Single-strand Conformation Polymorphism Band Research Articles

DEVELOPMENT AND CHARACTERIZATION OF POLYMORPHIC EST MARKERS IN COMMON CARP

Expressed sequence tags(ESTs) are of high value in genome mapping and marker-assisted selection(MAS).This study aims to develop EST markers for genome mapping and other studies of common carp(Cyprinus carpio L).A total of 7381 EST sequences(400—3500bp) were downloaded from the EST database of the GenBank as the source data of the present analyses.Sixty-seven pairs of primers(designated as HB1-67) were designed in the 3′ untranslated region(UTR) of the common carp ESTs,47 of which can successfully amplify specific products from genomic DNA.Twelve polymorphic EST markers were identified in a backcross mapping family of common carp by single-strand conformation polymorphism(SSCP) analysis.Six of the 12 polymorphic EST markers were derived from common carp genes of known function,and the rest were from common carp ESTs homologous to putative genes in zebrafish(Danio rerio) or channel catfish(Ictalurus punctatus).Allele distribution of these polymorphic EST markers was found to follow the Mendelian segregation(1∶1 or 3∶1) in both the backcross family and gynogenetic haploids,and the number of SSCP bands in haploid population was half of that in diploid family.The gene diversity(H=0.437) of Dongting Lake population of common carp revealed by 4 polymorphic EST markers is much lower than that by previous microsatellite analysis(close to 1).The results of present study indicated that EST-SSCP markers,with codominant inheritance,can be used as a powerful tool in the genetics of not only diploid but also haploid common carp.As Type I marker derived from coding region with relatively lower genetic variability,EST-SSCP markers are still of high potentials in such studies as genome mapping(including genetic linkage maps and gene maps,as well as comparative mapping),genetic structure,and genetic adaptability of fish populations.

Rapid Discrimination of Salmonella Isolates by Single-Strand Conformation Polymorphism Analysis

A molecular typing technique was developed for the differentiation of Salmonella isolates based on single-strand conformation polymorphism (SSCP) analysis of amplicons generated by PCR. Amplicons from parts of the fimA (both the 5′ and 3′ ends), mdh, invA, and atpD genes were generated separately from a panel of Salmonella strains representing Salmonella bongori, and four subspecies and 17 serovars of Salmonella enterica. These amplicons were subjected to SSCP analysis for differentiation of the salmonellae on the basis of different conformational forms arising due to nucleotide sequence variations in the target genes. Several distinct SSCP banding patterns (a maximum of 14 each for atpD and fimA 3′ end) were observed with this panel of Salmonella strains for amplicons generated from each target gene. The best discrimination of Salmonella subspecies and serovar was achieved from the SSCP analysis of a combination of at least three gene targets: atpD, invA, and either mdh or fimA 3′ end. This demonstrates the applicability of SSCP analysis as an important additional method to classical typing approaches for the differentiation of foodborne Salmonella isolates. SSCP is simple to perform and should be readily transferable to food microbiology laboratories with basic PCR capability.

Leptin gene polymorphism in Indian Sahiwal cattle by single strand conformation polymorphism (SSCP) (Short communication)

Leptin, a 16-Kilo dalton protein produced by the obesity (ob) gene, plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction in cattle. The genetic variation of the leptin gene in Sahiwal cattle (Bos indicus) was investigated using an optimized non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of 13 amplified fragments covering almost the entire gene. Twenty-eight SSCP band patterns were detected from 10 of these fragments in a sample of 202 Sahiwal cattle. Polymorphisms were detected in the samples, indicating that Sahiwal cattle have high genetic variability in the entire leptin gene. This result opens interesting prospects for future breeding programmes and conservation strategies. These leptin gene variants can be sequenced and screened in the entire population to develop single nucleotide polymorphisms (SNPs) for association studies with different productive and reproductive performances and marker assisted selection.

Dynamic characteristics of bacterial community in a sulphate-reducing bioreactor fed with acetate and intermittent ethanol

Acetotrophic Sulphate-Reducing Bacteria (ASRB) were enriched in a sulphidogenic Continuous Stirred Tank Reactor (CSTR) fed with acetate/ethanol and diagnosed by Single-Strand Conformation Polymorphism (SSCP) and 16S rRNA gene clone library. The CSTR was run for 65 days with synthetic wastewater containing sulphate and acetate with intermittent ethanol at hydraulic retention time of 10 h. The Sulphate-Removal Rate (SRR) reached to 3.8 g/(L?day) after 35 days of start-up. The SSCP profiles of bacterial community changed rapidly at the beginning of start-up before Desulphococcus sp., Desulphomicrobium sp., Aminomonas and Anaerolinea formed a stable community. Bacterial diversity decreased when the ethanol in the influent was replaced by acetate of equal Chemical Oxygen Demand (COD). Desulphomicrobium sp. was enriched and found to have a significant role in acetate utilisation. When ethanol was re-added instead of acetate of equal COD strength, microbial diversity increased and the Desulphomicrobium band of SSCP profiles became weak. At the later start-up stage, the 16S rRNA gene clone library indicated the presence of bacteria belonging to six different known phyla and sequences with similarities to those of sulphate-reducing bacteria accounted for 22%. The SSCP band sequences revealed that the characteristics of bacterial community populations resembled those of clone library sequences. The present study showed the addition of ethanol to influent would enhance bacterial diversity and SRR, ASRB could be enriched and Desulphomicrobium sp. was the primary source of acetate oxidation.

Enrichment of anaerobic benzene-degrading microorganisms by in situ microcosms

Microcosms filled with different solids (sand, lava, Amberlite XAD-7) were exposed for 67 days in the sulfidic part of a groundwater monitoring well downstream of the source zone of a benzene-contaminated aquifer and subsequently incubated in the laboratory. Benzene was repeatedly degraded in several microcosms accompanied by production of sulfide, leading to stable benzene-degrading enrichment cultures. In control microcosms without filling material, benzene was initially degraded, but the benzene-degrading capacity could not be sustained. The results indicate that long-term physiologically active benzene-degrading microorganisms were attached to surfaces of the solids. The biodiversity and attachment behavior of microorganisms in the in situ microcosms was assessed by confocal laser scanning microscopy and single-strand conformation polymorphism (SSCP) analysis, followed by sequencing of dominant SSCP bands. The microbial community was composed of several different Bacteria, representing members of Clostridia, Bacteroidales, all subgroups of the Proteobacteria, Verrucomicrobia, Nitrospira, Chloroflexi and Chlorobi. Only a few archaeal sequences could be retrieved from the communities. The majority of phylotypes were affiliated to bacterial groups with a possible functional relationship to the bacterial sulfur cycle, thus indicating that the microbial community in the investigated aquifer zone depends mainly on inorganic sulfur compounds as electron donors or acceptors, a finding that corresponds to the geochemical data.

Characterization of the predominant bacterial population of different mangrove rhizosphere soils using 16S rRNA gene-based single-strand conformation polymorphism (SSCP)

Variations in chemical parameters and bacterial populations in mangrove rhizosphere samples were noted for different sites. The C, N, P and K contents as well as pH, EC and salinity showed variation between sites. Significant differences in soil properties were also found in sampling sites. Two types of soil were noted among sites. Guesthouse had significantly higher organic matter and nutrient content (N) than other three sites suggesting that human discharges, litter deposition and surface runoff were major nutrient inputs. This contaminated site was located at the landward edges. Positive correlations between organic matter, N, P and K contents were found suggesting that these nutrients were from similar input sources. Effects of sampling sites on microbial diversity were also analyzed via SSCP. Porteresia coarctata and Rhizophora mucronata did not show any variation in the banding patterns among replicates sampled in short distance within site. But Sonneratia apetala showed variation among replicates sampled in distance within site. A significant variation was noted in the SSCP profile among replicates between sites. The majority of dominant SSCP band sequences were related to bacterial genera of root and root-free soil environments, namely Bacillus, Planococcus, Planomicrobium, low G+C Gram-positive bacterium, glacial ice bacterium and unidentified bacteria. In the analysis of 16S rRNA sequences, members belonging to the phylum Firmicutes dominated the sequence collection. The phylogenetic analysis of 16S rRNA gene sequences showed close relationships to a wide range of clones or bacterial species of phylum Firmicutes and unidentified bacteria.

Evaluation of PCR–Single-Strand Conformational Polymorphism Analysis for Identification of Gram-Negative Histamine-Producing Bacteria Isolated from Fish

In this study, the previously established PCR–single-strand conformation polymorphism (SSCP) system for detecting and identifying gram-negative histamine-producing bacteria was evaluated. This system can detect and identify histamine-producing bacteria directly from seafood by the use of sequence polymorphisms of the histidine decarboxylase gene (hdc). First, we isolated 81 histamine-producing strains of bacteria from fish samples and analyzed the hdc gene by the PCR-SSCP system. The 22 newly obtained SSCP banding patterns were added to our database, and the utility of our modified database was tested in a second experiment consisting of 18 strains of histamine-producing bacteria isolated from 25 fish samples. Approximately 80% of the histamine-producing strains corresponded to those in the new database. Use of the database for PCR-SSCP analysis, including the band patterns newly added in this study, for the hdc gene makes it possible to more accurately identify histamine producers.

Hepatitis C virus quasispecies in HIV‐infected women

Despite the high frequency of HCV and HIV coinfection, little is known about HCV quasispecies in HIV-positive patients. The current analysis included 236 HIV+/anti-HCV+ women enrolled in the Women's Interagency HIV Study (WIHS). Hypervariable region 1 of the second envelope gene was analyzed by single-strand conformation polymorphism (SSCP). The relationship between the HCV quasispecies and clinical and demographic features were analyzed in multivariate models. Age over 40 years and high HCV RNA load were the only factors significantly associated with quasispecies complexity, assessed as the number of SSCP bands. High HIV and HCV plasma loads were associated with quasispecies stability over time, as reflected by stable SSCP band patterns. However, women who were actively injecting drugs were 3 times more likely to experience quasispecies changes than their noninjecting counterparts. No affect on HCV quasispecies dynamics was noted in relation to CD4 count or highly active antiretroviral therapy (HAART). among HIV/HCV coinfected patients, HCV quasispecies complexity and dynamics correlate more closely with HIV and HCV plasma loads than with CD4+ cell counts. Active drug use is associated with quasispecies changes probably due to repeated superinfections with new HCV strains. This needs to be considered when planning treatment and prevention strategies for HCV in coinfected individuals.

The same mutation Glu208Lys in the GJB1 gene was detected in 2 families with X-linked Charcot-Marie-Tooth disease

Mutation of GJB1 gene was investigated in two families with X-linked Charcot-Marie-Tooth disease. Genomic DNA from venous blood samples was prepared. The coding sequence of the GJB1 gene was amplified from genomic DNA. PCR products were analyzed by single strand conformational polymorphism (SSCP) method. The PCR product having an abnormal pattern was sequenced to detect the mutation. It was found that the samples of all patients and one little girl with normal phenotype showed an abnormal SSCP band, but not detected in the other unaffected members in the first large family. In the second small family, an abnormal SSCP band was found in all the patients, but not detected in the unaffected member. The result of DNA sequencing demonstrated that both families had a same mutation of 622G-->A, which resulted in a substitution of Glu208Lys. This mutation has not been reported previously in China.

Study of genetic mutation locus in a family with congenital aniridia

The aim of this study was to determine the genetic mutation locus of congenital aniridia. Peripheral vein blood (2-5 ml) was collected from all members of a congenital aniridia family. DNA was extracted, then, the primers of polymorphic microsatellite genetic markers were synthesized, followed by polymerase chain reaction (PCR) analysis. Polyacrylamide gel electrophoresis (PAGE) was used to analyze the denatured PCR products and haplotype linkage analysis was applied according to the relationship between bands and family members to determine the association between the phenotype of aniridia and PAX6 gene. Fourteen exons of human PAX6 gene were amplified by PCR and allele specific variations were detected by single strand conformation polymorphism (SSCP). By comparing the difference of bands between patients and the unaffected members, the mutated exons was detected. The mutated locus was detected by direct DNA automated sequencing of the PCR products with different SSCP bands. PAX6 mutation was linked to the occurrence of aniridia in this family. An extra band corresponding to exon 9 in PAX6 was found by single strand conformational polymorphism analysis in all aniridia patients in this family, but not detected in the unaffected members. A mutation of C to T was detected by sequencing at the nucleotide 1080 that converts the Arg codon (CGA) to a termination codon (TGA). Mutation of PAX6 gene can result in the occurrence of congenital aniridia.

Identification of a Thiomicrospira denitrificans -Like Epsilonproteobacterium as a Catalyst for Autotrophic Denitrification in the Central Baltic Sea

Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier Thiomicrospira denitrificans. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of T. denitrificans. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to T. denitrificans could be relevant for the carbon budget of the central Baltic Sea because T. denitrificans exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of Thiobacillus denitrificans hypothesized previously for this function.

Picoplankton culture assessment using single strand conformation polymorphism and partial 18S sequencing

Environmental water samples were taken throughout 2001 from fractioned water samples at the Helgoland time series site. The less than 3-µm fraction was inoculated into various media. Serial dilutions from these inoculations produced a large number of rough cultures from which several hundred well-growing picoplankton cultures were started. We established a combination of crude DNA extraction, single strand conformation polymorphism (SSCP) fingerprinting and subsequent sequence analysis to screen these large numbers of cultures, assess their purity/clonality and determine their identity. Picoplankton species (i.e., cells smaller than 3 µm) are difficult to distinguish because of their small size and the lack of morphological characters. Therefore, molecular techniques provide the most reliable method to achieve their identification. Cultures were enriched for photoautotroph cells, i.e., no carbon source was added, and cultures were grown in the light. From these cultures, crude DNA was extracted, which was used for partial 18S polymerase chain reaction (PCR). On average, 50% of the cultures produced a PCR product. SSCP analysis of PCR products revealed the clonality of a given culture. If clonal, then there was only a single SSCP band; if not clonal, then there were multiple SSCP bands. Single SSCP bands were subsequently sequenced, and sequences were used to identify the culture. For this study, 300 cultures were screened resulting in the identification of 63 potentially clonal cultures. These methods proved to be relatively easy to apply to assess the clonality and purity of the cultures.

PCR-SSCP as a molecular tool for the identification of Benedeniinae (Monogenea: Capsalidae) from marine fish

PCR-based single strand conformation polymorphism (SSCP) analysis was used to characterize monogenean specimens of the subfamily Benedeniinae of morphologically uncertain specific status from different marine fish species, using Neobenedenia melleni, N. girellae and Entobdella corona for comparison. The first internal transcribed spacer (ITS-1) and the 5' terminal variable region (D1-D3 domains) of the large subunit ribosomal DNA (lsrDNA) were amplified separately from individual monogeneans, and the amplicons were subjected to PCR-SSCP analyses, followed by direct sequencing. Both SSCP patterns and the ITS-1 sequences data allowed specimens representing Entobdella spp. from the host Taeniura melanospilos to be unequivocally distinguished from those representing Neobenedenia spp. and those representing Benedenia spp. Neobenedenia girellae, a morphologically controversial species, had identical SSCP banding pattern and ITS-1 sequence to that of N. melleni, supporting the proposal that N. girellae is a synonym of N. melleni. Neobenedenia spp. and Benedenia spp. had identical SSCP patterns and ITS-1 sequences. These findings and the PCR-SSCP approach taken should have implications for the accurate identification and assessment of taxonomic validity of other important monogenean groups of the marine fish.

Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion

Studies on hepatitis C virus (HCV) quasispecies dynamics in the natural course of infection are rare owing to difficulties in obtaining samples from the early phase of infection. We studied 15 patients from the Transfusion-Transmitted Viruses Study who seroconverted to anti-HCV after receiving infected blood. Follow-up serum samples were collected every 2-3 weeks for 6 months, at 10 months, and at 11-16 years. Viral quasispecies in the second envelope hypervariable region 1 (E2/HVR1) and 5' untranslated region (5'UTR) were analyzed with single-strand conformation polymorphism (SSCP) and heteroduplex mobility assay (HMA). Seven patients cleared infection within 7-24 weeks (mean, 14.0 wk) and 3 patients eventually became anti-HCV negative. In 6 patients with resolving hepatitis the SSCP band pattern remained stable, whereas in one patient minor changes appeared before clearance. In contrast, in all 8 patients progressing to chronicity, major changes in the E2/HVR1 quasispecies developed at 8-22 weeks (mean, 13.1 wk). Shannon entropy and medium mobility shift values derived from HMA gels remained stable in patients with resolving hepatitis but changed in those who developed chronic infection. Only 2 patients showed minor changes in 5'UTR. A decrease in E2/HVR1 complexity at the time of transmission (bottleneck) was found in 5 patients altogether. Changes in E2/HVR1 quasispecies 8-22 weeks after infection, likely caused by mounting immune pressure, were predictive of ensuing chronic infection, whereas stability was associated with resolution. Our study also showed that composition of HCV quasispecies may be preserved during transmission from host to host.

Diversity of bacteria associated with Collembola – a cultivation-independent survey based on PCR-amplified 16S rRNA genes

The bacterial communities found in eight different soil-inhabiting microarthropod species of the Class Collembola (springtails) were analyzed by analysis of PCR-amplified 16S rRNA genes obtained without cultivation from total DNA. To characterize the bacteria associated with the parthenogenetically-reproducing Folsomia candida, the almost complete 16S rRNA genes were amplified with three universal primers, i.e., the forward primer F27 and the reverse primer R1492 or R1525. With the reverse primer R1492, 100% of 49 cloned PCR products were identical to the 16S rRNA gene of the intracellular reproduction parasite Wolbachia pipientis (Supergroup E). However, no Wolbachia was detected with the reverse primer R1525 when two different breeding stocks of F. candida were analyzed. Clone libraries from one breeding stock were composed exclusively of a sequence related closely to intracellular bacteria of the genus Rickettsiella ( Gammaproteobacteria) (93 clones). In contrast, this sequence was not detected in the other F. candida breeding stock. Instead, sequences of 95 clones originated from different phylogenetic groups ( Alphaproteobacteria, Gammaproteobacteria, Firmicutes and Planctomycetes). Several were closely related to bacteria, which are known to live in soil and interact with insects. Genetic profiles based on PCR-amplified partial 16S rRNA genes and single-strand conformation polymorphism (SSCP) indicated that different Collembola species harbored different bacterial communities. SSCP bands indicating Wolbachia pipientis supergroup E were detected in profiles from four of the five parthenogenetic species included in this study. Other dominant bands in the SSCP profiles were related to bacteria from the phyla Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. One sequence ( AJ605704) indicated the presence of a not-yet-described intracellular symbiont from the Gammaproteobacteria, but several other sequences may represent less specific interactions between environmental bacteria and Collembola.

Genetic Differentiation of Phytoplasma Isolates by DNA Heteroduplex Mobility Assay and Single-Strand Conformation Polymorphism Analysis

Heteroduplex mobility assay (HMA) and single-strand conformation polymorphism (SSCP) analyses combined with PCR were developed for genetic differentiation of various phytoplasma isolates. In the HMA and SSCP analyses, differences in the mobility shifts and the SSCP band patterns identified three distinct types of phyto-plasmas: Type Ⅰ, jujube witches'-broom (JWB) and ligustrum witches'-broom (LiWB); Type Ⅱ, mulberry dwarf（MD) and sumac witches'-broom (SuWB); and Type Ⅲ, paulownia witches'-broom (PaWB). Results of the sequence analyses revealed that phytoplasmas of JWB and MD had 100％ homology with LiWB and SuWB, respectively. On the other hand, PaWB phyto-plasma had 97.8% homology with MD phytoplasma. The PCR-HMA and SSCP techniques were very useful in determining variations in sequence among several isolates of phytoplasmas. Furthermore, the methods were rapid, economical, highly sensitive, and easy to handle with the gels.

Application of a yeast assay to detect functional p53 mutations in archival prostate cancer tissue.

Detection and functional evaluation of mutant p53 alleles using a yeast assay could yield significant information for predicting the prognosis of patients with prostate cancer (CaP). Since the current version of this yeast assay is not applicable to archival tissues, we developed a modified assay for use on formalin-fixed, paraffin-embedded tissue and have applied it to the study of patient samples. Using this modified assay, we examined archival CaP samples from 10 patients for mutations in exons 5-8 of p53 gene. Mutations were detected in four samples: three resulted in the formation of red yeast colonies indicating complete loss of function, while one gave pink yeast colonies, indicating that this mutant retained partial function. In parallel, we analyzed these samples for p53 abnormalities using a single-strand conformational polymorphism (SSCP) approach. Only three of the four yeast-positive samples gave abnormal SSCP bands. In each case where abnormal p53 was found by both methods, DNA sequencing revealed the identical base change. These results suggest that the modified yeast assay may be more sensitive than SSCP for detection of p53 mutations, and demonstrate that the modified method can be used to detect and evaluate the function of p53 mutants present in archival tissue.

PCR-based Single-strand Conformation Polymorphism (SSCP) Analysis to Clone Nine Aquaporin Genes in Cucumber

Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes.

Mutation analysis of the human CYP3A4 gene 5′ regulatory region: population screening using non-radioactive SSCP

Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5′ regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5′ proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at −7.9 kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4 ∗1B allele in nine subjects, three novel alleles were found: CYP3A4 ∗1E (having a T→A transversion at −369 in one subject), CYP3A4 ∗1F (having a C→G tranversion at −747 in 17 subjects) and CYP3A4 ∗15B containing a nine-nucleotide insertion between −845 and −844 linked to an A→G transition at −392 and a G→A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

Detection of exon polymorphisms in the human lactoferrin gene.

We previously demonstrated that lactoferrin gene polymorphisms occur in cancer cells of patients with leukemia and breast cancer. In this study, we established a non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, one of the most sensitive and simplest methods to detect polymorphisms and mutations of the human lactoferrin gene. We optimized the PCR conditions for nine different DNA templates and 16 pairs of exon primers for SSCP analysis. The DNA templates used in the analyses were prepared from a cosmid clone (CT6-1) that contains the human lactoferrin gene, human placental tissue, leukocytes from 10 normal volunteers, leukemic cells of two patients, and previously established three breast and two leukemic cell lines. With the appropriate exon-primer sets, PCR products from exon I to exon 16 of the lactoferrin gene were generated from the DNA templates and analyzed by SSCP. Compared with the homogenous cloned DNA, lactoferrin gene polymorphisms were detected within exons 2, 5, 7, 9, 13, 14, and 15 of the normal placental and leukocyte DNA. In addition, abnormal migration patterns of the lactoferrin gene in cancer cells were detected in exons 4, 5, 13, 14, and 15. The PCR-SSCP band migration patterns can be attributed either to gene polymorphism in normal cells or to DNA mutations in cancer cells and the employed method cannot distinguish between them. Nonetheless, the present analysis suggests that genetic polymorphisms of the lactoferrin gene exist in selected exons and additional mutations of the lactoferrin gene do occur in the cancer cells.