Arbitrarily Primed PCR Fingerprinting Research Articles

Transmission of mutans streptococci in mother-child pairs.

Background & objectives:Dental caries is an infectious, transmissible disease. Maternal transfer of mutans streptococci (MS) has been a subject of research. The aim of this study was to evaluate the transmission of MS from mother to children through genetic analysis.Methods:Thirty mother–child pairs were included and divided into three groups according to the age of the children. Saliva samples were collected and MS colonies from each mother-child pair were isolated. After inoculation and incubation, MS colonies were submitted to amplification technique by polymerase chain reaction (PCR) for identification and arbitrarily primed PCRs (AP-PCRs) to determine various MS genotypes.Results:From birth to six months of age, 30 per cent of children exhibited MS colonization, and by the age of 30 months, 100 per cent harboured the bacteria (P < 0.001). Factors associated with MS colonization were eruption of teeth (P < 0.001), feeding habits with mean colony count being significantly lower in breast-fed as compared to bottle-fed children (P < 0.001) and a significant association between mean MS count of child and mother's practice of sharing spoon with child (P < 0.001). The AP-PCR fingerprinting profile analysis showed 17 MS groups (clusters) containing identical or highly related isolates in mother-child pairs with a high level of similarity (77.27 %).Interpretation & conclusions:The presence of matching MS genotypes suggested vertical transmission from mothers to children. Feeding habits, gum cleaning and number of erupted teeth in children had significant effect on MS colonization. There is a need to develop strategies to present MS colonization in children.

Comparative Study of PCR-Based Approaches for the Genetic Characterization of Three Strains of Acidithiobacillus caldus Isolated from Different Sites in China

Comparative study of the genetic characteristics among three Acidithiobacillus caldus strains isolated from different typical environments in China was performed using a combination of molecular methods, namely sequencing analysis of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers (ITS), repetitive element PCR (rep-PCR), arbitrarily primed PCR (AP-PCR) fingerprinting and random amplified polymorphic DNA (RAPD). Both of the 16S rRNA gene and 16S-23S rRNA gene intergenic spacers sequences of the three strains exhibited small variations, with 99.9-100%, 99.7-100% identity respectively. In contrast, according to the analysis of bacterial diversity based on rep-PCR and AP-PCR fingerprinting, they produced highly discriminatory banding patterns, and the similarity values between them varied from 61.97% to 71.64%. RAPD analysis showed that banding profiles of their genomic DNA exhibited obvious differences from each other with 53.44-75% similarity. These results suggested that in contrast to 16S rRNA genes and 16S-23S rRNA gene intergenic spacers sequencing analysis, rep-PCR, AP-PCR fingerprinting and RAPD analysis possessed higher discriminatory power in identifying these closely related strains. And they could be used as rapid and highly discriminatory typing techniques in studying bacterial diversity, especially in differentiating bacteria within Acidithiobacillus caldus.

A 22‐mer primer enhances discriminatory power of AP‐PCR fingerprinting technique in characterization of leptospires

To evaluate the discriminatory power and usefulness of arbitrarily primed-polymerase chain reaction (AP-PCR) characterization of leptospires with M16 primer. AP-PCR fingerprints of 20 reference strains of Leptospira representing 20 different serovars belonging to seven genospecies (Leptospira interrogans, 11; L. noguchii, 2; L. borgpetersenii, 1; L. santarosai, 2; L. biflexa, 2; L. kirschneri, 1; L. weilii, 1) were generated by employing M16 primer. Fingerprints generated with this primer were compared with those generated with two other commonly used primers PB1, and L10. An attempt was also made to type 20 leptospiral isolates with the M16 primer. Fingerprints with M16 primer could not only differentiate between strains of different genospecies, but also between strains of the same genospecies belonging to different serovars. While two commonly used primers (PB1 and L10) failed to discriminate between some of the different serovars belonging to the same genospecies, this primer was able to generate discriminatory fingerprints for all strains tested. All 20 Leptospira isolates, recovered from patients in Andaman Islands, could also be typed by fingerprints generated with the M16 primer. The discriminatory power of M16 primer adds more specificity to the rapidity of this system of characterization and can be used as an excellent tool in epidemiological studies on Leptospira.

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.

Identification of novel breast tumor-specific mutation(s) in the q11.2 region of chromosome 17 by RAPD/AP-PCR fingerprinting

Analysis of genetic instability in breast cancer tissues compared to uninvolved breast tissues from the same individuals by RAPD (random amplified polymorphic DNA)/AP-PCR (arbitrarily primed PCR) fingerprinting using 30 arbitrary primers revealed 190 amplified DNA fragments. Presumably, each of these represents a gene locus in a different region of the genome of breast cancer tissues. Among these amplified DNA fragments, 65 (34.2%) exhibited presence and absence or reductions and enhancements in the intensity in breast cancer tissues compared to uninvolved breast tissues from the same individuals, and 11 amplified DNA fragments (5.7%) represented polymorphisms in the uninvolved human breast tissues. Reductions and enhancements in the intensity of some of the amplified fragments were observed indicating allelic gains or losses in the breast tumor genome compared to the matched uninvolved tissue genome. The presence or absence of some of the amplified DNA fragments were observed in this study indicating homozygous deletions or insertions in the breast tumor DNA compared to the matched uninvolved tissue DNA. Notably, an insertion of a 1270 bp amplified fragment was observed in 81% (17 of 21) of the tumor samples using the primer, OPC04. This amplified fragment resolved into two, 1200 and 1300 bp, single-stranded amplified fragments on the denaturing sequencing gel. This separation into single-stranded fragments suggests that the amplified fragment contains a conformation that is semistable. The 1270 bp amplified fragment localizes to the q11.2 region of chromosome 17. Sequence analysis of this fragment showed a significant DNA base sequence similarity (93%) with one of the breast tumor-specific human EST. The similarity with EST sequences and RT-PCR analysis showed that a part of this amplified fragment is from the coding region of the genome. Any one of the events observed in this study could play an important role in the development of breast cancer or could occur during the clonal expansion of the genetically unstable breast cells.

Polycolonization of Helicobacter pylori among Chinese subjects

To investigate the situation among Chinese patients with regard to infection with multiple strains of Helicobacter pylori. Biopsy specimens for culture of H. pylori were obtained from gastric antrum, body and fundus of 20 patients during endoscopic investigation of upper gastrointestinal symptoms. H. pylori was identified by culture from one site in 16 and two or more sites in 10 of the 16 patients. Five isolated colonies of six strains of H. pylori from gastric antrum were subcultured and used for further analysis. Antibiotic susceptibility to metronidazole and clarithromycin was determined by disk diffusion test. Protein profiles of isolates were compared by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). DNA diversity of the isolates was determined by arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting. Of the 10 patients with multiple isolates, 70% (7/10) exhibited variation in susceptibility to metronidazole and 20% (2/10) to clarithromycin between different sites. In 83% of (5/6) single colonies, no variability was seen in metronidazole and clarithromycin susceptibility; they were either susceptible or resistant. Protein profiles of all isolates by SDS-PAGE were similar. Isolates from different patients produced clearly different AP-PCR fingerprints. In 50% of H. pylori strains isolated from different sites of the stomach, genetic diversity was demonstrated by different AP-PCR fingerprints. In 67% (4/6) strains, five single-colony fingerprints were similar. Genetic variability has been found in H. pylori strains. Individual patients are infected with a single predominant genotype at a single site but can be colonized by multiple strains, and they may show different antibiotic susceptibilities. Individual colonies of the H. pylori population from a single site may not always yield identical DNA fingerprints and antibiotic sensitivities.

AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level γ-radiation

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-doses of γ-radiation was investigated. Male BALB/c mice exposed to 10–50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of “non-parental bands” in DNA-fingerprints from the progeny of males chronically exposed to γ-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F1 generations.

AP-PCR Fingerprinting for Detection of Genetic Damage Caused by Polycyclic Aromatic Hydrocarbons

Exposure of organisms to genotoxic chemicals results in the formation of stable, covalently bound adducts between the chemical (or one of its metabolites) and the DNA, these adducts may cause mutations and cytogenetic changes. The primary effects of such exposure (i.e. adduct formation) and subsequent effects on the DNA (cytogenetic damage, mutation) may be monitored using a number of assays of varying sensitivity and specificity. Recent developments in molecular biology offer new possibilities for detecting DNA damage. We examined whether DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) can reveal differences in the DNA fingerprints of rats and shore crabs exposed to benzo[a]pyrene in the laboratory and of crabs from control and from polluted areas. The results indicate that differences between control and exposed animals were detectable and that DNA fingerprinting by AP-PCR offers a useful alternative biomarker assay for the detection of the genotoxic effects of environmental pollutants.

Site–Specific Oral Colonization of Mutans Streptococci Detected by Arbitrarily Primed PCR Fingerprinting

The clonal diversity and colonization pattern of mutans streptococci within an individual were studied by using a total of 50 tooth site samples obtained from seven 3– to 7–year–old children. Five children contributed saliva samples. From the 7 subjects, 90, 74, 81, 53, 83, 103, and 114 isolates (a total of 598) representing Streptococcus mutans and Streptococcus sobrinus were included for arbitrarily primed polymerase chain reaction (AP–PCR) fingerprinting. The children harbored one to four different AP–PCR types of mutans streptococci. In 3 of the 7 children, the distribution of AP–PCR types of S. mutans differed depending on tooth site (chi–square test for heterogeneity, p<0.001), suggesting that mutans streptococcal clones may selectively colonize specific hard–tissue sites.

Comparison of Arbitrarily Primed PCR, Antibiotic Resistance and Plasmid Profiling for Differentiating Salmonella enteritidis Isolated from Fish

A total of 28 isolates of Salmonella enteritidis isolated from Tilapia (Tilapia mossambica) were investigated through arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting using three primers, plasmid profiling and their antibiotic resistance patterns. All 28 isolates carried at least one plasmid ranging in size from 1.4 to 38 megadalton that enabled the S. enteritidis to be grouped into nine plasmid profiles. Isolates were resistant to nalidixic acid (21.1%), penicillin (39.3%) and streptomycin (71.4%), and were susceptible to carbenicillin, cephalothin, kanamycin, rifampicin and tetracycline. Seventeen isolates (60.7%) were resistant to a single antibiotic and eleven (39.3%) were resistant to two antibiotics; thus separating the isolates into six antibiotic resistance patterns. The AP-PCR results showed that the collection of isolates were genetically very heterogenous. AP-PCR enab led us to differentiate the isolates into 28 AP-PCR types. Analysis derived from these data in combination (AP-PCR, plasmid profiling and antibiotic resistance patterns) showed that all 28 isolates were distinct and were grouped into 28 individual groups. Our results demonstrate that AP-PCR fingerprinting method is more sensitive than plasmid profiling and antibiotic resistance patterns with respect to the individualization of the isolates used in this study.

Amplotyping of microdissected, methanol-fixed lung carcinoma by arbitrarily primed polymerase chain reaction.

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to detect somatic genetic alterations in lung carcinomas. DNA fingerprints generated by a single arbitrary primer were compared between normal and tumor tissues of the same individuals. We adapted the technique to the use of tissue fixed with methanol, which allowed the analysis of small areas of tissue by microdissection. This improvement of the fingerprinting technique permitted the study of tumors at early stages of progression. Loss of sequences from chromosome 7 was detected in 41.7% of adenocarcinomas and from chromosome 22 in 84.6% of small-cell carcinomas. Gains of sequences from chromosomes 1, 8 and 13 were detected in more than 40% of adenocarcinomas and in chromosome 2 in 63.3% of squamous-cell carcinomas. Our results indicate that allelic imbalances at these chromosomal regions are common genetic abnormalities in lung carcinomas. Loss of sequences from chromosome 22q13.3, found in 11 of 13 small-cell carcinomas, were confirmed by microsatellite PCR analysis. We show that the use of our improved AP-PCR fingerprinting permits the detection of both losses and gains of novel chromosomal regions early during lung cancer development. Our results indicate that early-stage tumors tend to have more allelic imbalances than relatively advanced tumors, suggesting a high tumor genetic heterogeneity in the early stages of lung tumor progression.

Incidence of Prevotella intermedia and Prevotella nigrescens carriage among family members with subclinical periodontal disease.

We established a typing system for Prevotella intermedia and Prevotella nigrescens using the combination of PCR ribotyping and arbitrarily primed PCR (AP-PCR) fingerprinting and applied this system to the study of intrafamilial incidence of these species in the oral cavity. PCR ribotyping followed by subtyping by AP-PCR fingerprinting was applied to each type strain of P. intermedia and P. nigrescens and 54 isolates (32 isolates of P. intermedia and 24 isolates of P. nigrescens) from extraoral infections, resulting in an excellent discriminatory power (discrimination index, 0.99) for both species. A total of 18 subjects from six families, with the subjects from each family comprising the mother, the father, and a child who had subclinical early-stage to moderate adult periodontitis or simple gingivitis and who carried P. intermedia or P. nigrescens, or both, were enrolled in the study of intrafamilial carriage. When 20 colonies per specimen of subgingival plaque, if available, were picked from primary culture, 115 P. intermedia and 178 P. nigrescens isolates were recovered from the 18 subjects. Among the subjects studied, family members shared the same subtype strain(s) but non-family members did not. Multiple subtypes were found in 8 (57%) of the 14 P. nigrescens-positive subjects but in only 3 (27%) of the 11 P. intermedia-positive subjects; the difference was, however, not statistically significant (P = 0.14). These results suggest that the combination of PCR ribotyping and AP-PCR fingerprinting is well suited for the epidemiological study of P. intermedia and P. nigrescens and that each family seems to carry a distinct subtype(s) of these species.

Detection of DNA Abnormalities by Arbitrarily Primed PCR Fingerprinting: Amplification of theMDM2Gene in a Mediastinum Fibrosarcoma

Arbitrarily primed PCR (AP-PCR) fingerprinting method is easy and useful for analysis of genetic alterations in anonymous chromosomal regions. We applied this technology to analysis of DNA from human primary cancers and found amplification of a DNA fragment in a mediastinum fibrosarcoma. PCR-based analysis of radiation hybrid panels following cloning and nucleotide sequence determination of the fragment revealed that it was derived from a region of chromosome 12q13-q15. In this region, theMDM2andIFNGgenes were noted as known genes that could be involved in human carcinogenesis. Southern blot analysis of genomic DNA of the tumor revealed the amplification of theMDM2gene together with the fragment locus, but not theIFNGgene. Our results demonstrated that detection of DNA alterations by AP-PCR fingerprinting without any previous knowledge of the genes and subsequent analysis of radiation hybrid panels could lead to easy identification of candidates for genes involved in carcinogenesis.

18] Identification of differentially expressed genes using RNA fingerprinting by arbitrarily primed polymerase chain reaction

Genomic fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a sensitive and efficient method for generating a large number of molecular markers. Based on the selective amplification of DNA sequences flanked, by chance, by sequences matching an arbitrarily chosen primer, AP-PCR reveals sequence polymorphisms between different template DNAs. The ability to detect differences between fingerprints of closely related organisms made this approach a useful tool for studying genetic diversity, population biology, epidemiology, and genetic mapping. The more recent application of AP-PCR fingerprinting to RNA, differential display, or RNA-arbitrarily primed PCR (RAP-PCR) has resulted in an interesting tool for the detection of differential gene expression. This chapter focuses on the use of RAP-PCR fingerprinting. It describes the method and provides the present protocols for the different steps from initial RNA preparation through sequence analysis and confirmation of differential expression of the transcripts identified.

Detection of DNA Abnormalities by Arbitrarily Primed PCR Fingerprinting: Allelic Losses in Chromosome 10q in Lung Cancers

DNA fingerprinting using arbitrarily primed PCR (AP-PCR) is useful for detecting cancer-specific DNA aberrations without targeting any particular genes or knowing any nucleotide sequences in advance. AP-PCR fingerprinting is an efficient method for finding loss of anonymous chromosomal regions in cancers. We analyzed DNA from 44 human non-small cell lung cancers by fingerprinting using a single primer and found a loss of signal intensity in a DNA fragment amplified from chromosome 10 (fragment F) in 15 tumors. The detailed location of the fragment F locus on chromosome 10q was determined by PCR-based analysis of radiation hybrid panels using a sequence-tagged site established for the fragment. In 12 of the 15 tumors, loss of the signal detected by AP-PCR fingerprinting was in agreement with the results obtained by analysis of allelic imbalances using 7 polymorphic CA-microsatellite DNA markers for loci around the fragment F locus (p=0.0009). We conclude that a hitherto unknown suppressor gene for lung cancer resides at 10q in the vicinity of fragment F.

Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR.

Reference strains from 30 serovars representing seven species of Leptospira and 48 recent isolates from human patients, dogs and rats, were characterised by polymerase chain reaction-restriction endonuclease analysis (PCR-REA), arbitrarily primed PCR (AP-PCR) and low stringency PCR (LS-PCR). PCR-REA analysis yielded seven groups among 29 serovars of pathogenic Leptospira; the non-pathogenic L. biflexa serovar patoc was not amplified with the primer pairs studied. AP-PCR and LS-PCR fingerprinting resulted in 25 and 21 distinct profiles, respectively, among the 30 reference strains. The results of the three PCR-based techniques were highly concordant and were in general agreement with those from previous DNA studies, confirming the high level of polymorphism among Leptospira species and serovars, and supported the concept of the serovar as the basic taxonomic unit of leptospiral classification. Results of the PCR-based typing methods for 11 randomised leptospiral strains, 36 clinical isolates from human patients and dogs and 12 survey isolates from trapped rats agreed with those from serological identification. With one exception, isolates of the same serovar gave identical profiles irrespective of the source. AP-PCR and LS-PCR are simple to perform and interpret, and appear to be useful for characterising isolates of Leptospira spp. for diagnostic and epidemiological purposes.

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune was studied using PEG-induced fusion. The fusion of protoplasts from auxotrophic mutant strains resulted in the formation of fusion hybrids in the frequencies of 3.6 to 7.3×10(-5). Most of these fusion hybrids were monokaryotic and sterile and no heterokaryosis occurred. Most fusants showed a significantly higher nuclear DNA content when compared to parental strains and no diploids (parent 1 genome plus parent 2 genome) were found. Some fusion hybrids revealed both parental fragments in nuclear and mitochondrial rDNA PCR profiles. AP-PCR (Arbitrarily-primed Polymerase Chain Reaction) fingerprints also indicated that most of the fusion products were recombinant hybrids.

Chromosomal Assignment of Human DNA Fingerprint Sequences by Simultaneous Hybridization to Arbitrarily Primed PCR Products from Human/Rodent Monochromosome Cell Hybrids

We have developed a technique for the simultaneous chromosomal assignment of multiple human DNA sequences from DNA fingerprints obtained by the arbitrarily primed polymerase chain reaction (AP-PCR). Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylon membrane. Human-specific hybridization bands in the human/rodent fingerprints unambiguously determine their chromosome of origin. We named this method simultaneous hybridization of arbitrarily primed PCR DNA fingerprinting products (SHARP). Using this approach, we determined the chromosomal origins of most major bands of human AP-PCR fingerprints obtained with two arbitrary primers. Altogether, the chromosomal localization of near 50 DNA fragments, comprehensive of all human chromosomes except chromosomes 21 and Y, was achieved in this simple manner. Chromosome assignment of fingerprint bands is essential for molecular karyotyping of cancer by AP-PCR DNA fingerprinting. The SHARP method provides a convenient and powerful tool for this purpose.

Detection of microsatellite instability in cancers by arbitrarily primed-PCR fingerprinting using a fluorescently labeled primer (FAP-PCR).

The microsatellite mutator phenotype (MMP), detected as a change in the number of repeating units in hundreds of thousands of microsatellite sequences in the tumor cell genome, underlies the carcinogenesis of a variety of tumors including sporadic and hereditary nonpolyposis colon cancers. This enhanced microsatellite instability was discovered using arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting of DNA from colon cancers. In this study, we found an arbitrary primer that can amplify multiple DNA fragments containing repeated sequences, including the poly A tracts found in the Alu repeats of the human genome. The combined use of primer labeling with fluorescence and an automated DNA sequencing analysis of AP-PCR products (FAP-PCR) detected alterations in fingerprint bands in all DNA samples previously determined to belong to the MMP. Fluorescent AP-PCR fingerprinting using this single arbitrary primer provides a convenient and efficient method for detecting tumor specific fingerprint alterations that are usually undetectable by conventional fingerprinting.

Nonradioactive Arbitrarily Primed Polymerase Chain Reaction: A Novel Technique for Detecting Genetic Defects in Skin Tumors

Initiation and progression of melanocytic and non-melanocytic skin tumors are accompanied and probably caused by a variety of genetic defects. In contrast to other human tumors, however, limited amounts of available tissue in skin cancer often hamper extensive genetic studies of native material of early lesions. Therefore, we applied a novel DNA fingerprinting technique based on polymerase chain reaction (PCR), the arbitrarily primed PCR (AP-PCR). This technique enabled us to scan large parts of the genome (about 30 kb/PCR reaction) for somatic mutations starting with minute amounts of tissue. In contrast to previous reports on AP-PCR, we were able to visualize PCR products by a rapid nonradioactive silver-staining technique using a simple device for staining of large polyacrylamide gels. In nine benign and malignant melanocytic skin tumors, the method provided a set of reproducible DNA fingerprints. Genetic defects were detected by comparing the fingerprints of tumor cells and constitutive DNA from blood leukocytes. Since nonradioactive AP-PCR fingerprinting also offers the unique capability to isolate and sequence polymorphic DNA fragments from fingerprint gels, we conclude that this technique seems to be important and practically feasible for elucidating the genetic roots of skin tumors.