Cleavase Fragment Length Polymorphism Research Articles

Vertical Transmission of Chlamydia trachomatis in Chongqing China

This is the first study to investigate vertical transmission of Chlamydia trachomatis in Chongqing China. For this study, 300 cervical swab samples from pregnant women and 305 nasopharygeal swab samples from their babies (605 specimens) were collected for nest polymerase chain reaction (nPCR) of the ompl gene, which encodes the major outer membrane protein (MOMP) and typed C. trachomatis using Cleavase fragment-length polymorphism (CFLP) labeled with digoxin. From these samples, 11% (33/300) of pregnant women samples were successfully amplified. The vertical transmission rate of C. trachomatis from mother to baby was 24% (8/33). The vertical transmission rates were 66.7% (6/9) for mothers with vaginal delivery and 8.3% (2/24) for those with cesarean section. The incidence of premature membrane rupture among C. trachomatis-positive pregnant women was 30.3% (10/33), which was greater than among those who were C. trachomatis-negative (13.5%, 36/267; chi(2) = 4.2; p < 0.05). Four genotypes including type E (3 pairs), type F (2 pairs), type H (2 pairs), and type D (1 pair) were observed by CFLP assay labeled with digoxin and confirmed by DNA sequencing in the 16 C. trachomatis-positive samples from eight pregnant women and their eight infants. Each pair of matched maternal-infantile samples showed identical CFLP. This study showed the incidence of C. trachomatis infection in pregnant women, the vertical transmission rate for C. trachomatis, and the genotypes of C. trachomatis in Chongqing, China. The CFLP assay labeled at the 5' end of the forward primer with digoxin was first used successfully to genotype of C. trachomatis. As a promising method for C. trachomatis genotyping, CFLP had good sensitivity, reproducibility, and simplicity and no radioactive contamination.

Diagnostic accuracy of methods for the detection of BRCA1 and BRCA2 mutations: a systematic review

As sequence analysis for BRCA1 and BRCA2 mutations is both time- and cost-intensive, current strategies often include scanning techniques to identify fragments containing genetic sequence alterations. However, a systematic assessment of the diagnostic accuracy has been lacking so far. Here, we report on a systematic review to assess the internal and external validity of current scanning techniques. Inclusion criteria were: controlled design, investigators blinded, and tests suitable as a scanning tool for the whole genes BRCA1 and BRCA2. Outcome parameters were sensitivity, specificity, and positive and negative predictive values compared to direct sequencing. Out of 3816 publications, 10 studies reporting on 12 methods met our inclusion criteria. The internal and external validity of most of these studies was limited. Sensitivities were reported to be 100% for enzymatic mutation detection (EMD), multiple-dye cleavase fragment length polymorphism (MD-CFLP), fluorescence-based conformation-sensitive gel electrophoresis (F-CSGE), RNA-based sequencing, restriction endonuclease fingerprinting-single strand conformation polymorphism (REF-SSCP), stop codon (SC) assay, and denaturing high-performance liquid chromatography (DHPLC). Sensitivity was 50-96% for SSCP, 88-91% for two-dimensional gene scanning (TDGS), 76% for conformation-sensitive gel electrophoresis (CSGE), 75% for protein truncation test (PTT), and 58% for micronucleus test (MNT). Specificities close to 100% were reported, except for MNT. PTT and SC assay are only able to detect truncating mutations. Most studies were designed to introduce new experimental approaches or modifications of established methods and require further evaluation. F-CSGE, REF-SSCP, RNA-based sequencing, EMD, and MD-CFLP will need further evaluation before their use in a routine setting can be considered. SSCP, MNT, PTT, CSGE, and TDGS cannot be recommended because of their low sensitivity. DHPLC outperforms all other methods studied. However, none of the four studies evaluating DHPLC was performed on BRCA2.

Comparative Evaluation of Cleavase Fragment Length Polymorphism With PCR-SSCP and PCR-RFLP to Detect Antimicrobial Agent Resistance in Mycobacterium tuberculosis.

Background: Several molecular methods potentially useful in the detection of Mycobacterium tuberculosis mutations, specifically in rpoB and katG, were compared. Methods and Results: DNA from 24 M. tuberculosis clinical isolates, with mutations associated with resistance to rifampin and/or isoniazid, was analyzed. A 128 bp amplicon, spanning the 81 bp rpoB region containing most mutations leading to rifampin resistance, was analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and a recently introduced mutation scanning method, cleavase fragment length polymorphism (CFLP) analysis. Also, a 350 bp amplicon encompassing that region was analyzed by the CFLP method. CFLP analysis of the 350 bp amplicon (23 isolates) identified 14 of 17 mutants; however, CFLP analysis of the 128 bp amplicon accurately identified all mutants as did PCR-SSCP with interpretative difficulty for two codon 513 mutations. CFLP and PCR-restriction fragment length polymorphism (RFLP) analyses of a 623 bp amplicon encompassing katG codons 315 and 463 showed that the CFLP method identified single and dinucleotide codon 315 substitutions with or without codon 463 (CGG-->CTG) changes, whereas PCR-RFLP (MspI) missed one codon 315 polymorphism (AGC-->ACA) in three isolates. Conclusion: Both PCR-SSCP and CFLP analyses were sensitive in identifying all mutations on short sequences in the rpoB mutants. CFLP appears to be more efficient than SSCP and RFLP for the detection of mutations in large amplicons.

Analysis of Four Common Salt-Wasting Mutations in CYP21 (Steroid 21-Hydroxylase) by Cleavase Fragment Length Polymorphism Analysis and Characterization of a Frequent Polymorphism in Intron 6.

Background: Congenital adrenal hyperplasia (CAH) owing to steroid 21-hydroxylase deficiency results from mutations in CYP21. The majority of patients with this disorder have salt-wasting because of mineralocorticoid deficiency, and most patients with salt-wasting have one of four common mutations: deletions of CYP21, or nondeletion mutations including a 3 < 9 premature splice mutation in intron 2; an 8-bp deletion in exon III; a cluster of mutations in exon VI; and R356W. Methods and Results: The authors analyzed these mutations using a new mutation-detection methodology, cleavase fragment length polymorphism (CFLP) analysis. All of these mutations were detectable using CFLP. The intron 2 splice mutation, exon III 8-bp deletion, and R356W mutation all resulted in novel CFLP bands not seen in subjects without those mutations. The exon VI cluster of mutations resulted in elimination of a CFLP band, which was easily detected in a patient with that mutation and a gene deletion on the homologous chromosome. The authors also report the characterization of a restriction fragment length polymorphism in intron 6 involving the RsaI site around nucleotide 1419. In 48 chromosomes from the parental generation of the Centre d'Étude du Polymorphisme Humain families, the frequency of the polymorphism that destroys the RsaI site was found to be 12.5%. Conclusions: CFLP is a potentially useful analytic method for analyzing frequent CYP21 mutations that cause salt-wasting CAH, although further development of the technique is required to clearly distinguish heterozygotes from homozygotes for mutations that generate novel CFLP bands and to identify heterozygotes for mutations that eliminate a CFLP band. The Rsa I polymorphism may be a useful marker for following the segregation of CYP21 alleles in genetic studies and is of use in showing gene deletions of CYP21 in informative families. The polymorphism may also be of use in genetic studies of the human major histocompatibility complex.

Detection of germline BRCA1 mutations by Multiple-Dye Cleavase Fragment Length Polymorphism (MD-CFLP) method.

We describe the Multiple-Dye Cleavase Fragment Length Polymorphism (MD-CFLP) method set up for a sensitive and preliminary rapid screening of BRCA1 mutations. We analysed exons 11 and 16, which are known to cover slightly more than 70% of the whole coding region of the gene, subdivided into 4 amplicons and labelled with different fluorescent dUTPs. MD-CFLP was first utilised on a panel of 30 DNA samples in which the presence of single-base substitutions or small deletions/insertions had been previously identified by direct sequencing as gold standard, in order to define the optimal conditions in terms of PCR amplification and temperature of digestion. In a second step, we blindly analysed 21 DNA samples by MD-CFLP to verify its reliability. The sensitivity and specificity of MD-CFLP were both 100% in the first study, and 80% and 94%, respectively, in the blind sample assay. Our results demonstrate the capability of the MD-CFLP method to detect DNA sequence alterations in fragments of more than 1 kb. We conclude that CFLP is a powerful tool in mutational analysis, offering reliable results in a shorter time and at a lower cost than conventional methods, and its potential can be enhanced when internal fluorescent labelling and laser detection are used. © 2001 Cancer Research Campaignhttp://www.bjcancer.com

Demonstration of focal p53 expression without genetic alterations in endometriotic lesions.

Their monoclonal origin (as indicated by recent investigations) indicates the neoplastic nature of most endometriotic lesions. p53, a representative tumor suppressor, regulates cell proliferation, and genetic alterations in p53 are involved in carcinogenesis in a wide variety of human cancers. The aim of this study was to examine endometriotic lesions for p53 expression and genetic alterations in p53. An immunohistochemical study revealed that 20% (13/64) of endometriotic lesions showed focal p53 expression in the epithelial cells. Using serial paraffin sections, we employed a microdissection method to extract DNA from the endometriotic tissues that showed p53 expression. No mutations were found in exons 5-8 in p53 by cleavase fragment length polymorphism scanning and polymerase chain reaction-DNA sequencing. Moreover, neither loss of heterozygosity nor microsatellite instability was detected at the microsatellite marker sites of p53. These results suggest that the focal p53 expression recognized in the endometriotic epithelia may be due to overproduction of wild-type p53 protein.

Identification of drug-resistantMycobacterium tuberculosis by single strand conformation polymorphism and cleavase fragment length polymorphism

Twenty isolates ofMycobacterium tuberculosis resistant to rifampicin(RIF), isoniazid(INH) and streptomycin(STR) were analysed by Polymerase Chain Reaction (PCR) amplification of rpoB, katG and rrs genes to evaluate comparative diagnostic significance of genetic assays. Mutations were identified by single strand conformation polymorphism (SSCP) and cleavase fragment length polymorphism (CFLP) and were confirmed by DNA sequencing. SSCP of 4 RIF resistant and 14 INH resistant isolates showed an extra peak at the level of 75-bp and 85-bp respectively, while 2 STR resistant isolates showed 2 peaks with 9 bases difference. CFLP showed a different pattern among RIF, INH and STR sensitive and resistant isolates Thus SSCP and CFLP can be used as alternative diagnostic methods for identification of mutations in RIF, INH and STR resistant strains of M.tuberculosis.

New Cleavase Fragment Length Polymorphism method improves the mutation detection assay.

Cleavase Fragment Length Polymorphism (CFLP) analysis is a convenient, accurate and highly sensitive method for the detection and localization of nucleic acid mutations. The assay is well suited for high-throughput screening and can be used to detect mutations in known and unknown nucleic acid samples. A recent improvement in the CFLP assay termed "temperature ramping" or "ramping" is reported here. This procedural improvement eliminates the need for time and temperature optimizations before the actual sample analysis. In this study, we compare the CFLP ramping procedure to the conventional CFLP optimization procedure and demonstrate equal, and in some cases improved, detection of point mutations. With ramping, CFLP reactions are identical for all DNA fragments analyzed, which allows for increased sample throughput, decreased assay time and lower overall cost.

Mutational analysis of human p53 gene in human gliomas by Cleavase® fragment length polymorphism

Cleavase® fragment length polymorphism (CFLP) is a newly developed method used for the detection of gene mutation. This study evaluates the usefulness of this method. Thirty‐three human glioma specimens obtained at surgery were analyzed for human p53 gene mutation using radiolabeled polymerase chain reaction (PCR)‐based CFLP analysis and the results were compared with results of direct sequencing. Three (one of five anaplastic astrocytomas and two of 24 glioblastomas) and five (one of four low‐grade gliomas, one of five anaplastic astrocytomas and three of 24 glioblastomas) samples were suspected as being mutations in exon 5–6 and 7–8 of the p53 gene, respectively. Further, two of three (one anaplastic astrocytoma and one glioblastoma) and two of five (both glioblastoma) samples were confirmed as having mutations in direct sequence (their positive rates were 66.7 and 40%, respectively). However, the false‐negative rate of CFLP analysis was very low (0%, 0/60). Therefore, it was concluded that although PCR‐CFLP using a radioisotope might be too sensitive given its superior false‐negative rate, it offers an alternative protocol for the screening of the mutation of human p53 gene.

Detection of p53 gene mutations in its full translational sequence by cleavase fragment length polymorphism analysis.

Detection of p53 gene mutations in tumour cells is of great clinical interest because p53 mutations have been shown to be closely related to chemotherapy resistance and poor prognosis.l-' However, in routine practice there is no efficient or convenient method to detect the mutations. Although many investigators use single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products followed by direct sequencing, the SSCP method is not practical because of several limitations: specimens with more than about 200 bp fragment size cannot be assayed; it is difficult to standardize the electrophoretic conditions such as temperature and compositions of gel and buffer; and the sensitivity is uncertain (up to 90%) and largely dependent on the conditions.v' Cleavase fragment length polymorphism (CFLP) is, like SSCP, primarily a method for detecting mutation-induced changes in secondary structure of single-stranded (ss) DNA, but has additional benefits: it is applicable for fragments up to 2 kb size; specific and reproducible patterns can be obtained; and it is able to detect abnormal signals indicative of mutation site. The endonuclease cleavase 15 recognizes and cleaves the 5' side of hairpin loops in selfannealed ssDNA derived from PCR products labelled at the 5' end, and generates end-labelled fragments of various sizes. Electrophoresis of the fragments reveals a sequence-specific CFLP pattern on polyacrylamide gels containing urea. We applied this method to detect mutations in the p53 gene after amplification of its full translational seq uence by reverse transcription (RT)-PCR. We obtained test samples from bone marrow aspirates from three patients with haematological disorders (two with acute myeloblastic leukaemia and one without malignancy) and

Detection of p53 gene mutations: analysis by single-strand conformation polymorphism and Cleavase fragment length polymorphism.

We have generated a collection of clones containing single point mutations within the exon 5-9 hot spot regions of the p53 gene by using polymerase chain reaction (PCR) to amplify select regions of the gene from characterized cell lines. These clones were then used to address the sensitivity of mutation detection using slab-gel single-strand conformation polymorphism (SSCP) and Cleavase fragment length polymorphism (CFLP) assay systems. Both methods exhibited high sensitivities for the detection of mutations in cloned p53 mutations in this study: 97% for CFLP and 94% for SSCP. In addition to resulting in higher sensitivity of mutation detection, CFLP has the capability to analyze longer fragments. In this study, CFLP identified five intronic mutations which were not investigated in the exon-specific SSCP assay. These results agree with those found elsewhere and demonstrate that CFLP scanning can have practical advantages when used for the identification of sequence alterations within the p53 gene.

Characterization of frequent polymorphisms in intron 2 of CYP21: application to analysis of segregation of CYP21 alleles

The gene encoding adrenal steroid 21-hydroxylase, CYP21, is located in the MHC class III region. Most cases of congenital adrenal hyperplasia (CAH) are caused by mutations in this gene, and most mutations appear to arise from gene conversion-like events involving the transfer of deleterious sequences from the pseudogene, CYP21P, which is located within 30 kb of CYP21. Approximately 20-30% of mutations are caused by deletions of CYP21. The second intron of CYP21 is polymorphic, and several base substitutions that include nt395, nt453, and nt601 have been reported; however, the frequencies of these polymorphisms are unknown. Using a combination of cleavase fragment length polymorphism analysis and direct sequencing, we examined the sequence of intron 2 in seven wild-type CYP21 genes and determined the frequency of polymorphisms at nt395, nt453, and nt601 in 48 chromosomes from the parental generation of Centre d'Etude du Polymorphisme Humain families. The observed frequencies of bases at these positions were as follows: 395C, 0.17; 395T, 0.83; 453C, 0.71; 453T, 0.29; 601A, 0.1; and 601C, 0.9. Using a PCR/restriction digestion approach to examine these intragenic markers, we could follow the segregation of alleles in informative families with 21-hydroxylase deficiency and identify deletions of CYP21. We emphasize that this method should be used in conjunction with other molecular genetic techniques for diagnosis of CAH. In addition to their potential use in families with CAH, these markers may be of use in genetic studies of the MHC in humans.

Analysis of four common salt-wasting mutations in CYP21 (steroid 21-hydroxylase) by cleavase fragment length polymorphism analysis and characterization of a frequent polymorphism in intron 6

Analysis of four common salt-wasting mutations in CYP21 (steroid 21-hydroxylase) by cleavase fragment length polymorphism analysis and characterization of a frequent polymorphism in intron 6

Mutation Analysis of the Glial Cell Line-Derived Neurotrophic Factor Gene in Parkinson's Disease

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic, central cholinergic, and motoneurons. GDNF also prevents the neuronal loss in experimental animal models for Parkinson's disease (PD). We have now investigated the GDNF gene for possible mutations in a group of nonfamilial PD and other patients. By cleavase fragment length polymorphism (CFLP) analysis and direct sequencing of the full coding region of GDNF gene we found a novel GDNF sequence variant in 1 of 30 PD patients. The alteration does not change the predicted amino acid sequence and it was also found in 1 of 20 patients without PD, suggesting that it represents a polymorphism in the gene. No other sequence variations were found. We conclude therefore that mutations in the GDNF coding region are not commonly contributing to the pathogenesis of PD.

Algorithmic approach to high-throughput molecular screening for alpha interferon-resistant genotypes in hepatitis C patients.

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5' untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634-639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.

Comparative evaluation of cleavase fragment length polymorphism with PCR-SSCP and PCR-RFLP to detect antimicrobial agent resistance in Mycobacterium tuberculosis

Comparative evaluation of cleavase fragment length polymorphism with PCR-SSCP and PCR-RFLP to detect antimicrobial agent resistance in Mycobacterium tuberculosis

Cleavase Fragment Length Polymorphism (CFLP): A Methodology to Further Exploit Polymorphisms from PCR Products of Plastid DNA (ptDNA) in Phaseolus

The CFLP methodology was applied for Cleavase I site detection within ptDNA intergenic regions (atpB-rbcL and rps14-psaB) at both interspecific and intraspecific levels in the genus Phaseolus. Optimal Cleavase I reaction temperature was 55 °C and the semi-dry electrophoretic transfer was more efficient than the original capillary one. Cleavase reactions yield a high number of fragments as compared to PCR-RFLP and allowed differentiation within and between landraces and wild forms of the Lima bean (Phaseolus lunatus L.) originating from Andean and Mesoamerican regions of Latin America. From sequencing data and using stemloop program (GCG, Madison), congruent numbers of hairpins/fragments were identified within the sequences, highlighting the robustness of the Cleavase I. Our results pointed out the ubiquity of short conserved motifs amongst a geographically localized group of species. In the vicinity of these motifs, synapomorphic-like substitutions were frequently observed. A phylogenetic tree based on these sequences is congruent with the CFLP pattern as well as with the widely accepted phylogeny of the genus. The usefulness of this new tool as alternative and/or complementary to PCR-RFLP technology on ptDNA is suggested and discussed.

Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis.

We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5'-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches.

Nuclear exclusion of wild-type p53 in immortalized human retinoblastoma cells.

Retinoblastoma is the most common childhood tumor of the eye, arising from cells that are defective in both copies of the retinoblastoma susceptibility gene (RB1). Most retinoblastoma tumor cells eventually undergo programmed cell death (i.e., apoptosis); however, some cells can acquire the ability to metastasize and become immortal. Transfection of immortal retinoblastoma cells with DNA sequences encoding wild-type p53 protein induces cell death, suggesting that the loss of both RB1 and p53 functions may be required for cell immortalization. We have examined this possibility by characterizing the p53 protein and messenger RNA in six independently isolated, immortalized retinoblastoma cell lines. Western blotting methods were used to assess p53 protein level in each cell line, and Cleavase Fragment-Length Polymorphism analysis of complementary DNAs was used to screen for mutations in p53 messenger RNA. Localization of p53 protein in cells of the immortalized lines and in specimens of retinoblastoma tumors was achieved by means of indirect immunofluorescence and immunocytochemistry, respectively. All six immortalized cell lines expressed wild-type p53 messenger RNA and high levels of p53 protein. Although p53 is normally a nuclear protein, the p53 in four of the six cell lines was located predominately in the cytoplasm; in the remaining two cell lines, p53 was localized in both the nucleus and the cytoplasm. Cytoplasmic localization of p53 in retinoblastoma tumor specimens was rare and usually restricted to cells that had invaded adjacent ocular tissues, indicative of the early stages of metastasis. Some immortalized retinoblastoma cells may exhibit p53 dysfunction through nuclear exclusion of wild-type p53 protein.