Neurotrophins in the ventral tegmental area: Role in social stress, mood disorders and drug abuse.

This review discusses the impact of neurotrophins and other trophic factors, including fibroblast growth factor and glial cell line-derived neurotrophic factor, on mood disorders, weight regulation and drug abuse, with an emphasis on stress- and drug-induced changes in the ventral tegmental area (VTA). Neurotrophins, comprising nerve growth factor, brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4/5 play important roles in neuronal plasticity and the development of different psychopathologies. In the VTA, most research has focused on the role of BDNF, because other neurotrophins are not found there in significant quantities. BDNF originating in the VTA provides trophic support to dopamine neurons. The diverse intracellular signaling pathways activated by BDNF may underlie precise physiological functions specific to the VTA. In general, VTA BDNF expression increases after psychostimulant exposures, and enhanced BDNF level in the VTA facilitates psychostimulant effects. The impact of VTA BDNF on the behavioral effects of psychostimulants relies primarily on its action within the mesocorticolimbic circuit. In the case of opiates, VTA BDNF expression and effects seem to be dependent on whether an animal is drug-naïve or has a history of drug use, only the latter of which is related to dopamine mechanisms. Social defeat stress that is continuous in mice or intermittent in rats increases VTA BDNF expression, and is associated with depressive and social avoidance behaviors. Intermittent social defeat stress induces persistent VTA BDNF expression that triggers psychostimulant cross-sensitization. Understanding the cellular and molecular substrates of neurotrophin effects may lead to novel therapeutic approaches for the prevention and treatment of substance use and mood disorders.

Intermittent social defeat stress enhances mesocorticolimbic ΔFosB/BDNF co-expression and persistently activates corticotegmental neurons: implication for vulnerability to psychostimulants.

Intermittent social defeat stress exposure augments behavioral response to psychostimulants in a process termed cross-sensitization. Brain-derived neurotrophic factor (BDNF) mediates synaptic plasticity and cellular responses to stress and drugs of abuse. We previously showed that repeated social defeat stress persistently alters BDNF and activates ΔFosB expression in mesocorticolimbic regions. Here, we hypothesized that social defeat stress would increase ΔFosB expression in BDNF-containing mesocorticolimbic neurons at a time when cross-sensitization is evident. Because the ventral tegmental area (VTA) is critical for cross-sensitization, we similarly hypothesized that repeated social defeat stress would induce ΔFosB in neurons of mesocorticolimbic terminal regions that innervate the VTA. We induced social defeat stress in rats by short confrontations with an aggressive resident rat every third day for 10 days. Control rats were handled according to the same schedule. Defeated rats exhibited sensitized locomotor response to amphetamine (1.0mg/kg, i.p.) 10 days after termination of stress exposure. Separate rats, which underwent stress procedures without amphetamine challenge, were used for histological assessments. Rats received intra-VTA infusion of the retrograde tracer, Fluorogold (FG), and brain tissue was collected 10 days after stress or handling for immunohistochemistry. Stress exposure increased BDNF immunoreactivity in anterior cingulate, prelimbic and infralimbic regions of the prefrontal cortex (PFC), medial amygdala (AMY), nucleus accumbens (NAc) and VTA; ΔFosB labeling in anterior cingulate cortex (ACG) and nucleus accumbens; and ΔFosB/BDNF co-expression in prelimbic cortex (PL), nucleus accumbens and medial amygdala. Infralimbic ΔFosB-labeling was enhanced by stress in neurons innervating the VTA. Increased ΔFosB/BDNF co-expression and persistent functional activation of corticolimbic neurons after stress may contribute to mechanisms underlying cross-sensitization to psychostimulants.

[DLys(6)]-luteinizing hormone releasing hormone-curcumin conjugate inhibits pancreatic cancer cell growth in vitro and in vivo.

Pancreatic ductal adenocarcinomas are invariably lethal, and developing effective treatments that have minimal side effects is a challenge. Previous studies from our laboratory have shown that conjugates of cell membrane disrupting lytic peptides and luteinizing hormone releasing hormone (LHRH) target and destroy human prostate and breast cancer cells in xenografts in the nude mouse model (Hansel et al., Mol Cell Endocrinol 2007;260-262:183-9; Hansel et al., Mol Cell Endocrinol 2007;269:26-33), which express LHRH receptors. The objectives of our study were to synthesize a bioconjugate of LHRH analog ([DLys(6)]-LHRH) and a dietary microchemical (curcumin) and test the hypothesis that [DLys(6)]-LHRH-curcumin targets and inhibits pancreatic cancer cell growth in vitro and in vivo. In in vitro studies, we determined by confocal microscopy, flow cytometry analysis and reverse transcriptase-polymerase chain reaction that MIAPaCa-2, Panc-1 and BxPC-3 pancreatic cancer cell lines express LHRH receptors. [DLys(6)]-LHRH-curcumin inhibited cell proliferation of pancreatic cancer cell lines and induced apoptotic cell death (p < 0.05). Apoptosis was induced by cleavage of polyadenosine-5'-diphosphate-ribose-polymerase and caspase-3. The activity of [DLys(6)]-LHRH-curcumin was equal to free curcumin at equimolar concentrations in vitro. Unlike curcumin itself, the [DLys(6)]-LHRH-curcumin conjugate is water soluble which allows its intravenous administration. In two in vivo studies, [DLys(6)]-LHRH-curcumin given intravenously caused a significant (p < 0.01) reduction in tumor weights and volumes, and free curcumin given by gavage at an equal dose failed to cause a significant reduction in tumor weights and volumes in the nude mouse pancreatic cancer model. [DLys(6)]-LHRH-curcumin treatment enhanced apoptosis compared to [DLys(6)]-LHRH and vehicle-treated controls in tumor tissue. In conclusion, [DLys(6)]-LHRH-curcumin may be useful in treating pancreatic cancer.

Short- and long-term effects of intermittent social defeat stress on brain-derived neurotrophic factor expression in mesocorticolimbic brain regions.

Social defeat stress is an ethologically salient stressor which activates dopaminergic areas and, when experienced repeatedly, has long-term effects on dopaminergic function and related behavior. The mechanism for these long-lasting consequences remains unclear. A potential candidate for mediating these effects is brain-derived neurotrophic factor (BDNF), a neurotrophin involved in synaptic plasticity and displaying alterations in dopaminergic regions in response to various types of stress. In this study, we sought to determine whether repeated social defeat stress altered BDNF mRNA and protein expression in dopaminergic brain regions either immediately after the last stress exposure or 4 weeks later. Male Sprague-Dawley rats were subjected to social defeat stress consisting of brief confrontation with an aggressive male rat every third day for 10 days; control rats were handled according to the same schedule. Animals were euthanized either 2 h or 28 days after the last stress or handling episode. Our results show that 2 h after stress, BDNF protein and mRNA expression increased in the medial prefrontal cortex. At this time-point, BDNF mRNA increased in the amygdala and protein expression increased in the substantia nigra. Twenty-eight days after stress, BDNF protein and mRNA expression were elevated in the medial amygdala and ventral tegmental area. Given the role of BDNF in neural plasticity, BDNF alterations that are long-lasting may be significant for neural adaptations to social stress. The dynamic nature of BDNF expression in dopaminergic brain regions in response to repeated social stress may therefore have implications for lasting neurochemical and behavioral changes related to dopaminergic function.

Genomic survey of prepulse inhibition in mouse chromosome substitution strains.

Prepulse inhibition (PPI) is a measure of sensorimotor gating, a pre-attentional inhibitory brain mechanism that filters extraneous stimuli. Prepulse inhibition is correlated with measures of cognition and executive functioning, and is considered an endophenotype of schizophrenia and other psychiatric illnesses in which patients show PPI impairments. As a first step toward identifying genes that regulate PPI, we performed a quantitative trait locus (QTL) screen of PPI phenotypes in a panel of mouse chromosome substitution strains (CSSs). We identified five CSSs with altered PPI compared with the host C57BL/6J strain: CSS-4 exhibited decreased PPI, whereas CSS-10, -11, -16 and -Y exhibited higher PPI compared with C57BL/6J. These data indicate that A/J chromosomes 4, 10, 11, 16 and Y harbor at least one QTL region that modulates PPI in these CSSs. Quantitative trait loci for the acoustic startle response were identified on seven chromosomes. Like PPI, habituation of the startle response is also disrupted in schizophrenia, and in the present study CSS-7 and -8 exhibited deficits in startle habituation. Linkage analysis of an F(2) intercross identified a highly significant QTL for PPI on chromosome 11 between positions 101.5 and 114.4 Mb (peak LOD = 4.54). Future studies will map the specific genes contributing to these QTLs using congenic strains and other genomic approaches. Identification of genes that modulate PPI will provide insight into the neural mechanisms underlying sensorimotor gating, as well as the psychopathology of disorders characterized by gating deficits.

Improved solid-phase synthesis of alpha,alpha-dialkylated amino acid-rich peptides with antimicrobial activity.

A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid-phase synthetic techniques. Each nonapeptide was rich in alpha,alpha-dialkylated amino acids [one 4-aminopiperidine-4-carboxylic acid (Api) and six alpha-aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9-fluorenylmethyloxycarbonyl (Fmoc)-Aib-Aib-OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3(10)-helical, amphipathic design of these peptides was born out most prominently in the N-terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations < or =100 microM) and the acetylated peptides (concentrations < or = 200 microM) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.

Long-term behavioral and neuronal cross-sensitization to amphetamine induced by repeated brief social defeat stress: Fos in the ventral tegmental area and amygdala.

Repeated exposure to stress induces cross-sensitization to psychostimulants. The present study assessed functional neural activation during social defeat stress-induced sensitization to a subsequent amphetamine challenge. Social defeat stress was induced in intruder rats during short confrontations with an aggressive resident rat once every third day during the course of 10 days. Rats received d-amphetamine injections (1 mg/kg, i.p.) 17 or 70 days after the first social defeat stress exposure. Amphetamine administration induced a significantly higher frequency of locomotor activity in stressed animals than in handled control rats, which was still evident 2 months after the last social stress exposure. Immunohistochemistry for Fos-like proteins was used to detect activated neural profiles in the striatum, nucleus accumbens (NAc), prefrontal cortex, amygdala, and ventral tegmental area (VTA). Repeated social defeat stress significantly increased Fos-like immunoreactive (Fos-LI) labeling 17 days after the start of stress exposure in the prelimbic and infralimbic cortical regions, NAc shell and core, medial, central and basolateral amygdala, and VTA, which probably represented the expression of chronic Fos-related antigens. Amphetamine augmented stress-induced Fos-LI labeling 17 days after the first stress episode in the dorsal striatum, NAc core, and medial amygdala, reflecting a cross-sensitization of Fos response. Amphetamine challenge 70 days after social stress exposures revealed sensitized Fos-LI labeling in the VTA and the amygdala. These data suggest that episodes of repeated social stress induce a long-lasting neural change that leads to an augmented functional activation in the VTA and amygdala, which might represent a neurobiological substrate for long-lasting cross-sensitization of repeated social defeat stress with psychostimulant drugs.

Peptides with indirect in vivo activity against an intracellular pathogen: selective lysis of infected macrophages.

A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50-80% alpha,alpha-dialkylated glycines (alpha,alpha-Dags) were synthesized on solid-phase resin as the C-terminus amides using N-alpha-fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in alpha,alpha-Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 microm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 microg for melittin, whereas 500 microg doses were nonlethal for many of the other peptides. Several of the alpha,alpha-Dag-rich peptides selectively destroy B. abortus-infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.

Sterically hindered C(alpha, alpha)-disubstituted alpha-amino acids: synthesis from alpha-nitroacetate and incorporation into peptides.

The preparation of sterically hindered and polyfunctional C(alpha,alpha)-disubstituted alpha-amino acids (alpha alpha AAs) via alkylation of ethyl nitroacetate and transformation into derivatives ready for incorporation into peptides are described. Treatment of ethyl nitroacetate with N,N-diisopropylethylamine (DIEA) in the presence of a catalytic amount of tetraalkylammonium salt, followed by the addition of an activated alkyl halide or Michael acceptor, gives the doubly C-alkylated product in good to excellent yields. Selective nitro reduction with Zn in acetic acid or hydrogen over Raney Ni gives the corresponding amino ester that, upon saponification, can be protected with the fluorenylmethyloxycarbonyl (Fmoc) group. The first synthesis of an orthogonally protected, tetrafunctional C(alpha,alpha)-disubstituted analogue of aspartic acid, 2,2-bis(tert-butylcarboxymethyl)glycine (Bcmg), is described. Also, the sterically demanding C(alpha,alpha)-dibenzylglycine (Dbg) has been incorporated into a peptide using solid-phase synthesis. It was found that once sterically congested Dbg is at the peptide N-terminus, further chain extension becomes very difficult using uronium or phosphonium salts (PyAOP, PyAOP/HOAt, HATU). However, preformed amino acid symmetrical anhydride couples to N-terminal Dbg in almost quantitative yield in nonpolar solvent (dichloroethane-DMF, 9:1).

Amphipathic control of the 3(10)-/alpha-helix equilibrium in synthetic peptides.

A series of short, amphipathic peptides incorporating 80% C(alpha),C(alpha)-disubstituted glycines has been prepared to investigate amphipathicity as a helix-stabilizing effect. The peptides were designed to adopt 3(10)- or alpha-helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mM SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3(10)-helix in peptides containing amino acids with large side-chains, as with 1-aminocyclohexane-1-carboxylic acid (Ac(6)c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3(10)-helix.

Reduction of zif268 messenger RNA expression during prolonged withdrawal following "binge" cocaine self-administration in rats.

Cocaine self-administration increases dopamine efflux and neuronal activity in the mesocorticolimbic dopamine system compared with experimenter-administered cocaine. Following a prolonged cocaine self-administration binge, dopamine efflux in the nucleus accumbens is attenuated and behaviors emerge that are indicative of anhedonia and anxiety. The neuronal correlates of these behavioral and neurochemical effects of a cocaine binge were assessed using in situ hybridization histochemistry to detect changes in zif268 messenger RNA expression. Rats were fitted with intravenous catheters; one group was trained to self-administer cocaine (0.5mg/injection), then allowed continuous access to cocaine during a 16h binge, while yoked animals received either saline or cocaine according to the same schedule. Measurement of tactile startle responses and ultrasonic distress calls either immediately after termination of cocaine access or one or 14 days later confirmed peak withdrawal at 24h after the binge. The level of zif268 messenger RNA was lower upon termination of cocaine self-administration than in both yoked treatment groups in the ventral tegmental area and hippocampus. In contrast, zif268 messenger RNA expression increased in the periaqueductal gray matter one day after termination of passive cocaine treatment, coincident with enhanced expression of ultrasonic vocalizations. Zif268 messenger RNA expression decreased over time in the nucleus accumbens core and infralimbic cortex, with reduced expression observed in the nucleus accumbens core, caudatoputamen, hippocampus and amygdala 14 days after termination of cocaine self-administration. The results suggest that withdrawal following a cocaine self-administration binge produces a long-lasting reduction of constitutive zif268 messenger RNA expression in mesocorticolimbic brain regions related to the nucleus accumbens. The relatively greater effect in animals that self-administered cocaine implies a relationship of certain regional responses to behavioral conditioning.

Inhibition of nitric oxide synthase in the ventral tegmental area attenuates cocaine sensitization in rats.

1. Male Sprague-Dawley rats were pretreated via bilateral infusion of the VTA with the selective neuronal nitric oxide synthase inhibitor, 7-nitroindazole (0, 8, or 40 ng/hemisphere), prior to each of 7 daily systemic cocaine (30 mg/kg, i.p.) or saline (1 ml/kg) treatments. 2. After a 7-day treatment withdrawal period, rats received a final systemic challenge with either cocaine (30 mg/kg, i.p.) or saline (1 ml/kg). 3. Locomotor and stereotypic activity were measured following the first and last treatments. 4. Daily cocaine treatment led to the development of sensitization to its stereotypic effects as revealed upon drug challenge. 5. The development of sensitization of cocaine-induced stereotypy was completely blocked by daily intra-VTA pretreatment with 7-nitroindazole. 6. In addition, attenuation of the locomotor effects of cocaine challenge was also observed in animals that received daily intra-VTA 7-nitroindazole (40 ng/hemisphere) infusions. 7. The results indicate that VTA nitric oxide is necessary for the development of sensitization of cocaine-induced stereotypic behavior, and that its repeated inhibition may produce lasting effects on the locomotor response to the drug.

The disruptive effect of cocaine on prepulse inhibition is prevented by repeated administration in rats.

These experiments tested the hypothesis that pretreatment with a behaviorally sensitizing regimen of cocaine alters the ability of cocaine to disrupt prepulse inhibition (PPI). Male Sprague-Dawley rats were treated with cocaine (30 mg/kg, i.p.) or saline vehicle for seven consecutive days followed by challenge treatment seven days later. Repeated cocaine treatment produced sensitization of stereotyped activity. Cocaine challenge following repeated vehicle treatment significantly reduced PPI, but this effect was completely abolished by repeated cocaine treatment. These data suggest that neuroadaptation following repeated treatment might prevent PPI disruption caused by psychomotor stimulants.

Expression of mu-opioid receptor mRNA in the medial preoptic area of juvenile rats.

Previous studies indicate that the latency to initiate parental behavior in both male and female rats increases with age; weanling (21 days old) rats display parental behavior 0-2 days after exposure to newborn pups, while older juveniles (30 days old) require 5-6 days of pup exposure before they express the behavior. Furthermore, activation of mu-opioid receptors inhibits parental behavior in juvenile and adult rats. We hypothesized that the age-related increase in behavioral latency could be modulated by the induction of mu-receptor expression in the medial preoptic area (MPOA), a region in which mu-receptors regulate parental behavior. In situ hybridization histochemistry was used to measure mu-receptor mRNA expression in the MPOA of male and female Sprague-Dawley rats that were 21, 24, 27, 30, or 33 days old. Using autoradiographic film analysis, we observed that neurons within part of the MPOA expressed very dense mu-receptor mRNA. Comparison of mRNA distribution with histological boundaries indicated that neurons within the medial preoptic nucleus (MPN), excluding the central part, exhibited the highest density of mu-receptor mRNA within the MPOA. High densities of mu-receptor mRNA extended dorsolaterally and caudally from the MPN toward the bed nucleus of the stria terminalis. MPN mu-receptor mRNA expression was not altered with age and no sex difference was observed. The dense presence of mu-receptor mRNA in the MPN suggests that ample substrate exists on which mu-receptor ligands could modulate the latency to begin parental behavior in juvenile rats, but such behavioral expression apparently is not mediated by a change in mu-receptor mRNA production.

Social defeat stress increases expression of mu-opioid receptor mRNA in rat ventral tegmental area.

Prompted by previous studies linking social defeat stress to changes in opioid antinociception, we evaluated the expression of mu-opioid receptor (MOR)-encoding mRNA in selected rat brain areas as a function of this type of stress. Because opioids mediate significant regulatory activities of brain dopamine neurons, dopaminergic loci in the ventral tegmental area (VTA) and substantia nigra (SN) were selected for analysis. Within 30 min after social defeat stress, the level of MOR-encoding mRNA, as detected and quantified by in situ hybridization histochemistry, increased in the lateral VTA and this increase was present for at least 6 h. In contrast, defeat stress was without effect on the expression of MOR-encoding mRNA in the SN. These data suggest that stress-induced alteration of MOR-encoding mRNA expression in the VTA may be involved in the consequences of social defeat stress.

Improved fidelity of thermostable ligases for detection of microsatellite repeat sequences using nucleoside analogs.

Microsatellite repeats consisting of dinucleotide sequences are ubiquitous in the human genome and have proven useful for linkage analysis, positional cloning and forensic identification purposes. In this study, the potential of utilizing the ligase detection reaction for the analysis of such microsatellite repeat sequences was investigated. Initially, the fidelity of thermostable DNA ligases was measured for model dinucleotide repeat sequences. Subsequently, the effect of modified oligonucleotides on ligation fidelity for dinucleotide repeats was determined using the nucleoside analogs nitroimidazole, inosine, 7-deazaguanosine and 2-pyrimidinone, as well as natural base mismatches. The measured error rates for a standard dinucleotide template indicated that the nitroimidazole nucleoside analogs could be used to increase the fidelity of ligation when compared to unmodified primers. Furthermore, use of formamide in the ligation buffer also increased ligation fidelity for dinucleotide repeat sequences. Using ligation-based assays to detect polymorphic alleles of microsatellite repeats in the human genome opens the possibility of using array-based typing of these loci for human identification, loss-of-heterozygosity studies and linkage analysis.

Universal DNA microarray method for multiplex detection of low abundance point mutations.

Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wild-type stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K- ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3x3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest.

Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations.

A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.

Nucleotide analogs facilitate base conversion with 3' mismatch primers.

We compared the efficiency of PCR amplification using primers containing either a nucleotide analog or a mismatch at the 3' base. To determine the distribution of bases inserted opposite eight different analogs, 3' analog primers were used to amplify four different templates. The products from the reactions with the highest amplification efficiency were sequenced. Analogs allowing efficient amplification followed by insertion of a new base at that position are herein termed 'convertides'. The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well as non-conversion control products with no base change. We compared the ability of natural bases to convert specific sites with and without a preconversion step using nucleotide analog primers. Conversion products were identified by a ligation detection reaction using primers specific for the converted sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conversion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3' base or analog transiently base paired to neighboring bases of the template. Nucleotide analogs could often reduce conversion artifacts and increase the yield of the expected product. While new analogs are needed to reliably achieve transversions, the current set have proven effective for creating transition conversions.