Polymerase Chain Reaction-restriction Fragment Length Polymorphism Method Research Articles

Rapid PCR-RFLP Method for the Identification of 5 Billfish Species

ABSTRACT: A rapid and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify billfish species Xiphias gladius (swordfish), Makaira nigricans (blue marlin), Makaira indica (black marlin), Istiophorus platypterus (sailfish), and Tetrapturus audax (striped marlin). After DNA extraction and amplifying, the 348-bp PCR products from gene encoding of cytochrome b were subjected to restriction enzyme analysis. No single enzyme tested was able to distinguish the 5 species at the same time, but the combination of results obtained from the digests of Bsa JI and Cac 81 could be used to differentiate the 5 billfish species. This sensitive, rapid, and valid method can be used to detect fraudulent substitutes.

Detection and Identification of cry1I Genes in Bacillus thuringiensis Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for detection and identification of cry1I genes from Bacillus thuringiensis (Bt) was established. Based on the analysis of conserved regions of the cry1I genes, 2 oligonucleotide primers were designed to amplify a 665-bp fragment of the genes. The amplification products were digested with restriction endonuclease HinfI or with RsaI in addition for specific detection of different variants from the known subclasses of cry1I genes. The PCR-RFLP pattern obtained revealed the detection of cry1I genes in 151 of 202 native Bt isolates. Furthermore, cry1I genes were detectable in 10 of 19 standard strains tested. The cry1Ia gene was the most abundant cry1I gene subclass present in 54 of 56 native Bt isolates and in 8 of 10 standard strains. Based on the results obtained, the PCR-RFLP method may be a valuable and reliable tool for specific detection and identification of cry1I genes.

Novel mutations in the cytochrome P450 2C19 gene: a pitfall of the PCR-RFLP method for identifying a common mutation

CYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2-5% of Caucasian populations, but at higher frequencies (18-23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.

Rapid PCR-RFLP method for discrimination of imported and domestic mackerel

With the ever-decreasing domestic fishery catch of Japanese mackerel Scomber japonicus, alternative Atlantic mackerel Scomber scombrus has been increasingly imported and currently accounts for approximately 34% of mackerel consumption in Japan. As there is no morphologic difference between the species after removal of their skin, not only fresh and frozen fillets but also processed seafood of S. scombrus are frequently marketed with mislabeling as S. japonicus. In this study, a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed to discriminate imported mackerel S. scombrus and domestic mackerel S. japonicus. PCR amplification for the nuclear 5S ribosomal DNA nontranscribed spacer was performed using Scomber-specific primers. Direct digestions of the PCR products using either PvuII or HaeIII restriction enzymes generated species-specific profiles, indicating that both enzymes enable the accurate identification of S. scombrus and S. japonicus. This robust and reproducible method can serve as molecular-based routine food inspection program to enforce labeling regulations.

Rapid assignment of the swine major histocompatibility complex (SLA) class I and II genotypes in Clawn miniature swine using PCR‐SSP and PCR‐RFLP methods

Inbred miniature swine with defined novel SLA haplotypes will be useful in allo- and xeno-transplantation studies, which can be carried out representing variable combinations of SLA haplotypes. In Clawn miniature swine, two haplotypes (c1 and c2) and one crossover haplotype (c3) have been assigned by nucleotide sequence determination of RT-PCR products of the three SLA classical class I genes and two SLA class II genes. To select SLA class I and II homozygotes in Clawn miniature swine individuals, we developed a rapid and simple SLA-class I- and II-DNA typing method by a combination of polymerase chain reaction-sequence specific primer (PCR-SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Seven allele specific primer pairs were designed for amplification of the second exons of three SLA class I genes, SLA-1, SLA-2, and SLA-3, and one SLA class II gene, DRB1. Furthermore, based on PCR-RFLP patterns in the SLA-DQB1 gene, two allelic variants were recognized in the second exon in the Clawn miniature swine. Three haplotypes, c1, c2 and c3, were simply identified by the combination of PCR-SSP and PCR-RFLP methods in 22 samples from five families. A single allele at each of the class I and II genes was also observed in seven samples as SLA class I and II homozygotes with either the c1 or c2 haplotype. The combination of PCR-SSP and PCR-RFLP methods facilitate the rapid identification of the three haplotypes and SLA class I and II homozygotes in individual Clawn miniature swine.

Chloroplast DNA variations of cultivated radish and its wild relatives

Chloroplast DNA sequences were investigated to further elucidate the evolution of cultivated radish. This is the first known attempt to analyze intrageneric DNA sequences of chloroplast DNA in Raphanus. The nucleotide sequence of the 5′- matK region (690 bp) was determined for 17 accessions including cultivated radish ( Raphanus sativus), wild radish ( R. raphanistrum), and East Asian wild radish ( Raphanus sativus var. hortensis f. raphanistroides Makino). Three parsimony-informative sites showed that cultivated radish has two distinctly diverged chloroplast DNA sequences. When considering the evolutionary rate of the matK sequences, it was difficult to hypothesize that a new haplotype had occurred in cultivated radish and then rapidly spread among the cultivated radish varieties. Alternatively, it is far more likely that cultivated radish received two distinct haplotypes from its wild ancestor(s). For more detailed analyses, five different chloroplast regions were amplified through the use of five newly constructed primers. The total length of the amplified regions was 32.8 kb, which covered both the coding and non-coding DNA sequences. In total, 137 restriction fragments were detected. The results of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses were consistent with that of the cpDNA sequences. We found that the combined data of the matK sequences and the PCR-RFLP analysis indicated that Raphanus raphanistrum was not the maternal ancestor of cultivated radish, and that the geographical distribution pattern of the haplotypes suggests that the East Asian wild radish has contributed to the establishment of the East Asian cultivated radish. The present PCR-RFLP method using the newly developed primers works for other plant species and for other purposes.

PCR and PCR–RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis

Background : We have focused on the DNA topoisomerase II genes of several pathogenic fungi, and developed polymerase chain reaction (PCR) and PCR–restriction fragment length polymorphism (RFLP) methods targeting this gene for identification of dermatophytes. Objective : To assess the availability of the PCR-based identification for an etiologic study of dermatophytosis, by testing these PCR and PCR–RFLP methods for stability and reproducibility. Methods : Three hundred and fifty-six dermatophyte strains were isolated from 305 patients with tinea, and their genomic DNAs were used as templates for the PCR using primer mixes (PsT, PsME, dPsD1 or dPsD2) composed of gene-specific primers for identification of dermatophytes to the species level. The genomic DNAs of Trichophyton rubrum were further subjected to subrepeat element analysis of the nontranscribed spacer (NTS) of ribosomal DNA (rDNA). Results : In this study, six dermatophyte species ( T. rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum) were obtained. In all cases, the identifications obtained from the PCR and PCR–RFLP targeting the DNA topoisomerase II gene coincided with those from the conventional morphological features-based identification technique. The sensitivity of the PCR-based identification was found to be a colony of approximately 3 mm in diameter. Furthermore, T. rubrum was divided into three groups (17 types) on the basis of the sizes and numbers of the products generated from the TRS-1 region, and three types from the TRS-2 region. Conclusion : The PCR and PCR–RFLP targeting the DNA topoisomerase II gene were rapid, stable, and reproducible for species identification of dermatophytes, and thus are convenient tools for an etiologic study of dermatophytosis.

Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction–restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR–RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR–RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR–RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.

Demonstration of interbreeding between virulent and avirulent populations of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) by PCR-RFLP method

The pinewood nematode, Bursaphelenchus xylophilus, is the causative agent of pine wilt disease. The virulence of nematodes significantly varies among pine trees, but does not vary within a single pine. To elucidate the reason for no variation in virulence within a single pine, a technique to investigate the population structure of nematodes of different virulence is needed. In this study, the demonstration of interbreeding between virulent and avirulent populations of B. xylophilus in vitro was attempted using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Ribosomal DNA containing the 5.8S gene, the internal transcribed spacer regions 1 and 2, and partial regions of 18S and 28S genes were used for analysis. First, PCR-RFLP patterns of offspring produced by interbreeding between two individuals of virulent and avirulent isolates were analyzed. The offspring from a single-pair interbreeding showed 3-digested fragment patterns with the restriction enzyme HhaI, patterns the same as the virulent isolate, the avirulent isolate and a hybrid pattern containing both fragments of the virulent and avirulent isolates. The virulent population was mixed with the avirulent one in vitro and propagated nematodes were individually analyzed by PCR-RFLP method. The nematodes showed the same 3 PCR-RFLP patterns as the offspring from a single-pair interbreeding. The detection of nematodes with a hybrid pattern demonstrates the occurrence of interbreeding between virulent and avirulent populations of B. xylophilus.

Chloroplast DNA study in sweet cherry cultivars (Prunus avium L.) using PCR-RFLP method

The chloroplast DNA of 96 sweet cherry cultivars (Prunus avium L.) and five cultivars of sour cherry (P. cerasus L.) were analysed to reveal their haplotypes using PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) method. The main advantages of the PCR-RFLP technique are: resolutive, time and cost effective and reproducible. Approximately 9.6% of the chloroplast genome was analysed, using six universal primer pairs and two restriction enzymes. All the mutations detected were insertion-deletions, ranging between 5–30 bp. The combination of all the mutations resulted in three haplotypes (H1, H2 and H3) in the 96 sweet cherry cultivars and a single haplotype (H4) in the five cultivars of sour cherry. The cpDNA polymorphism determined by PCR-RFLP markers helped to understand the maternal inheritance of chloroplast genome in sweet cherry, clearly distinguished the sour from the sweet cherry, and supported that P. avium is not the maternal species of P. cerasus.

Chloroplast and nuclear DNA studies in a few members of the Brassica oleracea L. group using PCR-RFLP and ISSR-PCR markers: a population genetic analysis.

A population genetic analysis of chloroplast and nuclear DNA was performed covering nine wild populations of Brassica oleracea. Three members of the n = 9 group, all close to B. oleracea, Brassica alboglabra Bailey, Brassica bourgeaui (Webb) O. Kuntze and Brassica montana Pourret, were also studied to better understand their relationship with B. oleracea. Chloroplast DNA was analysed using the PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) method. The ISSR-PCR (inter-simple sequence repeat - polymerase chain reaction) technique was adopted to study nuclear DNA. Twelve primer pairs of chloroplast DNA showed very good amplification. The amplified product of each primer pair, digested by three restriction enzymes, revealed no variation of cpDNA among the taxa studied. This indicates they may have the same chloroplast genotype. Seven selected ISSR primers have detected genetic variation, both within and among the populations/taxa surveyed. The information obtained on the intra- and inter-populational genetic diversity of wild populations of B. oleracea neatly defined the individual plants. It could provide important guidelines for backing management and conservation strategies in this species. The study confirms a close relationship between B. alboglabra, B. bourgeaui and B. montana, which is parallel to their morphological similitude.

Linkage disequilibrium between HLA-DPB1 alleles and retinoid X receptor β haplotypes

The human retinoid X receptor β (RXRB) gene maps to the major histocompatibility complex (MHC) region, between KE4 and COL11A2, approximately 130-kb centromeric to HLA-DPB1. We have recently reported a new polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method to detect the G to T single nucleotide polymorphism (SNP) located seven nucleotides after the tenth exon of the RXRB gene, or 3′end+7 position according to existing nomenclature. We also reported strong linkage disequilibrium between the HLA-DPB1*0401 and RXRB+7*T alleles. In the present study, we describe two PCR-RFLP methods to detect additional SNPs in the RXRB gene, T to A, at exon10+378 and A to T at 3′end+140. This new methodology permitted the unambiguous assignment of three distinct SNPs at RXRB exon10+378, 3′end+7 and 3′end+140 to form an “RXRB haplotype.” The data generated from this study were used to determine linkage disequilibrium between several MHC markers and the RXRB alleles and haplotypes. Family studies revealed significant linkage disequilibrium between the RXRB alleles and a number of HLA-DPB1 alleles.

RHC/c genotyping based on polymorphism in the promoter region of the RHCE gene

Designing of PCR tests for the RHC allele is difficult because of the high DNA sequence homology between RHC and RHD genes, which differ by only a one-nucleotide substitution at position 48 in exon 1 of the RHCE gene. We sequenced the promoter region of the RHCE gene, and compared our results with the reported sequence. Genomic DNA was prepared from blood samples collected from 656 Japanese donors. The DNA segment encompassing the promoter region and exon 1 of the RHCE gene from 30 donors was amplified by PCR and analyzed by DNA sequencing. Four nucleotide differences between RHC/c and RHD were found at positions −468, −304, −58, and −46. On the basis of the nucleotide differences at positions −468 ( RHCE vs. RHD) and −292 ( RHC vs. RHc), we then developed a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for RHC/c genotyping. Analysis of the genomic DNA from the 656 donors revealed that this method could discriminate RHC from RHc, irrespective of the RHD genotype, with only a few exceptions. The combination of our system and the intron 2-based PCR-RFLP method previously reported may prove to be more accurate than either of the methods alone, and therefore, useful and valuable for RHC/c genotyping.

Comparison of a nested polymerase chain reaction – restriction fragment length polymorphism method, the PATH antigen detection method, and microscopy for the detection and identification of malaria parasites

A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, the PATH antigen detection method, and light microscopy were compared for their capacity to detect and identify Plasmodium species. One hundred and thirty-six blood specimens obtained from patients suspected of having malaria were examined by each of the three methods. Forty-four specimens were positive for malaria using microscopy as the "gold standard". The sensitivity for nested PCR was 100%, and the specificity was 98%. For the detection of Plasmodium falciparum, the antigen detection method had a sensitivity of 100% and a specificity of 97%. Species identification obtained using PCR-RFLP was identical or superior to light microscopy in 42 cases (96%). Although the nested PCR-RFLP method was more sensitive and specific, the rapid turnaround time and high sensitivity of the antigen detection method makes it a useful adjunct to standard microscopy.

Analytical Evaluation of Primer Engineered Multiplex Polymerase Chain Reaction–Restriction Fragment Length Polymorphism for Detection of Factor V Leiden and Prothrombin G20210A

Factor V Leiden and prothrombin G20210A are clinically relevant genetic risk factors for venous thrombosis. Analysis for both mutations is increasingly being performed on patients exhibiting hypercoagulability. The goal of the current study was to evaluate the performance of primer-engineered multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the simultaneous detection of factor V Leiden and prothrombin G20210A. Primer-engineered multiplex PCR-RFLP methods for the detection of factor V Leiden and prothrombin G20210A from the medical literature were reviewed. A modified method was optimized in which both mutations generate HindIII RFLPs and the prothrombin amplicon contains an invariant HindIII recognition site to assess the completeness of endonuclease digestion. Digested amplification products were analyzed by agarose gel electrophoresis in a single gel lane and visualized by ethidium bromide. Primer-engineered multiplex PCR-RFLP was used to analyze 205 human genomic DNA samples whose factor V Leiden genotypes had been previously determined by MnlI PCR-RFLP. Complete concordance for factor V Leiden genotypes was observed between the two methods in the 205-sample cohort comprising 139 wild-type, 62 heterozygous mutant, and four homozygous mutant individuals. For prothrombin G20210A, primer-engineered multiplex PCR-RFLP identified 196 wild-type and nine heterozygous mutant individuals in the 205-sample cohort. To independently verify prothrombin genotypes, the nine heterozygous mutants and an additional 11 wild-type patient samples (representing 10% of patient samples) were subjected to DNA sequencing. Complete concordance was observed between DNA sequencing and primer-engineered multiplex PCR-RFLP results. In further validation, 123 of the DNA samples consisting of four heterozygous mutant and 119 wild type individuals were genotyped with the Invader Assay for Factor II (prothrombin G20210A). Results showed 100% concordance between the Invader Assay and primer-engineered multiplex PCR-RFLP. A primer-engineered multiplex PCR-RFLP based on single restriction endonuclease digestion has been evaluated and shown to simultaneously and accurately detect factor V Leiden and prothrombin G20210A mutations. The method is robust and readily adaptable to the clinical molecular diagnostic laboratory.

Quantitative assessment of apolipoprotein E genotypes by image analysis of PCR–RFLP fragments

Apolipoprotein E (APOE) genotyping usually involves polymerase chain reaction (PCR) and assessment of restriction fragment length polymorphism (RFLP) by gel electrophoresis. We made determination of HhaI restriction endonuclease digestive patterns more objective and improved diagnostic accuracy with a quantitative approach using sensitive DNA stain (SYBR Green) and image analysis of gel patterns. For distinguishing true and partially-digested restriction fragments, band ratios were calculated for the staining intensity of gel patterns from 116 sample runs of 63 human blood specimens. Each of these specimens was independently genotyped for APOE by at least two (and most of them by three) different PCR–RFLP methods. Based on the distribution of band ratios, decision levels were established and used for developing a program for computer-aided interpretation of APOE genotypes (Microsoft Excel software). Appropriateness of the decision levels for band ratios was validated by APOE genotyping of additional 61 specimens. The approach described here is applicable to a variety of other molecular diagnostic techniques that are based on PCR–RFLP or sequence-specific signal amplifications.

New DRB1* alleles (HLA-DRB1*1135, DRB1*1430 and DRB1*1433) and a confirmatory sequence (DRB1*1133).

Three new DRB1 alleles (DRB1*1135, DRB1*1430 and DRB1*1433) and a confirmatory sequence (DRB1*1133) have been identified after following up unusual or novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns during routine typing of the DRB1*03,*08,*11,*12,*13 and *14 allele groups. Of the new alleles found and described in this paper, two alleles were initially detected by the PCR-RFLP method which produced unexpected restriction polymorphism (DRB1*1133 and DRB1*1135) while the remaining two were found after following up rare allele typings from this technique (DRB1*1430 and DRB1*1433).

Screening for variants of the uncoupling protein 2 gene in Japanese patients with non—insulin-dependent diabetes mellitus

We examined genetic mutations in the coding regions of the uncoupling protein 2 (UCP2) gene in 100 patients with non—insulin-dependent diabetes mellitus (NIDDM). The sequences of each exon-intron boundary were detected by polymerase chain reaction (PCR) using specific primer pairs designed in the cDNA sequence of UCP2 and a cycle-sequence method. Using the specific primer pairs in the intron 5′- or 3′-untranslated region, each exon with its exon-intron boundaries was amplified with the PCR method, and the PCR products were analyzed using a single-strand conformation polymorphism (SSCP) method. One nucleotide substitution in exon 4 was found, which exchanged Ala (gcc) at position 55 of the amino acid sequence for Val (gtc), previously reported in Denmark by Urhammer et al in 1997. The polymorphism was reanalyzed in all patients and 120 normal subjects using a PCR—restriction fragment length polymorphism method. There was no difference in the genotype distribution between patients and normal subjects, and our genotype distribution was similar to the Danish study. Furthermore, there were no clinical differences between genotype groups among the patients. No other mutation including the exon-intron boundary was found in these patients. Genetic mutations of UCP2 may not be commonly associated with obesity or diabetes in Japanese subjects.

Origin of regenerated epithelium in cryopreserved tracheal allotransplantation

Background. Our previous study showed that a cryopreserved tracheal allograft could be transplanted using omentopexy without immunosuppression. The present study investigated, by the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method, whether the regenerated epithelia were of recipient origin or donor origin in a cryopreserved tracheal allotransplantation model. Methods. Twenty-nine mongrel dogs were classified by preoperative peripheral blood PCR-RFLP analysis. The cryopreserved tracheal allografts were implanted into recipient animals that showed a different phenotype from donor animals. A small specimen of epithelia excised from the allograft of animals postmortem was analyzed with the modified PCR-RFLP method. Results. The animals were separated into 16 phenotypes by preoperative PCR-RFLP results, and cryopreserved tracheal allografts transplanted into 8 animals. PCR-RFLP analysis of graft epithelia at 10 days after transplantation showed the donor blood phenotype and analysis of graft epithelia taken from the animals that survived more than 20 days after operation showed the recipient blood and epithelial phenotype. Conclusions. The donor epithelia in the grafts were no longer present within about 20 days after transplantation. The recipient epithelia migrated gradually from the anastomotic site, and the regenerated epithelia that are of recipient origin covered the allograft within about 50 days after transplantation.

Codon 12 Ki-ras mutation in non-small-cell lung cancer: comparative evaluation in tumoural and non-tumoural lung.

Ki-ras activation by point mutation on codon 12 has been reported in non-small-cell lung carcinomas and in various models of experimental lung tumours induced by chemical carcinogens. The cellular targets for carcinogenic compounds of tobacco smoke are usually considered to be the cells of the bronchial mucosa or alveolar epithelium. However, little is known about preneoplastic events in bronchopulmonary carcinogenesis. The hypothesis of the presence of widespread target cells containing Ki-ras mutation was investigated by evaluating concurrent neoplastic and non-neoplastic bronchial and alveolar samples from 51 patients with non-small-cell lung carcinomas. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method used can detect one cell with a mutation on codon 12 among 10(2) normal cells. In tumour samples, a mutation was detected in 20% of adenocarcinomas, but in none of the adenosquamous or squamous cell carcinomas. No mutation was detected in the non-neoplastic bronchial or parenchymal samples. When using an enriched PCR-RFLP method detecting one mutated allele among 10(3) normal alleles a mutation was detected in 23% of adenocarcinomas. In conclusion, Ki-ras activation by mutation on codon 12 was not observed in non-neoplastic bronchial or parenchymal tissues in patients with bronchopulmonary cancers and does not appear to be a genetic event present in non-malignant epithelial target cells exposed to tobacco smoke.