Single Restriction Fragment Length Polymorphism Research Articles

RT-PCR-RFLP for genetic diversity analysis of the citrus tatter leaf virus strain of Apple stem grooving virus

A method for detecting genetic diversity of the Citrus tatter leaf virus (CTLV) strain of Apple stem grooving virus was developed based on restriction fragment length polymorphisms (RFLP) of the 889 nt 3′ sequence amplified by RT-PCR, and nine Hinf I RFLP patterns were defined. This RFLP assay, together with biological indexing and sequencing, was applied to characterize 18 CTLV isolates collected from seven provinces of China. The results indicated that the majority of these isolates (16/18) presented as single well-defined RFLP patterns, among which RFLPIand RFLPII were dominant. In contrast, two samples exhibited as a mixture of two RFLP patterns, suggesting a mixed infection of CTLV variants. Of the 18 isolates, six of eight samples with RFLPIIor RFLP III induced mild to moderate symptoms on indexing plants (Citrus. sinensis × Poncirus trifoliata cv. Rusk), while four isolates conforming to RFLPIand six samples to RFLP IV ~ IX induced severe symptoms. In the phylogenetic tree constructed according to the 3′ nucleotide sequence of the 18 CTLV isolates, RFLPIIand RFLP III samples clustered into phylogenetic group A, whereas the others gathered into phylogenetic group B. These results revealed correlation among mild (moderate) symptoms, phylogenetic group A and RFLPIIor RFLP III restrictotypes, suggesting the RFLP assay may be useful in quick identification of CTLV strains.

Population structure of Sclerotinia sclerotiorum on sunflower in Australia

Sunflower (Helianthus annuus) is host to infections by Sclerotinia sclerotiorum originating from either homothallic sexually-derived ascospores (stem and head rots) or asexually-derived sclerotia (root rot). While sunflower can be infected by either ascospores or sclerotia this study found no association between the genotypes found in lesions and the type of infection (stem, head or root rot). Multicopy Restriction Fragment Length Polymorphisms (RFLPs) showed individual sclerotia comprised of only one genotype, and that all eight ascospores within an ascus also had only one genotype. Mycelial Compatibility Groups (MCGs), Random Amplified Polymorphic DNAs (RAPDs), single and multicopy RFLP analyses all showed the majority of sunflower plants were infected by only one genotype. A sample of 250 isolates collected hierarchically from sunflowers in Queensland and New South Wales were shown to belong to one large genetic population of S. sclerotiorum. Temporal studies revealed genetic uniformity was maintained across years, further confirming one genetic population. A range of molecular markers were used to genotype 120 isolates, resulting in differing levels of resolution of a genotype. Between 13 and 24 genotypes were identified with similarities and differences in the assemblages of isolates within each genotype depending on the marker used.

Preliminary Studies on CPG/ Hinf I RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus (CTV) causes economically important losses to the citrus industry worldwide. Mild strain cross protection (MSCP) against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces (Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi) were tested by direct tissue blot immuno-assay (DTBIA), and 158 of them were tested positively, which therefore were subjected to coat protein gene (CPG)/ Hinf I restriction fragment length polymorphism (RFLP) analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/ Hinf I RFLP groups III and I are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/ Hinf I RFLP groups III and I caused severe stem-pittings in sweet oranges in China.

PCR-RFLP using Ssu-rDNA amplification: applicability for the diagnosis of mixed infections with different trypanosome species in cattle

The use of a single restriction fragment length polymorphism (RFLP)-PCR assay which is able to characterise all important bovine trypanosome species was evaluated for the detection of mixed infections with Trypanosoma brucei brucei, Trypanosoma theileri, Trypanosoma congolense and Trypanosoma vivax. Results showed that mixed infections are detectable at a minimum ratio of 2%/98% of standardised DNA solutions with a concentration of 10 ng ml −1. All mixed infections gave clear profiles that could be easily differentiated except with T. theileri and T. congolense where the T. theileri band was concealed by the T. congolense profile.

Molecular analysis of incHI1 antimicrobial resistance plasmids from Salmonella serovar Typhi strains associated with typhoid fever.

The first outbreak of multidrug-resistant (MDR) typhoid fever in Vietnam was in 1993, and by 1995 nearly 90% of cases were MDR. Plasmid HCM1, sequenced in full, is an incHI1 plasmid from Salmonella enterica serovar Typhi strain CT18, isolated in Vietnam in 1993. Restriction analysis shows that pHCM1 shares a restriction fragment length polymorphism (RFLP) pattern with plasmids isolated from the first outbreak and 10 of 17 MDR plasmids isolated from sporadic cases occurring at the same time in Vietnam. A core region of pHCM1 has significant DNA sequence similarity to plasmid R27, isolated in 1961 from S. enterica in the United Kingdom. There are five regions of DNA in pHCM1 which are not present in R27. Two of these are putative acquisition regions; the largest is 34.955 kbp in length and includes sequences of several antibiotic resistance genes and several insertion sequences. The borders of this region are defined by two identical IS10 left elements, associated with an inversion of DNA or with a truncated Tn10 element. The second, smaller region is 14.751 kbp and carries a trimethoprim resistance gene dfr14A cassette associated with a class 1 integrase. In 1993 to 1994, restriction analysis revealed some variations in the structures of Salmonella serovar Typhi MDR plasmids which were mapped to the two putative acquisition regions and three smaller variable regions. In 1996 a single RFLP type, RFLP7, was found to carry the dfrA7 and sul-1 genes, which were not present on R27 or pHCM1. This plasmid type appears to have a selective advantage over other plasmids with the same resistance phenotype.

Fine analysis of the Pneumocystiscarinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis

Pneumocystiscarinii is a general designation for a group of unusual unicellular fungal parasites responsible of pneumopathy in animal hosts. Divided into several subgroups termed the ‘special forms’, P. carinii is prone to an extensive karyotype variation. In previous studies, the nuclear genome of these organisms has been considered to be haploid and a set of 16 chromosomes has been assigned to P. carinii f. sp. carinii, a special form known to infect rats. We report the analysis of the genome of an isolate representative of the karyotype 1 of this special form, using two-dimensional pulsed-field gel electrophoresis procedures. The ‘karyotype and restriction display’ (KARD) fingerprints indicated the presence of 17 different chromosomes. The haploid genome size was estimated to be 8.4 Mbp. Some homologous chromosomes were distinguished on the basis of a single restriction fragment length polymorphism, which raises the possibility of a diploid nucleus. A restriction map of the chromosome 15, characterized by two homologues with a size difference of 7 kb, was constructed. Hybridization data indicated that insertion/deletion events may have occurred within subtelomeric regions which carry genes encoding the major surface glycoprotein (MSG) of Pneumocystis.

A new DNA extraction method for high‐throughput marker analysis in a large‐genome species such as Triticum aestivum

AbstractGene mapping and marker‐assisted selection in complex, polyploid genomes still relies strongly on restriction fragment length polymorphism (RFLP) analysis, as conversion of RFLP to polymerase chain reaction (PCR) markers can be very difficult. DNA extraction in amounts suitable for RFLP analysis represents the most time‐consuming and labour‐intensive step in molecular marker analysis of plant populations. In this paper, a new flexible method for plant DNA extraction is presented. It allows a high‐throughput of samples in a short time without the need for freezing or lyophilizing the plant material. The method allows the isolation of genomic DNA with a yield of 100 μg for a minimal amount of 200 mg of leaf material. This is sufficient for work with large‐genome plant species such as hexaploid wheat, where 20 μg of genomic DNA are required for a single RFLP analysis.

Differentiation of the sudden death syndrome pathogen of soybean, Fusarium solani f.sp. glycines, from other isolates of F. solani based on cultural morphology, pathogenicity, and mitochondrial DNA restriction fragment length polymorphisms

Forty-four isolates consisting of Fusarium solani (Mart.) Sacc. f.sp. glycines Roy, Fusarium solani f.sp. phaseoli (Burkholder) W.C. Snyder & H.N. Hans., and F. solani, collected from a variety of hosts and locations, were compared based on pathogenicity on soybean and mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs). The 24 isolates of F. solani f.sp. glycines caused more severe sudden death syndrome (SDS) foliar symptoms and root rot on soybean compared with all other isolates. All isolates of F. solani f.sp. glycines belonged to a single mtDNA RFLP haplotype. The other isolates of F. solani belonged to nine mtDNA RFLP haplotypes distinct from that of the SDS pathogen and did not cause significant levels of SDS. Three isolates of F. solani f.sp. phaseoli in a single mtDNA RFLP haplotype were very similar to isolates of F. solani f.sp. glycines in culture. One of these isolates consistently produced SDS-like symptoms in pathogenicity tests, although at a low frequency. Fusarium solani f.sp. glycines represent a genetically distinct subgroup within F. solani but may be related to F. solani f.sp. phaseoli.Key words: Fusarium solani f.sp. phaseoli, Glycine max, sudden death syndrome (SDS), mtDNA.

Genomic variability of equine herpesvirus-5.

Seventeen New Zealand isolates of equine herpesvirus 5 (EHV-5) were compared to the Australian prototype strain. PCR primers were designed to amplify EHV-5 glycoprotein B (gB) gene, and Restriction Fragment Length Polymorphism (RFLP) was used to detect differences between cloned PCR products. EHV-5 isolates from different horses showed a high degree of heterogeneity. However, EHV-5 isolates from individual horses remained homogeneous when examined over a period of time or isolated from different sites. A single EHV-5 gB RFLP profile was detected in isolates from each individual horse but one. Two or possibly three different genotypes of EHV-5 were detected in cultures inoculated with a nasal swab of this horse. The heterogeneity observed between EHV-5 isolates from different horses suggests that the use of RFLP may provide a useful epidemiological approach to gain more knowledge about the biology of EHV-5.

The genetic algorithm applied to haplotype data at the LDL receptor locus

Conventional statistical methods based upon single restriction fragment length polymorphisms often prove inadequate in studies of genetic variation. Cladistic analysis has been suggested as an alternative, but requires basic assumptions that usually cannot be met. We wanted to test whether it could be a workable approach to apply the genetic algorithm, an artificial intelligence method, to haplotype data. The genetic algorithm creates in-computer artificial ‘individuals’, all having ‘genes’ coding for solutions to a problem. The individuals are allowed to compete and ‘mate’, individuals with genes coding for better solutions mating more often. Genes coding for good solutions survive through generations of the genetic algorithm. At the end of the run, the best solutions can be extracted. We applied the genetic algorithm to data consisting of cholesterol values and haplotypes made up of seven restriction sites at the LDL receptor locus. The persons included were 114 FH (familial hypercholesterolemia) patients and 61 normals. The genetic algorithm found the restriction sites 1 (Sph1 in intron 6), 2 (StuI in exon 8), and 7 (ApaLI site in the 3′ flanking region) were associated with high cholesterol levels. As a validity check we used runs of the genetic algorithm applied to ‘artificial patients’, i.e. artificially generated haplotypes linked to artificially generated cholesterol values. This demonstrated the genetic algorithm consistently found the appropriate haplotype. We conclude that the genetic algorithm may be a useful tool for studying genetic variation.

Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli

Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692–7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca 2+-binding motifs predicted from the primary sequence, together with the Ca 2+-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.

Isolation, Characterization, and Distribution of a Biocontrol Fungus from Cysts of Heterodera glycines

ABSTRACT Seventy-six populations of Heterodera glycines were collected from 33 counties in 10 states of the United States along the Mississippi and Missouri Rivers in 1992 and 1993. A sterile hyphomycete fungus of an unnamed taxon, designated ARF18 and shown to be a parasite of eggs of H. glycines, was isolated from eggs and cysts of 10 of the populations from Kentucky, Louisiana, Mississippi, and Tennessee. Ten isolates of ARF18 obtained in this study and seven isolates obtained in earlier studies were characterized for cultural morphology on several growth media, the ability to produce sclerotium-like structures (SLS) on cornmeal agar, growth rates, pathogenicity to eggs of H. glycines in vitro, and mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs). All 17 isolates of ARF18 readily grew on potato dextrose agar, cornmeal agar, and nutrient agar. Based on colony morphology and SLS appearance on cornmeal agar, the isolates could be grouped into two morphological phenotypes. Isolates that produced SLS that were composed of a compact mass of hyphae were designated ARF18-C, whereas isolates that produced SLS composed of a mass of loosely clumped hyphae were designated ARF18-L. Only minor differences in growth rates were detected among the ARF18-C and ARF18-L isolates. All 10 ARF18-C isolates, which were from Arkansas, Louisiana, Mississippi, and Tennessee, belonged to a single mtDNA RFLP haplotype. The seven ARF18-L isolates shared many comigrating mtDNA restriction fragments with one another, but belonged to three distinct mtDNA RFLP haplotypes. Ability to infect eggs of H. glycines in vitro varied considerably among the various isolates of ARF18. In particular, several of the ARF18-C isolates were consistently able to infect over 50% (mean = 70.0%, standard deviation = 16%) of the eggs of H. glycines, whereas ARF18-L infected eggs to a lesser degree (mean = 25%, standard deviation = 27%). ARF18-C was isolated only from H. glycines populations from below 37 degrees N latitude. The presence of ARF18 was associated with soils with Mg levels <314 kg/ha, cyst numbers >4.5 per 100 cm(3), and iron levels >203.5 kg/ha; or with Mg levels >314 kg/ha and Na levels <121 kg/ha. The widespread distribution of ARF18 and the ability of some isolates to aggressively colonize eggs of H. glycines are indications that it has potential as a biological control agent for H. glycines.

Genetic variability in white leghorns revealed by chicken liver expressed sequence tags

A total of 92 expressed sequence tags from chicken liver (CLEST) were searched for homology with known genes. Among the CLEST, 29% had no sequence similarities with known genes, 34% showed sequence similarity to rRNA, 9% to mitochondrial genes, 23% to known nuclear genes, and 5% to human expressed sequence tags. Among the nuclear CLEST (excluding rRNA), clones with sequence similarity to aldolase B were represented four times, whereas all the other clones represented unique genes. The presence of MspI and TaqI restriction fragment length polymorphisms (RFLP) associated with CLEST were analyzed by bulk Southern blotting in 16 strains of White Leghorn chickens derived from five different genetic bases. No RFLP were observed with rRNA CLEST and a single MspI RFLP was observed with mitochondrial CLEST. The nuclear CLEST with sequence similarity to known nuclear genes were grouped into two classes on the basis of their involvement in intermediary metabolism. Among the nine genes coding for metabolic enzymes, all but one were polymorphic at MspI and/or TaqI sites in at least one of the strains, whereas among the other genes six of nine were polymorphic. The average frequency of clones revealing RFLP per cDNA clone and restriction enzyme for the two classes were 0.7 and 0.3, respectively. The analysis indicated that in White Leghorns, RFLP markers in the vicinity of nuclear CLEST are relatively frequent. Further, RFLP in the vicinity of genes coding for metabolic enzymes were significantly more frequent than near genes coding for other proteins.

Trait association of genetic markers in the growth hormone and the growth hormone receptor gene in a White Leghorn strain

Alleles of the growth hormone (GH) gene and GH receptor (GHR) gene were analyzed for association with juvenile body weight (HBWT), age at first egg (AFE), the hen-day rate of egg production (HDR), egg specific gravity (SPG), and egg weight (EWT) in a strain of White Leghorns. The particular strain segregated at near equal frequencies for two GH alleles defined by differences at three restriction fragment length polymorphisms (RFLP) and for two GHR alleles defined by a single RFLP. The GH genotype was significantly associated with AFE (P < or = 0.04) as well as HDR from 274 to 385 d (P < or = 0.04) and 386 to 497 d (P < or = 0.0003). The GHR genotype (haploid in female chickens) had trends for association with HBW (P < or = 0.06) and HDR from AFE to 273 d (P < or = 0.07). The effects on the egg quality traits SPG and EWT were not significant. Regression analysis revealed that HDR was associated negatively with AFE and positively with HBWT. The slope of the regression line of HDR on AFE varied with the GH genotype, with the effect that the differences in HDR between GH genotypes was relatively small in chickens with early AFE and large in chickens with late AFE. Similarly, the slope of the regression of HDR on HBWT varied between GHR genotypes, with the result that the effect of the GHR genotype on HDR in chickens with low HBWT was opposite to its effect in chickens with high HBWT. The complex relationship between genotypes and traits may reflect gene interaction and indicates that simple models based on additive gene effects may not be adequate for the dissection of the genetic architecture of quantitative traits.

Desaturase multigene families of Brassica napus arose through genome duplication

This paper reports the estimated gene copy number and restriction fragment length polymorphism (RFLP) map locations of five different desaturase cDNA clones from Brassica napus (oilseed rape). The desaturase enzymes encoded by four of these genes catalyze successive reactions that insert double bonds into lipid-linked fatty acid residues. Delta-12 (e2) and delta-15 (e3) desaturases are active in the endoplasmic reticulum, while omega-6 (p2) and omega-3 (p3) desaturases catalyze analogous desaturation reactions via a parallel pathway located in plastids. The fifth cDNA clone (b5) contains a desaturase-like domain bound to a cytochrome b5 segment. Estimates of gene copy number based on Southern blot analysis of 16 oilseed rape varieties and three different resynthesized Brassica napus lines indicated that e2 had 4–6 gene copies and e3, p2, p3 and b5 each had 6–8 gene copies per haploid genome. Estimates of the gene copy number for the two progenitor species, Brassica oleracea and Brassica rapa, supported the premise that all these genes were at least duplicated or triplicated in the two progenitor species before they combined to form B. napus. RFLP mapping results showed that the e2 probe detected 4 distinct loci, the e3 probe 6 loci and p2, p3 and b5 each detected 8 loci, with pairs of loci often mapping to homoeologous regions on 2 different linkage groups. The 28 mappable loci were distributed across 12 linkage groups of the B. napus map (Parkin et al. 1995) and were usually represented by single RFLP fragments. A collinear segment containing the e2 and p3 loci was positioned on B. napus linkage groups N1, N11, N3, N13, N5 and N15. This segment was collinear with a 30-cM region of Arabidopsis thaliana chromosome 3 that contains the homologous fad2 (e2) and fad7(p3) genes. This suggests that the desaturase multigene families arose as the result of duplication of large chromosome segments rather than duplication of individual genes.

Pod Dehiscence of Soybean: Identification of Quantitative Trait Loci

The dehiscence of pods (shattering) prior to harvest is an undesirable trait of soybean, Glycine max (L.) Merr. Pod dehiscence (PD) is relatively uncommon in modern North American soybean cultivars, but is often observed when unimproved germplasm or the wild species, G. soja Siebold & Zucc., are used as parents to introgress useful genes or to develop genetically diverse breeding populations. In light of the potential for efficient selection using DNA markers, the objective of this study was to identify quantitative trait loci (QTL) that condition resistance to PD. A map of 140 linked restriction fragment length polymorphism (RFLP) markers was constructed using 120 F4-derived lines from a soybean population (Young x PI 416937) that segregated for resistance to PD. These lines were scored for PD on a visual scale of 1 to 10 at both Athens, Georgia, and Windblow, North Carolina, in 1994. Heritability of pod dehiscence was 92%. Associations of marker loci with QTL that condition resistance to PD were tested using homozygous RFLP class means in a single-factor ANOVA. A total of five putatively independent RFLP markers were associated with PD at both locations and in a combined analysis over locations. A single RFLP locus on linkage group J of the USDA/Iowa State University map accounted for 44% of the variation in PD score. Epistasis was observed between one pair of significant marker loci. These results establish the genomic location of one major and a few minor QTL, identify an epistatic interaction, and indicate transgressive segregation which is plausibly the result of susceptibility alleles contributed by the resistant parent.

DNA Analysis on Forensic Science

Since DNA analysis technology was introduced to forensic science field in 1985, the effective application of this technology to evidential samples such as bloodstains, body fluid stains and the tissue samples, has been a matter of great interest of forensic science laboratories in the world. As a first step of the application of the DNA analysis to evidential samples, single locus VNTR (Variable Number of Tandem Repeat) was investigated using specific probes to each locus (YNH24, CMM101, MS1, MS32 etc.) and hybridization methods. Since Saiki et al. reported the PCR (Polymerase Chain Reaction) amplification techniques using Taq polymerase, the adoption of the PCR-based DNA typing systems such as HLADQα, AMPFLP (Amplified Fragment Length Polymorphisms), and STR (Short Tandem Repeat) has become very common in the forensic community. Now, many reports concerning single locus RFLP (Restriction Fragment Length Polymorphism)-VNTR, AMPFLP (MCT118), HLADQα, Polymarker and STR have been widely published in the scientific journal and a reasonable combination of these systems has commonly been used for identifying evidential samples. Moreover, the guideline of a quality assurance for DNA analysis has been published from the national forensic science laboratory of each country (FBI in USA, FSS in UK etc.). Recently, the validation studies and the population studies have been performed with respect to STRs such as TH01, CSF1PO, vWA, FES/FPS etc. Studies on the technical strategy for applying mitochondrial DNA analysis to evidential samples and on the application of DNA analysis to ABO blood group are now progressing in the forensic science laboratories in the world.

DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex

Nineteen reference and 156 clinical strains of the genus Nocardia belonging to 12 taxonomic groups were studied for restriction fragment length polymorphism (RFLP) by using an amplified 439-bp segment of the 65-kDa heat shock protein gene. Of 30 restriction endonucleases, digestion with MspI and then digestion with BsaHI produced RFLP band patterns which separated all 12 groups except N. asteroides type IV from 6 of 12 N. transvalensis isolates and N. carnea from the N. asteroides type VI isolates. Commonly encountered species such as N. nova, N. farcinica, N. brasiliensis sensu stricto, and N. otitidiscaviarum were easily separated. Each taxon resulted in a single RFLP band pattern that included > or = 96% of all biochemically grouped isolates for 9 of 12 taxa with MspI and for 8 of 12 taxa with BsaHI. With the use of both patterns, only 6 of 175 (3.4%) isolates failed to fit the biochemically defined group patterns. These studies provide the first evidence for the separate identities of four antibiogram-defined (but currently unnamed) groups within the N. asteroides complex (types I, II, IV, and VI) and the presence of two subgroups within N. transvalensis. They also provide genotypic evidence for the separate identities of N. nova and N. farcinica. The lack of BstEII recognition sites in amplicons obtained from nocardiae provides a simple and rapid method for the differentiation of nocardiae from mycobacteria. DNA amplification with RFLP analysis is the first rapid method that distinguishes all clinically significant taxa and recognized species within the genus Nocardia.

Analysis of methicillin-resistant and methicillin-susceptible Staphylococcus aureus by a molecular typing method based on coagulase gene polymorphisms.

A molecular typing method for Staphylococcus aureus based on coagulase gene polymorphisms (coagulase gene typing) was evaluated by examining a total of 240 isolates which comprised 210 methicillin-resistant S. aureus (MRSA) and 30 methicillin-susceptible S. aureus (MSSA) collected from a single hospital. By AluI restriction enzyme digestion of the PCR-amplified 3'-end region of the coagulase gene including 81-bp repeated units, the MRSA and MSSA isolates examined were divided into 6 and 12 restriction fragment length polymorphism (RFLP) patterns, respectively, whereas five patterns were commonly detected in MRSA and MSSA. MRSA isolates that showed a particular RFLP pattern were considered to be predominant in the hospital. Coagulase typing with type-specific antisera was also performed for all S. aureus isolates for comparison. Coagulase types II and VII were most frequently detected and included isolates with four and five different AluI RFLP patterns, respectively, whereas each of the other coagulase types corresponded to a single RFLP pattern. These results indicated that RFLP typing was more discriminatory than serological typing, for typing S. aureus and demonstrated its utility in epidemiologic investigation of S. aureus infection in hospitals.

DNA Typing in Forensic Science

In the last few years, DNA typing procedures have become increasingly important in the fields of forensic science and forensic medicine. This paper reviews background information on DNA and human genetics, and addresses how molecular techniques such as restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analysis have been used to detect genetic polymorphism in human populations. The systems discussed include single locus RFLP, HLA DQ-alpha, amplified fragment length polymorphism (AMP-FLPs), short tandem repeats (STRs), and mitochondrial DNA typing. Several DNA typing methods have been thoroughly validated for forensic use. With proper control measures, DNA analysis should be considered reliable. At this time, DNA evidence/testimony is generally accepted by the courts and greatly assists in the resolution of criminal and civil investigations.