Wild-type Primer Research Articles

Allele-specific PCR with a novel data processing method based on difference value for single nucleotide polymorphism genotyping of ALDH2 gene

Single nucleotide polymorphism (SNP) analysis based on allele-specific polymerase chain reaction (AS-PCR) is a relatively effective and economical method compared with other genotyping technologies such as DNA sequencing, DNA hybridization and isothermal amplification strategies. But AS-PCR is limited by its labor-intensive optimization of reaction parameters and time-consuming result assessment. In this study, we put forward a novel idea of data processing to address this problem. SNP analysis was accomplished by AS-PCR with endpoint electrochemical detection. For each sample, two separate reactions were run simultaneously with two sets of allele-specific primers (wild-type primers for W system and mutant primers for M system). We measured their redox current signals on screen-printed electrodes once AS-PCR finished and calculated the difference value of current signals between two systems to determine the genotyping result. Based on the difference value of fluorescent signals, real-time fluorescent PCR was used to study reaction parameters in AS-PCR. With screened parameters, we obtained the genotyping results within 50 min. 36 hair-root samples from volunteers were analyzed by our method and their genotypes of ALDH2 gene (encoding aldehyde dehydrogenase 2) were totally identical with data from commercialized sequencing. Our work first employed difference value between two reaction systems to differentiate allele and provided a novel idea of data processing in AS-PCR method. It is able to promote the quick analysis of SNP in the fields of health monitor, disease precaution, and personalized diagnosis and treatment.

Detection of 4977-bp deletion of mitochondrial DNA in in vitro fertilization failure women: A case-control study

The quality of oocyte is often considered as a limiting factor for fertility, especially IVF. Some mitochondrial mutations, particularly the 4977-bp deletion increase with the age. Thus, this mutation can serve as a marker for cell aging, which indicates the reduced quality of the oocytes for fertilization. It has been suggested that this can also be investigated in the blood cells of women with IVF failure. 1-Determination of the frequency of 4977-bp deletion in women with IVF failure, 2-Investigation of the relationship between 4977-bp deletion and the age of patients. Polymerase chain reaction was used to detect the 4977-bp deletion in blood samples of 52 IVF failure women and 52 women who had at least one healthy child. After polymerase chain reaction with deleted and wild-type primers, the products were examined using agarose gel electrophoresis. 48.07% of women with IVF failure and 34.62% of healthy women had a mitochondrial 4977-bp deletion, with p=0.163 and OR: 1.749. Also, in association with the age of these patients and the frequency of 4977-bp mutation, p and OR were obtained 0.163 and 1.749, respectively and frequency of this mutation was higher in patients over 35 yr old compared to other subgroups (Patients ≥35: 57.69). According to the findings of this study, there is no a significant relationship between the frequency of mitochondrial 4977-bp mutation and failure in IVF.

BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing

Amplicon-based next-generation sequencing (NGS) has been widely adopted for genetic variation detection in human and other organisms. Conventional data analysis paradigm includes primer trimming before read mapping. Here we introduce BAMClipper that removes primer sequences after mapping original sequencing reads by soft-clipping SAM/BAM alignments. Mutation detection accuracy was affected by the choice of primer handling approach based on real NGS datasets of 7 human peripheral blood or breast cancer tissue samples with known BRCA1/BRCA2 mutations and >130000 simulated NGS datasets with unique mutations. BAMClipper approach detected a BRCA1 deletion (c.1620_1636del) that was otherwise missed due to edge effect. Simulation showed high false-negative rate when primers were perfectly trimmed as in conventional practice. Among the other 6 samples, variant allele frequencies of 5 BRCA1/BRCA2 mutations (indel or single-nucleotide variants) were diluted by apparently wild-type primer sequences from an overlapping amplicon (17 to 82% under-estimation). BAMClipper was robust in both situations and all 7 mutations were detected. When compared with Cutadapt, BAMClipper was faster and maintained equally high primer removal effectiveness. BAMClipper is implemented in Perl and is available under an open source MIT license at https://github.com/tommyau/bamclipper.

A new nested allele-specific multiplex polymerase chain reaction method for haplotyping of VKORC1 gene to predict warfarin sensitivity.

The vitamin K epoxide reductase complex 1 gene (VKORC1) is commonly assessed to predict warfarin sensitivity. In this study, a new nested allele-specific multiplex polymerase chain reaction (PCR) method that can simultaneously identify single nucleotide polymorphisms (SNPs) at VKORC1 381, 861, 5808, and 9041 for haplotype analysis was developed and validated. Extracted DNA was amplified in the first PCR DNA, which was optimized by investigating the effects of varying the primer concentrations, annealing temperature, magnesium chloride concentration, enzyme concentration, and the amount of DNA template. The amplification products produced from the first round of PCR were used as templates for a second PCR amplification in which both mutant and wild-type primers were added in separate PCR tubes, followed by optimization in a similar manner. The final PCR products were resolved by agarose gel electrophoresis and further analysed by using a VKORC1 genealogic tree to infer patient haplotypes. Fifty patients were identified to have H1H1, one had H1H2, one had H1H7, 31 had either H1H7 or H1H9, one had H1H9, eight had H7H7, and one had H8H9 haplotypes. This is the first method that is able to infer VKORC1 haplotypes using only conventional PCR methods.

Frequency Of NOTCH1 Mutations In Brazilian Patients With CLL

NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis, and since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutation (c.7544_7545delCT), represents roughly 75% of all PEST domain mutations in CLL we designed a test that can, at the same time, detect the presence of this mutation specifically and also any insertion or deletion in exon 34.We designed a PCR reaction using one FAM-labeled forward primer anchored at codon 2407 and two reverse primers. One specific for the c.7544_7545delCT mutation anchored at codon 2414 yielding a product of 356 base pairs (bp) and one anchored at codon 2425, yielding a product of 391 bp, comprising the hot spot for mutations in the NOTCH1 PEST domain. Primers were designed with Primer3 software (http://frodo.wi.mit.edu/) and the specificity of the reaction evaluated using the tool “PCR in silico” (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start). The test yields three possible outputs:a) A single 391 bp peak: wild type samplesb) Three peaks (391 bp, 389 bp and 356 bp): heterozygous for c.7544_7545delCTc) Two peaks (391 bp and another bigger or smaller, depending on the size of insertion / deletion): another insertion or deletion, but not c.7544_7545delCT.We have studied 91 blood samples from CLL patients in diverse disease stages and found NOTCH1 mutations in 16 patients, (17.5%). When only patients with trisomy 12, isolated or with other cytogenetic abnormalities, were analyzed (N=16), we found an incidence of NOTCH1 mutations of 43% (7/16). At the same time, the frequency of NOTCH1 mutations in patients without trisomy 12 was 12% (9/75), in agreement with previous reports. All mutated cases (N=13) had the mutation c.7544_7545delCT.In conclusion, we have designed a robust, fast and cost effective assay for routine identification of NOTCH1 PEST domain mutations, suitable for implementation in the clinical setting. Additionally we have found that the incidence and distribution among cytogenetic groups of NOTCH1 mutation in Brazilian patients with CLL is similar to the incidence described in other cohorts.A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak.B – Presence of heterozygous c.7544_7545delCT mutation evidenced by the presence of a 356 bp peak,corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Simple method for differentiating measles vaccine from wild‐type strains using loop‐mediated isothermal amplification

Because of increasing measles vaccine coverage, the proportion of patients with modified measles has been increasing. Such patients have low-grade fever with very mild eruptions similar to vaccine-related adverse events. Differentiation between these two pathogenic conditions is required to improve the quality of laboratory-based measles surveillance. In this study, vaccine-specific and wild-type specific primer sets were designed for loop-mediated isothermal amplification in the N gene, and vaccine strains, C1, D3, D4, D5, D8, D9, G3 and H1 wild strains were examined. Three vaccine strains were efficiently amplified using a vaccine-specific primer set with an approximately 10-times higher sensitivity than wild-type primer. Modified measles was differentiated from vaccine-associated cases by this system, but limitations were encountered with the other genotypes.

Diagnostic accuracy of an allele-specific reverse transcriptase-PCR assay targeting the H275Y oseltamivir resistant mutation in 2009 pandemic influenza A/H1N1 virus

Oseltamivir resistant 2009 pandemic influenza A/H1N1 viruses (pH1N1) are emerging and rapid molecular assays identifying these strains are needed for clinical management. Development and evaluation of an allele-specific, real-time reverse transcriptase-PCR assay (ASPCR) targeting the H275Y oseltamivir resistant mutation in pH1N1 virus. ASPCR uses two allele-specific forward primers (wild-type and mutant) and a common reverse primer and probe. Wild-type and mutant genotypes were defined by the difference in PCR Ct values (DeltaCt(mut-wt)) between the mutant primer and wild-type primer amplification curves for the same sample. Mixtures of wild-type and mutant genotypes were analyzed to evaluate sensitivity and determine assay cut-off values. ASPCR results were confirmed using an allelic discrimination assay (AD) and pyrosequencing. Mixtures containing 5-95% mutant genotype could be detected. A DeltaCt(mut-wt)>or=3.5 identified wild-type genotype (<10% mutant); between 3.5 and -3.5 identified mixed genotypes (10-90% mutant); and <or=-3.5 identified fully mutant genotype (>90% mutant). Among 264 clinical samples, 171 were wild-type, 10 were mixed, and 29 were fully mutant. The 39 samples with mixed or mutant results were from 11 patients. Of 107 samples with sufficient volume tested by ASPCR and AD, 12 were indeterminate by AD due to low viral load, 86 were wild-type by both assays, and 9 were mutant by both assays. Thirteen samples were confirmed by pyrosequencing and one discrepant sample was mixed by ASPCR and fully mutant by pyrosequencing. ASPCR is sensitive, quantitative and specific for H275Y mutation analysis and provides an accurate approach for detecting pH1N1 oseltamivir resistance in clinical samples.

ANTICANCER ACTIVITY OF CALANONE ON HeLa CELL LINE

Calanone (coumarin derivate compound), isolated from Calophyllum sp. had been shown to have cytotoxic activity on leukemia L1210 cell line with IC50 = 59.40 mg/mL. Calanone presumed have anticancer activity on HeLa cervical carcinoma cell. This study was conducted to investigate the cytotoxic and apoptotic activity of calanone and its effect to p53 and p21 expression on HeLa cervical carcinoma cell. Cytotoxic assay of calanone was performed on HeLa cell line, using MTT assay. Apoptotic assay was performed on HeLa cell line incubated with calanone for 24 h, by immunofluororescence method, using fluorochromes ethidium bromide and acridine orange. Expression of p53 was examined on HeLa cell line, by PCR with p53 wild-type primer. Expression of p21 was examined on HeLa cell line, by immunohistochemistry method. 5-fluorourasil was used as positive control in cytotoxic, apoptotic assay, and p53 expression. The result showed that calanone has cytotoxic activity on HeLa cell line, with IC50 = 22.887 mg/mL, caused cytotoxicity through apoptotic mechanism, increase p53 tumor suppressor gene expression, while the p21 expression test showed a negative result. Keywords: Calanone, cytotoxic, HeLa cell line

P53 is required for chloroquine-induced atheroprotection but not insulin sensitization

An intact genotoxic stress response appears to be atheroprotective and insulin sensitizing. ATM, mutated in ataxia telangiectasia, is critical for the genotoxic stress response, and its deficiency is associated with accelerated atherosclerosis and insulin resistance in humans and mice. The antimalarial drug chloroquine activates ATM signaling and improves metabolic phenotypes in mice. p53 is a major effector of ATM signaling, but it is unknown if p53 is required for the beneficial effects of chloroquine. We tested the hypothesis that the cardiometabolic effects of chloroquine are p53-dependent. ApoE-null mice with or without p53 were treated with low-dose chloroquine or saline in the setting of a Western diet. After 8 weeks, there was no p53-dependent or chloroquine-specific effect on serum lipids or body weight. Chloroquine reduced plaque burden in mice wild-type for p53, but it did not decrease lesion extent in p53-null mice. However, chloroquine improved glucose tolerance, enhanced insulin sensitivity, and increased hepatic Akt signaling regardless of the p53 genotype. These results indicate that atheroprotection induced by chloroquine is p53-dependent but the insulin-sensitizing effects of this agent are not. Discrete components of the genotoxic stress response might be targeted to treat lipid-driven disorders, such as diabetes and atherosclerosis.

A Rapid PCR‐based Method for the Identification of ob Mutant Mice

With increasing incidence of obesity, there is greater demand for suitable research and therapeutic models. The ob/ob mouse model develops obesity by 5 weeks of age. Previously, a method using DNA purification, PCR, and restriction digestion of products was devised to identify mice bearing the ob allele. Here, we describe a direct PCR method that requires no DNA purification. Wild-type and ob-specific primers are used under the same conditions in two separate and simultaneously run three-primer PCRs. Standard PCR using the wild-type primer mix produces 191 bp and 104 bp bands in +/+ and ob/+ and only the control 191 bp band in ob/ob animals. The ob-specific primer reaction produces 191 bp and 123 bp bands in ob/+ and ob/ob and only the control 191 bp band in +/+ animals. Phenotypic weight gain in offspring of heterozygous intercrosses was used to validate genotypes. This primer-specific PCR method allows simultaneous identification of +/+, ob/+, and ob/ob genotypes prior to breeding age to facilitate breeding and research studies in an important model of clinical obesity.

Characterization of the Molecular Basis of Group II Intron RNA Recognition by CRS1-CRM Domains

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Constitutive Activation of Ras and Mitogen-Activated Protein Kinase (MAPK/ERK) in LGL Leukemia Unrelated to Activating Mutations in N and KRas Genes.

Background: Patients with Large Granular Lymphocyte (LGL) leukemia have defects in the CD95/CD95L (Fas-Ligand)-apoptotic pathway and a clinical syndrome with similarities to autoimmune lymphoproliferative diseases (ALPS). Patients with ALPS have autoimmunity and lymphocyte accumulation of CD3+CD4−CD8− aβ T-cells; whereas, the expanded population of lymphocytes in LGL leukemia are characterized by a CD3+CD4−CD8+ T-cell or a CD3−CD56+CD16+ NK-cell phenotype. Patients with ALPS usually possess mutations in genes involved in the CD95/CD95L apoptosis pathway but it was recently shown that a germline activating mutation in NRas exon 1 (Gly13Asp) resulted in clinical pathologic features of ALPS. In our previous studies, we found leukemic NK-LGLs to have evidence of constitutively activated extracellular-regulated kinase (ERK) and Ras that contributed to survival. Ablation of this signaling pathway with a dominant-negative form of Ras (N17Ras) and pharmacologic inhibition with the farnesyltransferase inhibitor FTI2153 resulted in ERK inhibition and enhanced apoptosis. Therefore, we hypothesize that expansion of leukemic LGL may be secondary to an acquired or germline mutation in the Ras gene. In this study, we performed allele-specific amplification of the NRas and KRas genes to determine the incidence of activating mutations in codons 12, 13, and 61.Methods: PBMCs were collected from 5 healthy controls and twelve patients with T-LGL and one patient with NK-LGL leukemia (n=13). Apoptosis assessed using annexin/7-AAD flow cytometry was performed in response to inhibition of Ras and MEK using FTI2153 and PD98059, respectively. Western blot analysis was performed for phosphorylated-activate MAPK (ERK1/2), total MAPK, Bcl-2, XIAP, and CD95-FasR in five patients. Allele-specific PCR was performed using primers that recognized mutated Ras bases at their 3′ end and multiplex reactions were performed that simultaneously detected the wild type sequence, as previously described (Bezieau et al., Hum Mutat 18:221, 2001). A total of 40 unique mutations were analyzed and primer sensitivity and specificity was determined in cell lines with confirmed mutations in each of these exons.Results: Apoptosis was induced in a time and dose-dependent fashion using both MEK and Ras inhibitors. Constitutively activated MAPK/ERK and higher amounts of CD95-FasR was detected in LGL patients compared to healthy controls; whereas, Bcl-2 showed variable expression in LGL leukemia. XIAP was expressed in patients but was not different than controls. After treatment with the MEK inhibitor (PD98059), activated MAPK was reduced along with Bcl-2 when expressed. We detected the appropriate mutations as expected in all tumor cell lines but there were no mutations detected in LGL leukemia cases including NRas exon1 (Gly13Asp), which was mutated in ALPS. Wild-type primers were capable of detecting wild-type Ras in each reaction validating the amplification procedure.Conclusions: These results show that LGL leukemia has dysregulated apoptosis in association with an activated Ras/MAPK signaling pathway unrelated to mutations in the NRas or KRas genes examined. Ras-inactivating drugs are currently being investigated in LGL leukemia.

Disruption of CXC Motif Chemokine Ligand-14 in Mice Ameliorates Obesity-induced Insulin Resistance

In obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle.

Assay to Detect H5N1 Oseltamivir Resistance

Assay to Detect H5N1 Oseltamivir Resistance

Development of an Oligonucleotide-Based SNP Detection Method on Lateral Flow Strips Using Hexapet Tags

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags (hexapet tags). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix (competitive format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Analysis of Polypurine Tract-associated DNA Plus-strand Priming in Vivo Utilizing a Plant Pararetroviral Vector Carrying Redundant Ectopic Priming Elements

Initiation of DNA plus-strand synthesis in most reverse-transcribing elements requires primer generation by reverse transcriptase-associated RNase H at one or more template polypurine tracts (PPTs). We have exploited infectious clones of the plant pararetrovirus cauliflower mosaic virus carrying redundant ectopic plus-strand priming elements to study priming in vivo. Ectopic priming generated an additional discontinuity in progeny virion DNA during infection of plants. We found that altering the length of the 13-base pair PPT by +/-25% significantly reduced priming efficiency. A short pyrimidine tract 5' to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5' end remained 3 nucleotides from the PPT 3' end in mutant primers that were longer or shorter than the wild-type primer. Use of an ectopic redundant primer to study replication-dependent priming was validated by demonstrating that it could rescue infectivity following destruction of the wild-type priming elements. We propose a model for plant pararetroviral plus-strand priming in which pyrimidines enhance PPT recognition during polymerase-dependent RNase H cleavages, and suggest that fidelity of primer maturation during polymerase-independent cleavages involves PPT length measurement and 3' end recognition by RNase H.

PCR mutagenesis-based method for generation of positive controls for SSCP analysis.

We have developed a primer-mediated PCR mutagenesis-based method for the generation of positive controls to test the sensitivity of single-strand conformation polymorphism (SSCP) or any other PCR-based mutation screening method. This technique is based on the incorporation of a third longer primer, containing a mismatched base, into the PCR along with the two wild-type primers normally used to amplify DNA fragments for SSCP analysis. The longer mismatch primer (LMP) shares the sequence of one of the wild-type primers and also contains 5 to 10 additional bases, which include the mismatched base. The resulting PCR product is identical in length and sequence to the wild-type template with the exception that the LMP base mismatch is incorporated into nearly 100% of the product. We have observed an altered SSCP mobility pattern in all cases where positive controls have been generated using this technique. We believe that the use of such in vitro-generated controls can contribute to the interpretation of band patterns and to the optimization of experimental conditions for SSCP to facilitate maximum detection of sequence variants.

HIV-1 Reverse Transcriptase Discriminates against Non-self tRNA Primers

The interactions between the Reverse Transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) and the natural tRNA Lys3primer for initiation of viral DNA synthesis were examined. We constructed a set of HIV-1 RNA templates in which the wild-type primer binding site (PBS Lys3) is replaced by sequences complementary to tRNA Ile, tRNA Lys1,2, tRNA Phe, tRNA Proor tRNA Trpand tested the ability of RT enzymes of different retroviral species to initiate cDNA synthesis from self versusnon-self tRNA primers. We demonstrate that initiation of HIV-1 reverse transcription is a specific process that is most efficient with the self tRNA Lys3primer. Interestingly, the property of HIV-1 RT to discriminate against non-self tRNA primers is lost upon extension of the tRNA by only two deoxyribonucleotides. Furthermore, selective tRNA priming by HIV-1 RT was not observed with viral RNA-tRNA Lys3duplexes isolated from HIV-1 virion particles, suggesting that the majority of tRNA Lys3primers annealed to viral RNA in particles is extended by a variable number of deoxyribonucleotides. This result indicates that reverse transcription is initiated relatively early in nascently assembled virions.

ARMS test for diagnosis of factor VLeiden mutation, a common cause of inherited thrombotic tendency

We developed a simple and rapid amplification-refractory mutation system (ARMS) assay for the factor V mutation [R506Q] (factor VLeiden), which results in the autosomal dominant thrombotic tendency, resistance to activated protein C (rAPC). PCR primers within Exon 10 of the factor V gene were designed. A common upstream primer was paired with either a mutant or wild-type-specific downstream primer. The 3'-most nucleotide of the specific primers recognized either the mutant or normal allele, and the 3' penultimate nucleotide was mismatched to enhance specificity of the reaction. The assay was validated using authentic factor VLeiden DNA samples. Seven of 103 hematologically normal children (6.8%) were found to be heterozygotes. Among 27 patients studied by the rAPC assay, ARMS assay and rAPC results were concordant in 26. Among these were a 1-year-old child with a calcified clot in the inferior vena cava. Both the patient and his father were heterozygous for the mutation and both had abnormal rAPC assays. rAPC and factor VLeiden assays were discordant in a young girl with a history of stroke. Biochemical rAPC assay was abnormal, while ARMS assay revealed amplification only with wild-type primers, suggesting a non-[R506Q] mechanism for rAPC. This assay will be a valuable tool for studying subjects with thromboses and their family members.

ARMS test for diagnosis of factor VLeiden mutation, a common cause of inherited thrombotic tendency.

We developed a simple and rapid amplification-refractory mutation system (ARMS) assay for the factor V mutation [R506Q] (factor VLeiden), which results in the autosomal dominant thrombotic tendency, resistance to activated protein C (rAPC). PCR primers within Exon 10 of the factor V gene were designed. A common upstream primer was paired with either a mutant or wild-type-specific downstream primer. The 3'-most nucleotide of the specific primers recognized either the mutant or normal allele, and the 3' penultimate nucleotide was mismatched to enhance specificity of the reaction. The assay was validated using authentic factor VLeiden DNA samples. Seven of 103 hematologically normal children (6.8%) were found to be heterozygotes. Among 27 patients studied by the rAPC assay, ARMS assay and rAPC results were concordant in 26. Among these were a 1-year-old child with a calcified clot in the inferior vena cava. Both the patient and his father were heterozygous for the mutation and both had abnormal rAPC assays. rAPC and factor VLeiden assays were discordant in a young girl with a history of stroke. Biochemical rAPC assay was abnormal, while ARMS assay revealed amplification only with wild-type primers, suggesting a non-[R506Q] mechanism for rAPC. This assay will be a valuable tool for studying subjects with thromboses and their family members.