, temperate species that experienced little mortality and substantial growth enhancement in response to warming are rare throughout southern boreal forest and unlikely to rapidly expand their density and distribution. Therefore, projected climate change will probably cause regeneration failure of currently dominant southern boreal species and, coupled with their slow replacement by temperate species, lead to tree regeneration shortfalls with potential adverse impacts on the health, diversity and ecosystem services of regional forests.

Effects of climate warming on photosynthesis in boreal tree species depend on soil moisture.

Climate warming will influence photosynthesis via thermal effects and by altering soil moisture1-11. Both effects may be important for the vast areas of global forests that fluctuate between periods when cool temperatures limit photosynthesis and periods when soil moisture may be limiting to carbon gain4-6,9-11. Here we show that the effects of climate warming flip from positive to negative as southern boreal forests transition from rainy to modestly dry periods during the growing season. In a three-year open-air warming experiment with juveniles of 11 temperate and boreal tree species, an increase of 3.4 °C in temperature increased light-saturated net photosynthesis and leaf diffusive conductance on average on the one-third of days with the wettest soils. In all 11 species, leaf diffusive conductance and, as a result, light-saturated net photosynthesis decreased during dry spells, and did so more sharply in warmed plants than in plants at ambient temperatures. Consequently, across the 11 species, warming reduced light-saturated net photosynthesis on the two-thirds of days with driest soils. Thus, low soil moisture may reduce, or even reverse, the potential benefits of climate warming on photosynthesis in mesic, seasonally cold environments, both during drought and in regularly occurring, modestly dry periods during the growing season.

Phenological responses of temperate and boreal trees to warming depend on ambient spring temperatures, leaf habit, and geographic range.

Changes in plant phenology associated with climate change have been observed globally. What is poorly known is whether and how phenological responses to climate warming will differ from year to year, season to season, habitat to habitat, or species to species. Here, we present 5 y of phenological responses to experimental warming for 10 subboreal tree species. Research took place in the open-air B4WarmED experiment in Minnesota. The design is a two habitat (understory and open) × three warming treatments (ambient, +1.7 °C, +3.4 °C) factorial at two sites. Phenology was measured twice weekly during the growing seasons of 2009 through 2013. We found significant interannual variation in the effect of warming and differences among species in response to warming that relate to geographic origin and plant functional group. Moreover, responses to experimental temperature variation were similar to responses to natural temperature variation. Warming advanced the date of budburst more in early compared to late springs, suggesting that to simulate interannual variability in climate sensitivity of phenology, models should employ process-based or continuous development approaches. Differences among species in timing of budburst were also greater in early compared to late springs. Our results suggest that climate change-which will make most springs relatively "early"-could lead to a future with more variable phenology among years and among species, with consequences including greater risk of inappropriately early leafing and altered interactions among species.

Boreal and temperate trees show strong acclimation of respiration to warming.

Plant respiration results in an annual flux of carbon dioxide (CO2) to the atmosphere that is six times as large as that due to the emissions from fossil fuel burning, so changes in either will impact future climate. As plant respiration responds positively to temperature, a warming world may result in additional respiratory CO2 release, and hence further atmospheric warming. Plant respiration can acclimate to altered temperatures, however, weakening the positive feedback of plant respiration to rising global air temperature, but a lack of evidence on long-term (weeks to years) acclimation to climate warming in field settings currently hinders realistic predictions of respiratory release of CO2 under future climatic conditions. Here we demonstrate strong acclimation of leaf respiration to both experimental warming and seasonal temperature variation for juveniles of ten North American tree species growing for several years in forest conditions. Plants grown and measured at 3.4 °C above ambient temperature increased leaf respiration by an average of 5% compared to plants grown and measured at ambient temperature; without acclimation, these increases would have been 23%. Thus, acclimation eliminated 80% of the expected increase in leaf respiration of non-acclimated plants. Acclimation of leaf respiration per degree temperature change was similar for experimental warming and seasonal temperature variation. Moreover, the observed increase in leaf respiration per degree increase in temperature was less than half as large as the average reported for previous studies, which were conducted largely over shorter time scales in laboratory settings. If such dampening effects of leaf thermal acclimation occur generally, the increase in respiration rates of terrestrial plants in response to climate warming may be less than predicted, and thus may not raise atmospheric CO2 concentrations as much as anticipated.

Asynchronous nitrogen supply and demand produce nonlinear plant allocation responses to warming and elevated CO2.

Terrestrial ecosystem responses to climate change are mediated by complex plant-soil feedbacks that are poorly understood, but often driven by the balance of nutrient supply and demand. We actively increased aboveground plant-surface temperature, belowground soil temperature, and atmospheric CO2 in a brackish marsh and found nonlinear and nonadditive feedbacks in plant responses. Changes in root-to-shoot allocation by sedges were nonlinear, with peak belowground allocation occurring at +1.7 °C in both years. Above 1.7 °C, allocation to root versus shoot production decreased with increasing warming such that there were no differences in root biomass between ambient and +5.1 °C plots in either year. Elevated CO2 altered this response when crossed with +5.1 °C, increasing root-to-shoot allocation due to increased plant nitrogen demand and, consequently, root production. We suggest these nonlinear responses to warming are caused by asynchrony between the thresholds that trigger increased plant nitrogen (N) demand versus increased N mineralization rates. The resulting shifts in biomass allocation between roots and shoots have important consequences for forecasting terrestrial ecosystem responses to climate change and understanding global trends.

Assessing the relevant time frame for temperature acclimation of leaf dark respiration: A test with 10 boreal and temperate species.

Plants often adjust their leaf mitochondrial ("dark") respiration (Rd ) measured at a standardized temperature such as 20°C (R20 ) downward after experiencing warmer temperatures and upward after experiencing cooler temperatures. These responses may help leaves maintain advantageous photosynthetic capacity and/or be a response to recent photosynthate accumulation, and can occur within days after a change in thermal regime. It is not clear, however, how the sensitivity and magnitude of this response change over time, or which time period prior to a given measurement best predicts R20 . Nor is it known whether nighttime, daytime, or 24-hour temperatures should be most influential. To address these issues, we used data from 1620 Rd temperature response curves of 10 temperate and boreal tree species in a long-term field experiment in Minnesota, USA to assess how the observed nearly complete acclimation of R20 was related to past temperatures during periods of differing lengths. We hypothesized that R20 would be best related to prior midday temperatures associated with both photosynthetic biochemistry and peak carbon uptake rates that drive carbohydrate accumulation. Inconsistent with this hypothesis, prior night temperatures were the best predictors of R20 for all species. We had also hypothesized that recent (prior 3-10 days) temperatures should best predict R20 because they likely have stronger residual impacts on leaf-level physiology than periods extending further back in time, whereas a prior 1- to 2-day period might be a span shorter than one to which photosynthetic capacity and Rd adjust. There was little to no support for this idea, as for angiosperms, long time windows (prior 30-60 nights) were the best predictors, while for gymnosperms both near-term (prior 3-8 nights for pines, prior 10-14 nights for spruce/fir) and longer-term periods (prior 45 nights) were the best predictors. The importance of nighttime temperatures, the relatively long "time-averaging" that best explained acclimation, and dual peaks of temporal acclimation responsiveness in some species were all results that were unanticipated.

Species-specific flowering phenology responses to experimental warming and drought alter herbaceous plant species overlap in a temperate-boreal forest community.

BACKGROUND AND AIMS: Warmer temperatures and altered precipitation patterns are expected to continue to occur as the climate changes. How these changes will impact the flowering phenology of herbaceous perennials in northern forests is poorly understood but could have consequences for forest functioning and species interactions. Here, we examine the flowering phenology responses of five herbaceous perennials to experimental warming and reduced summer rainfall over 3 years. METHODS: This study is part of the B4WarmED experiment located at two sites in northern Minnesota, USA. Three levels of warming (ambient, +1.6 °C and +3.1 °C) were crossed with two rainfall manipulations (ambient and 27 % reduced growing season rainfall). KEY RESULTS: We observed species-specific responses to the experimental treatments. Warming alone advanced flowering for four species. Most notably, the two autumn blooming species showed the strongest advance of flowering to warming. Reduced rainfall alone advanced flowering for one autumn blooming species and delayed flowering for the other, with no significant impact on the three early blooming species. Only one species, Solidago spp., showed an interactive response to warming and rainfall manipulation by advancing in +1.6 °C warming (regardless of rainfall manipulation) but not advancing in the warmest, driest treatment. Species-specific responses led to changes in temporal overlap between species. Most notably, the two autumn blooming species diverged significantly in their flowering timing. In ambient conditions, these two species flowered within the same week. In the warmest, driest treatment, flowering occurred over a month apart. CONCLUSIONS: Herbaceous species may differ in how they respond to future climate conditions. Changes to phenology may lead to fewer resources for insects or a mismatch between plants and pollinators.

Experimental warming advances phenology of groundlayer plants at the boreal-temperate forest ecotone.

PREMISE OF THE STUDY: Changes to plant phenology have been linked to warmer temperatures caused by climate change. Despite the importance of the groundlayer to community and forest dynamics, few warming experiments have focused on herbaceous plant and shrub phenology. METHODS: Using a field study in Minnesota, United States, we investigated phenological responses of 16 species to warming over five growing seasons (2009-2013) at two sites, under two canopy covers, and in three levels of simultaneous above- and belowground warming: ambient temperature, ambient +1.7°C and ambient +3.4°C. We tested whether warming led to earlier phenology throughout the growing season and whether responses varied among species and years and depended on canopy cover. KEY RESULTS: Warming extended the growing season between 11-30 days, primarily through earlier leaf unfolding. Leaf senescence was delayed for about half of the species. Warming advanced flowering across species, especially those flowering in August, with modest impacts on fruit maturation for two species. Importantly, warming caused more than half of the species to either converge or diverge phenologically in relation to each other, suggesting that future warmed climate conditions will alter phenological relationships of the groundlayer. Warm springs elicited a stronger advance of leaf unfolding compared to cool spring years. Several species advanced leaf unfolding (in response to warming) more in the closed canopy compared to the open. CONCLUSIONS: Climate warming will extend the growing season of groundlayer species in the boreal-temperate forest ecotone and alter the synchrony of their phenology.

Biogeographic variation in evergreen conifer needle longevity and impacts on boreal forest carbon cycle projections.

Leaf life span is an important plant trait associated with interspecific variation in leaf, organismal, and ecosystem processes. We hypothesized that intraspecific variation in gymnosperm needle traits with latitude reflects both selection and acclimation for traits adaptive to the associated temperature and moisture gradient. This hypothesis was supported, because across 127 sites along a 2,160-km gradient in North America individuals of Picea glauca, Picea mariana, Pinus banksiana, and Abies balsamea had longer needle life span and lower tissue nitrogen concentration with decreasing mean annual temperature. Similar patterns were noted for Pinus sylvestris across a north-south gradient in Europe. These differences highlight needle longevity as an adaptive feature important to ecological success of boreal conifers across broad climatic ranges. Additionally, differences in leaf life span directly affect annual foliage turnover rate, which along with needle physiology partially regulates carbon cycling through effects on gross primary production and net canopy carbon export. However, most, if not all, global land surface models parameterize needle longevity of boreal evergreen forests as if it were a constant. We incorporated temperature-dependent needle longevity and %nitrogen, and biomass allocation, into a land surface model, Community Atmosphere Biosphere Land Exchange, to assess their impacts on carbon cycling processes. Incorporating realistic parameterization of these variables improved predictions of canopy leaf area index and gross primary production compared with observations from flux sites. Finally, increasingly low foliage turnover and biomass fraction toward the cold far north indicate that a surprisingly small fraction of new biomass is allocated to foliage under such conditions.

Is it getting hot in here? Adjustment of hydraulic parameters in six boreal and temperate tree species after 5 years of warming.

Global temperatures (T) are rising, and for many plant species, their physiological response to this change has not been well characterized. In particular, how hydraulic parameters may change has only been examined experimentally for a few species. To address this, we measured characteristics of the hydraulic architecture of six species growing in ambient T and ambient +3.4 °C T plots in two experimentally warmed forest sites in Minnesota. These sites are at the temperate-boreal ecotone, and we measured three species from each forest type. We hypothesized that relative to boreal species, temperate species near their northern range border would increase xylem conduit diameters when grown under elevated T. We also predicted a continuum of responses among wood types, with conduit diameter increases correlating with increases in the complexity of wood structure. Finally, we predicted that increases in conduit diameter and specific hydraulic conductivity would positively affect photosynthetic rates and growth. Our results generally supported our hypotheses, and conduit diameter increased under elevated T across all species, although this pattern was driven predominantly by three species. Two of these species were temperate angiosperms, but one was a boreal conifer, contrary to predictions. We observed positive relationships between the change in specific hydraulic conductivity and both photosynthetic rate (P = 0.080) and growth (P = 0.012). Our results indicate that species differ in their ability to adjust hydraulically to increases in T. Specifically, species with more complex xylem anatomy, particularly those individuals growing near the cooler edge of their range, appeared to be better able to increase conduit diameters and specific hydraulic conductivity, which permitted increases in photosynthesis and growth. Our data support results that indicate individual's ability to physiologically adjust is related to their location within their species range, and highlight that some wood types may adjust more easily than others.

Design and performance of combined infrared canopy and belowground warming in the B4WarmED (Boreal Forest Warming at an Ecotone in Danger) experiment.

Conducting manipulative climate change experiments in complex vegetation is challenging, given considerable temporal and spatial heterogeneity. One specific challenge involves warming of both plants and soils to depth. We describe the design and performance of an open-air warming experiment called Boreal Forest Warming at an Ecotone in Danger (B4WarmED) that addresses the potential for projected climate warming to alter tree function, species composition, and ecosystem processes at the boreal-temperate ecotone. The experiment includes two forested sites in northern Minnesota, USA, with plots in both open (recently clear-cut) and closed canopy habitats, where seedlings of 11 tree species were planted into native ground vegetation. Treatments include three target levels of plant canopy and soil warming (ambient, +1.7°C, +3.4°C). Warming was achieved by independent feedback control of voltage input to aboveground infrared heaters and belowground buried resistance heating cables in each of 72-7.0 m(2) plots. The treatments emulated patterns of observed diurnal, seasonal, and annual temperatures but with superimposed warming. For the 2009 to 2011 field seasons, we achieved temperature elevations near our targets with growing season overall mean differences (∆Tbelow ) of +1.84°C and +3.66°C at 10 cm soil depth and (∆T(above) ) of +1.82°C and +3.45°C for the plant canopies. We also achieved measured soil warming to at least 1 m depth. Aboveground treatment stability and control were better during nighttime than daytime and in closed vs. open canopy sites in part due to calmer conditions. Heating efficacy in open canopy areas was reduced with increasing canopy complexity and size. Results of this study suggest the warming approach is scalable: it should work well in small-statured vegetation such as grasslands, desert, agricultural crops, and tree saplings (<5 m tall).

Acclimation of photosynthetic temperature optima of temperate and boreal tree species in response to experimental forest warming.

Rising temperatures caused by climate change could negatively alter plant ecosystems if temperatures exceed optimal temperatures for carbon gain. Such changes may threaten temperature-sensitive species, causing local extinctions and range migrations. This study examined the optimal temperature of net photosynthesis (Topt ) of two boreal and four temperate deciduous tree species grown in the field in northern Minnesota, United States under two contrasting temperature regimes. We hypothesized that Topt would be higher in temperate than co-occurring boreal species, with temperate species exhibiting greater plasticity in Topt , resulting in better acclimation to elevated temperatures. The chamberless experiment, located at two sites in both open and understory conditions, continuously warmed plants and soils during three growing seasons. Results show a modest, but significant shift in Topt of 1.1 ± 0.21 °C on average for plants subjected to a mean 2.9 ± 0.01 °C warming during midday hours in summer, and shifts with warming were unrelated to species native ranges. The 1.1 °C shift in Topt with 2.9 °C warming might be interpreted as suggesting limited capacity to shift temperature response functions to better match changes in temperature. However, Topt of warmed plants was as well-matched with prior midday temperatures as Topt of plants in the ambient treatment, and Topt in both treatments was at a level where realized photosynthesis was within 90-95% of maximum. These results suggest that seedlings of all species were close to optimizing photosynthetic temperature responses, and equally so in both temperature treatments. Our study suggests that temperate and boreal species have considerable capacity to match their photosynthetic temperature response functions to prevailing growing season temperatures that occur today and to those that will likely occur in the coming decades under climate change.

Nematode community shifts in response to experimental warming and canopy conditions are associated with plant community changes in the temperate-boreal forest ecotone.

Global climate warming is one of the key forces driving plant community shifts, such as range shifts of temperate species into boreal forests. As plant community shifts are slow to observe, ecotones, boundaries between two ecosystems, are target areas for providing early evidence of ecological responses to warming. The role of soil fauna is poorly explored in ecotones, although their positive and negative effects on plant species can influence plant community structure. We studied nematode communities in response to experimental warming (ambient, +1.7, +3.4 °C) in soils of closed and open canopy forest in the temperate-boreal ecotone of Minnesota, USA and calculated various established nematode indices. We estimated species-specific coverage of understory herbaceous and shrub plant species from the same experimental plots and tested if changes in the nematode community are associated with plant cover and composition. Individual nematode trophic groups did not differ among warming treatments, but the ratio between microbial-feeding and plant-feeding nematodes increased significantly and consistently with warming in both closed and open canopy areas and at both experimental field sites. The increase in this ratio was positively correlated with total cover of understory plant species, perhaps due to increased predation pressure on soil microorganisms causing higher nutrient availability for plants. Multivariate analyses revealed that temperature treatment, canopy conditions and nematode density consistently shaped understory plant communities across experimental sites. Our findings suggest that warming-induced changes in nematode community structure are associated with shifts in plant community composition and productivity in the temperate-boreal forest ecotones.

Reduced feeding activity of soil detritivores under warmer and drier conditions.

Anthropogenic warming is projected to trigger positive feedbacks to climate by enhancing carbon losses from the soil1. While such losses are, in part, owing to increased decomposition of organic matter by invertebrate detritivores, it is unknown how detritivore feeding activity will change with warming2, especially under drought conditions. Here, using four year manipulation experiments in two North American boreal forests, we investigate how temperature (ambient, +1.7 °C, +3.4 °C) and rainfall (ambient, -40% summer precipitation) perturbations influence detritivore feeding activity. In contrast to general expectations1,3, warming had negligible net effects on detritivore feeding activity at ambient precipitation. However, when combined with precipitation reductions, warming decreased feeding activity by ~14%. As across all plots and dates, detritivore feeding activity was positively associated to bulk soil microbial respiration, our results suggest slower rates of decomposition of soil organic matter, and thus reduced positive feedbacks to climate under anthropogenic climate change.

A molecular fraction collecting tool for the ABI 310 automated sequencer.

Several methods exist to retrieve and purify DNA fragments after agarose or polyacrylamide gel electrophoresis for subsequent analyses. However, molecules present in low concentration and molecules similar in size to their neighbors are difficult to purify. Capillary electrophoresis has become popular in molecular diagnostic laboratories because of its automation, excellent resolution, and high sensitivity. In the current study, the ABI Prism 310 Genetic Analyzer was reconfigured into a fraction collector by adapting the standard gel block to accommodate a collection tube at the distal end of capillary. The time to collect the desired peaks was estimated by extrapolating from standard capillary electrophoresis using the original gel block. Fraction collection from a mixture of DNA fragments amplified from wild type and several internal tandem duplication mutations of the FMS-like tyrosine kinase 3 (Flt3) gene yielded highly purified DNA fragments containing internal tandem duplication mutations and predictable electrokinetics using the reconstructed gel block. The reconfigured instrument could successfully isolate DNA amplicons from extremely low-amplitude peaks (110 relative fluorescent units), which were undetectable using polyacrylamide gel electrophoresis. In addition, we successfully isolated bands that were only three bases apart that comigrated on polyacrylamide gel electrophoresis. DNA sequencing was used to confirm that the correct peaks were recovered at sufficient purity.

Warming alters the energetic structure and function but not resilience of soil food webs.

Climate warming is predicted to alter the structure, stability, and functioning of food webs1-5. Yet, despite the importance of soil food webs for energy and nutrient turnover in terrestrial ecosystems, warming effects on these food webs-particularly in combination with other global change drivers-are largely unknown. Here, we present results from two complementary field experiments testing the interactive effects of warming with forest canopy disturbance and drought on energy fluxes in boreal-temperate ecotonal forest soil food webs. The first experiment applied a simultaneous above- and belowground warming treatment (ambient, +1.7°C, +3.4°C) to closed canopy and recently clear-cut forest, simulating common forest disturbance6. The second experiment crossed warming with a summer drought treatment (-40% rainfall) in the clear-cut habitats. We show that warming reduces energy fluxes to microbes, while forest canopy disturbance and drought facilitates warming-induced increases in energy flux to higher trophic levels and exacerbates reductions in energy flux to microbes, respectively. Contrary to expectations, we find no change in whole-network resilience to perturbations, but significant losses of ecosystem functioning. Warming thus interacts with forest disturbance and drought, shaping the energetic structure of soil food webs and threatening the provisioning of multiple ecosystem functions in boreal-temperate ecotonal forests.

Consistent leaf respiratory response to experimental warming of three North American deciduous trees: a comparison across seasons, years, habitats and sites.

Most vascular plants acclimate respiration to changes in ambient temperature, but explicit tests of these responses in field settings are rare, and how acclimation responses vary in space and time is relatively unstudied, hindering our ability to predict respiratory release of carbon under future climatic conditions. We measured temperature response curves of leaf respiration for three deciduous tree species from 2009 to 2012 in a field warming experiment (+3.4 °C above ambient) in both open and understory conditions at two sites in the southern boreal forest in Minnesota, USA. We analyzed the effects of warming on leaf respiration, and how the effects varied among species, times of season (early, middle and late parts of the growing season), sites, habitats (understory, open) and years. We hypothesized that the respiration exponent (Q10) of the short-term temperature response curve and the degree of acclimation would be smaller under conditions where plants were more likely to be substrate limited, such as in the understory or the margins of the growing season. However, in contrast to these predictions, stable Q10 and strong respiratory acclimation were consistently observed. For each species, the Q10 did not vary with experimental warming, nor was its response to warming influenced by time of season, year, site or habitat. Strong leaf respiratory acclimation to warming occurred in each species and was consistent across most sources of variation. Most of the leaf traits studied were not affected by warming, while the Q10-leaf nitrogen and R25-soluble carbohydrate relationships were observed, and shifted with warming, implying that acclimation may be associated with the adjustment in respiratory capacity and its relation to leaf nitrogen and soluble carbohydrate content. Consistent Q10 and acclimation across habitats, sites, times of season and years suggest that modeling of temperature acclimation may be possible with relatively simple functions.

Warming shifts 'worming': effects of experimental warming on invasive earthworms in northern North America.

Climate change causes species range shifts and potentially alters biological invasions. The invasion of European earthworm species across northern North America has severe impacts on native ecosystems. Given the long and cold winters in that region that to date supposedly have slowed earthworm invasion, future warming is hypothesized to accelerate earthworm invasions into yet non-invaded regions. Alternatively, warming-induced reductions in soil water content (SWC) can also decrease earthworm performance. We tested these hypotheses in a field warming experiment at two sites in Minnesota, USA by sampling earthworms in closed and open canopy in three temperature treatments in 2010 and 2012. Structural equation modeling revealed that detrimental warming effects on earthworm densities and biomass could indeed be partly explained by warming-induced reductions in SWC. The direction of warming effects depended on the current average SWC: warming had neutral to positive effects at high SWC, whereas the opposite was true at low SWC. Our results suggest that warming limits the invasion of earthworms in northern North America by causing less favorable soil abiotic conditions, unless warming is accompanied by increased and temporally even distributions of rainfall sufficient to offset greater water losses from higher evapotranspiration.

Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations.

PCR detection of chromosomal translocations and small insertion/deletion mutations is challenging when potential amplicon size varies greatly. Molecular diagnostic laboratories face such difficulties with the BCL2-IGH translocation in follicular lymphoma and with internal tandem duplication mutation of the FLT3 gene in leukemia, where breakpoints are widely distributed, mutations may be multiple, signal strength is low, and background noise is elevated. We developed a strategy, called Δ-PCR, that ensures PCR specificity and identifies individual breakpoints. Δ-PCR uses two forward primers (external and internal) and a reverse primer simultaneously. The internal primer functions as a probe with a defined distance Δ from the external primer. For follicular lymphoma, we prepared upstream, BCL2-specific primers for potential breakpoints to pair with a common, downstream VLJH primer. Multiplexed PCR amplicons are sized by capillary electrophoresis. Each of the upstream pairs has a defined interval separating them that uniquely identifies the breakpoint. The presence of two amplicons with a defined size difference confirms validity of the rearrangement and identity of the specific breakpoint, even if signal strength is low. By testing 40 follicular lymphoma and 12 control specimens from formalin-fixed, paraffin-embedded (FFPE) blocks, we showed that multiplex Δ-PCR is a simple, sensitive strategy to identify translocations with multiple breakpoints or partners. The strategy was also applied to detect minor leukemic clones with internal tandem duplication mutations and could have broader applications for other insertion/deletion and duplication mutations.

Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.

Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively.