Common Restriction Fragment Length Polymorphism Research Articles

Association of Matrix Metalloproteinase-2 Gene Promoter Polymorphism and the Associated Phenotype Variation with Myocardial Infarction

In many developing countries Myocardial infarction has Becoming a major problem in public health 1,2. MI is a multifactorial Disease caused by genetic and environmental factors.The major cause of Death in the world is Myocardial infarction 3. The high plasma lipid Levels, high plasma glucose levels,high blood pressure, obesity, smoking, And family history of cardiac disease are the most important risk factors For MI. MI is mainly due to atherosclerosis of the coronary arteries . The structural changes, which permits the accumulation of cells, Extracellular matrix and lipids in the intimate layer of the diseased artery And allows the growth of atherosclerotic plaque. The fibrous cap lining Atheromatous plaque gets ruptured, gives rise to thrombosis, and its Complications4. Pathophysiology of MI involves a wide variety of proteins, Including the matrix metalloproteinases (mmps).Atheromatous plaque Formation is facilitated by the action of mmps. Major extra cellular Components of the basal lamina around blood vessels such as type 1V Collagen, laminin, and fibronectin are degraded by MMP-2. Mmps Also weakens the arterial wall, resulting destabilizing of atheromatous Plaque and dissolution of fibrous cap leading to MI5. Matrix metalloproteinases are zinc dependent endopeptidases That degrade components of the extracellular matrix (ECM). 72kda type IV collagenase also known as MMP-2, is an ubiquitous Metalloproteinase involved in various functions such as vascular Remodeling, atheromatous plaque rupture, and degrading matrix Proteins. MMP-2, also known as gelatinase A . In human, it is Encoded by the MMP-2 gene6. MMP-2 is produced as zymogen, after the production pro MMP- 2 is thought to be bound to its specific inhibitor, called as tissue Inhibitor of matrix metalloproteinases-2 . There are several pathways for Activation of the proenzyme, but mainly the most important pathway is Activation by membrane type metalloproteinases-1 (MT1-MMP)7. MTIMMP Binds to TIMP-2,this complexed structure comes near to the Active site of the MT-MMP enzyme. This results to removal of two Specific pro peptides from pro MMP-2 and the production of an Active 72 kda MMP-2 enzyme8. The gene for human MMP-2 contains 27,862 basepair genomic DNA and is composed of 13 exons. It has been localized on chromosome 16q219. Several common restriction fragment length polymorphisms (rflps) have been reported in the MMP-2 gene locus. MMP-2 gene – 1306C>T promoter region is linked with development of MI10. This Base transition is situated in CCACC box of the sp1 binding site. Increased MMP-2 levels have been found in the plasma of Patients with MI11. Elevated MMP-2 has also been found in Atherosclerotic plaques of coronary arteries12. In view of this we have evaluated the distribution of MMP-2 Promoter gene polymorphism by PCR- RFLP and the concerned Phenotype (MMP-2) was analyzed by using ELISA.

Correlation of restriction fragment length polymorphism genotyping with internal transcribed spacer sequence, randomly amplified polymorphic DNA and multilocus sequence groupings for Candida parapsilosis

Epidemiological studies of Candida parapsilosis have been performed by molecular methods. To compare two prominent methods, 29 isolates, typed by multilocus sequence typing (MLST), were typed by restriction fragment length polymorphism (RFLP). Of the 19 proposed Candida parapsilosis sensu stricto isolates [group I by internally transcribed spacer (ITS1) sequence], the most commonly encountered species, 17 were RFLP type VII-1. The species Candida orthopsilosis (eight isolates) and Candida metapsilosis (two isolates) consisted of five and one other RFLP types, respectively; none were VII-1. None of the non-VII-1 types were in more than one ITS group. VII-1 is the most common RFLP type (176/203 in continuing studies), and C. parapsilosis sensu stricto is similarly dominant in other studies, and cannot be subtyped by RFLP or MLST. RFLP subtype VII-1 and C. parapsilosis sensu stricto appear to be nearly identical; C. orthopsilosis, which can be subtyped by MLST, can also be subtyped by RFLP. C. metapsilosis appears rarely.

Comparison of the community structure of planktonic bacteria in ballast water from entry ships and local sea water in Xiamen Port

In this study, the bacterial community structures in samples of ballast water collected from a ship from Singapore and of local sea water collected from Xiamen Port were compared using restriction fragment length polymorphism (RFLP) and 16S rDNA sequence analysis. Except for dominant α-Proteobacteria that are common to both systems, the bacterial community structures of the two systems were quite different. Most of the clones derived from the different systems were grouped into different phylogenetic clusters, and the systems share only one common RFLP pattern. The ballast water, which is likely from clean offshore waters, contains sequences specific to α- and γ-Proteobacteria. Phylogenetic analysis revealed that the ballast water contained sequences belonging to attached bacteria and bacteria commonly found in the open sea, as well as many novel sequences. In addition, no known pathogenic bacteria were detected in the ballast water samples. Conversely, water samples from Xiamen Port were apparently affected by the near shore environments. Specifically, in addition to α- and γ-Proteobacteria, water from Xiamen Port contained β- and δ-Proteobacteria, Synechococcus, Bacteroidetes and Actinobacteria, which are common in coastal environments. Additionally, four pathogenic bacterial sequences and one plasmid sequence of a potential red tide forming alga were detected in the water from Xiamen Port, which suggests that the local sea water is polluted. The results of this study can be used as background information to assess the risk associated with the introduction of non-indigenous species to local systems and to establish ballast water management systems.

Mutations in the mitochondrial DNA D-loop region and breast cancer risk

Mitochondrial genome alterations have been suggested to play an important role in carcinogenesis. The D-loop region of mitochondrial DNA (mtDNA) contains essential transcription and replication elements, and mutations in this region may serve as a potential sensor for cellular DNA damage and a marker for cancer development. Using data and samples from the Shanghai Breast Cancer Study, we investigated MnlI restriction sites located between nucleotides 16,106 and 16,437 of the mtDNA D-loop region to evaluate restriction fragment length polymorphism (RFLP) patterns in tumor tissue from 501 primary breast cancer patients when compared with tumor tissue from 203 women with benign breast disease (BBD). RFLP patterns in correspondingly paired, adjacent, non-tumor tissues taken from 120 primary breast cancer patients and 59 BBD controls were also evaluated. Five common RFLP patterns were observed, and no significant differences were observed in the distribution of these patterns between tumor and adjacent non-tumor tissue samples from breast cancer patients and tissue samples from BBD controls. On the other hand, somatic MnlI site mutations, defined as a difference in MnlI RFLP pattern between tumor tissue and the corresponding, adjacent, non-tumor tissue, occurred more frequently in breast cancer patients (28.3%) than in BBD patients (15.3%) (P = 0.05) and more frequently in proliferative BBD (13.0%) than in non-proliferative BBD (7.1%). Our data suggest that somatic MnlI site mutations may play a role in the pathogenesis of breast cancer.

Chromosomal rearrangements differentiating the ryegrass genome from the Triticeae, oat, and rice genomes using common heterologous RFLP probes

An restriction fragment length polymorphism (RFLP)-based genetic map of ryegrass (Lolium) was constructed for comparative mapping with other Poaceae species using heterologous anchor probes. The genetic map contained 120 RFLP markers from cDNA clones of barley (Hordeum vulgare L.), oat (Avena sativa L.), and rice (Oryza sativa L.), covering 664 cM on seven linkage groups (LGs). The genome comparisons of ryegrass relative to the Triticeae, oat, and rice extended the syntenic relationships among the species. Seven ryegrass linkage groups were represented by 10 syntenic segments of Triticeae chromosomes, 12 syntenic segments of oat chromosomes, or 16 syntenic segments of rice chromosomes, suggesting that the ryegrass genome has a high degree of genome conservation relative to the Triticeae, oat, and rice. Furthermore, we found ten large-scale chromosomal rearrangements that characterize the ryegrass genome. In detail, a chromosomal rearrangement was observed on ryegrass LG4 relative to the Triticeae, four rearrangements on ryegrass LGs2, 4, 5, and 6 relative to oat, and five rearrangements on ryegrass LGs1, 2, 4, 5, and 7 relative to rice. Of these, seven chromosomal rearrangements are reported for the first time in this study. The extended comparative relationships reported in this study facilitate the transfer of genetic knowledge from well-studied major cereal crops to ryegrass.

Mapping of yield-related QTLs in pepper in an interspecific cross of Capsicum annuum and C. frutescens.

An advanced backcross QTL study was performed in pepper using a cross between the cultivated species Capsicum annuum cv. Maor and the wild C. frutescens BG 2816 accession. A genetic map from this cross was constructed, based on 248 BC(2) plants and 92 restriction fragment length polymorphism (RFLP) markers distributed throughout the genome. Ten yield-related traits were analyzed in the BC(2) and BC(2)S(1) generations, and a total of 58 quantitative trait loci (QTLs) were detected; the number of QTLs per trait ranged from two to ten. Most of the QTLs were found in 11 clusters, in which similar QTL positions were identified for multiple traits. Unlike the high percentage of favorable QTL alleles discovered in wild species of tomato and rice, only a few such QTL alleles were detected in BG 2816. For six QTLs (10%), alleles with effects opposite to those expected from the phenotype were detected in the wild species. The use of common RFLP markers in the pepper and tomato maps enabled possible orthologous QTLs in the two species to be determined. The degree of putative QTL orthology for the two main fruit morphology traits-weight and shape-varied considerably. While all eight QTLs identified for fruit weight in this study could be orthologous to tomato fruit weight QTLs, only one out of six fruit shape QTLs found in this study could be orthologous to tomato fruit shape QTLs.

The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity, low mitochondrial diversity, regular recombination, and gene flow

A total of 1673 Mycosphaerella graminicola strains were assayed for DNA fingerprints and restriction fragment length polymorphism (RFLP) markers in the nuclear and mitochondrial genomes. The isolates were collected from 17 wheat fields located in 11 countries on five continents over a six year period (1989–1995). Our results indicate that genetic diversity in the nuclear genome of this fungus was high for all but three of the field populations surveyed and that populations sampled from different continents had similar frequencies for the most common RFLP alleles. Hierarchical analysis revealed that more than 90% of global gene diversity was distributed within a wheat field, while ∼5% of gene diversity was distributed among fields within regions and ∼3% was distributed among regions on different continents. These findings suggest that gene flow has occurred on a global scale. On average, each leaf was colonized by a different nuclear genotype. In contrast, only seven mtDNA haplotypes were detected among the 1673 isolates and the two most common mtDNA haplotypes represented approximately 93% of the world population, consistent with a selective sweep. Analysis of multilocus associations indicated that all field populations were in gametic equilibrium, suggesting that sexual recombination is a regular occurrence globally.

Epidemiological studies of human and animal Salmonella typhimurium DT104 and DT104b isolates in Ireland.

A total of 122 human and animal Salmonella Typhimurium DT104 isolates and 6 epidemiologically related DT104b isolates from human and animal products were analysed by pulsed-field gel electrophoresis (PFGE). Genomic DNA was subjected to macrorestriction with three enzymes, SpeI, SfiI and XbaI. A total of 14 restriction fragment length polymorphism (RFLP) profiles were identified when the PFGE patterns from the three enzymes were combined. The majority of isolates (81.2%) exhibited the same RFLP profile. Six animal DT104 isolates, susceptible to enrofloxacin and resistant to naladixic acid, were identified from the antibiotic susceptibility test. Four of these isolates had a different PFGE profile from the common RFLP. In addition, 4 of the 6 isolates were geographically clustered in one region. It was concluded that there was one predominant strain of S. Typhimurium DT104 in Ireland and that the potential and selection pressures for emergence of fluoroquinolone-resistant isolates were present.

A genetic linkage map of durum wheat

A genetic linkage map of tetraploid wheat [Triticum turgidum (L.) Thell.] was constructed using segregation data from a population of 65 recombinant inbred lines (RILs) derived from a cross between the durum wheat cultivar Messapia and accession MG4343 of T. turgidum (L.) Thell. ssp dicoccoides (Korn.) Thell. A total of 259 loci were analysed, including 244 restriction fragment length polymorphisms (RFLPs), one PCR (polymerase chain reaction) marker (a sequence coding for a LMW (low-molecular-weight) glutenin subunit gene located at the Glu-B3 locus), seven biochemical (six seed-storage protein loci and one isozyme locus) and seven morphological markers. A total of 213 loci were mapped at a LOD≥3 on all 14 chromosomes of the A and B genomes. The total length of the map is 1352 cM and the average distance between adjacent markers is 6.3 cM. Forty six loci could not be mapped at a LOD≥3. A fraction (18.6%) of the markers deviated significantly from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on chromosomes 1B, 3AL, 4AL, 6AL and 7AL. The durum wheat map was compared with the published maps of bread wheat using several common RFLP markers and general features are discussed. The markers detected the known structural rearrangements involving chromosomes 4A, 5A and 7B as well as the translocation between 2B-6B, but not the deletion on 2BS. This map provides a useful tool for analysing and breeding economically important quantitative traits and for marker-assisted selection, as well as for studies of genome organisation in small grain cereal species.

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean (Vigna radiata L. Wilczek).

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC20 generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC20F2 population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC20F2 population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene.

Characterization of large linear plasmids in mycobacteria.

Linear plasmids were found in Mycobacterium xenopi, M. branderi, and M. celatum. These elements represented a wide size range (from 20 to 320 kb), had Streptomyces-like terminal structures, and defined five hybridization groups. Cross-hybridization and common restriction fragment length polymorphism patterns of plasmids from the different species suggest either genetic exchange or a common ancestry of the species.

Comparative linkage maps for the mosquitoes, Aedes albopictus and Ae. aegypti, based on common RFLP loci

Aedes albopictus and Aedes aegypti are members of the mosquito family Culicidae and share a haploid chromosome complement of three. Although a genetic linkage map based on restriction fragment length polymorphism (RFLP), markers exists for Ae. aegypti, the extent of synteny and linkage order conservation between the two species was unknown. A comparative linkage map for Ae. albopictus was constructed based mainly on cDNA clones from Ae. aegypti. Nearly all Ae. aegypti probes hybridized to Ae. albopictus DNA at high stringency. For eighteen RFLP markers tested, the linkage group and linear order appears to be identical for the two species. 78% of the loci tested exhibited significant deviations from the expected segregation ratio in at least one of the test crosses. An excess of heterozygote genotypes was recovered with most loci. This probably reflects the effects of lethal loci on survival of F2 progeny homozygous for the parental genotypes. These results demonstrate that comparative linkage maps based on common DNA markers provide a basis for rapidly developing linkage maps for various mosquito species, and the opportunity to examine the significance and function of orthologous quantitative trait loci associated with mosquito vector competence for disease transmission.

Chromosomal localization and detection of DNA polymorphisms in the bovine polymeric immunoglobulin receptor gene

Polymeric immunoglobulin receptor (PIGR) mediates transcellular transport of secretory antibodies in glandular and mucosal epithelial cells. By use of a bovine-rodent somatic cell hybrid panel the bovine PIGR locus has been assigned to syntenic group U1. Using in situ hybridization, PIGR was localized to bovine chromosome 16, segment q13, thus confirming the recent assignment of syntenic group U1 to this chromosome. Two common restriction fragment length polymorphisms (RFLPs) with the enzymes BamHI and MspI were detected using the PIGR cDNA as probe. Direct PCR sequencing of a segment in the PIGR coding region (nucleotides 162-413) from 13 bulls of Norwegian Cattle revealed single nucleotide exchanges at two positions. An efficient PCR-RFLP method for detection of these mutations was developed.

Characterization of Neisseria meningitidis by polymerase chain reaction and restriction endonuclease digestion of the porA gene.

Subtype classification based on the use of monoclonal antibodies to the class 1 outer membrane protein combined with techniques such as multilocus enzyme electrophoresis remains the standard method of characterizing isolates during outbreaks of invasive meningococcal disease. We developed a rapid typing method based on the restriction fragment length polymorphisms (RFLPs) within the polymerase chain reaction (PCR) product of the porA gene, which encodes the class 1 outer membrane protein, reflecting genotypic rather than phenotypic variability between strains. Forty-five isolates of invasive Neisseria meningitidis obtained from October 1990 to April 1992 were studied after randomization and coding. Included among these were isolates from a local outbreak that resulted in a mass vaccination program. PCR amplification for each isolate was followed by restriction digestion with the following enzymes in the indicated sequence: HaeIII, RsaI, HinfI, HpaII, and AluI. Eighteen different patterns were demonstrated on the basis of RFLPs, whereas only seven groups were identified after standard subtyping. The most common isolate identified by serosubtyping was serogroup C, serotype 2a, subtype P1.2 (C:2a:P1.2) (38%). Thirteen (76%) of these group C isolates shared a common RFLP pattern after digestion with the five restriction enzymes. We were able to further differentiate strains of C:2a:P1.2 with electrophoretic type 5 from electrophoretic types 1, 9, and 15 that occurred during an apparent outbreak. We were also able to characterize 15 isolates (33%) which could not be subtyped with monoclonal antibodies. Our method offers a convenient alternative to standard subtyping procedures and is particularly useful in outbreak situations in which rapid characterization of N. meningitidis is essential so that rational public health policy regarding preventative measures can be formulated.

Application of DNA probes for rRNA and vanA genes to investigation of a nosocomial cluster of vancomycin-resistant enterococci

DNA probes specific for genes encoding rRNA and the glycopeptide resistance gene vanA were used to investigate a cluster of vancomycin-resistant (MICs, > 512 mg/liter) Enterococcus faecalis and Enterococcus faecium isolated from separate patients in a renal unit in a London hospital. When digested with BamHI, 12 of 13 vancomycin-resistant E. faecalis isolates exhibited a common restriction fragment length polymorphism pattern of rRNA genes (ribotype). A vanA probe hybridized with chromosomal DNA in these 12 isolates. The other isolate of vancomycin-resistant E. faecalis had a different ribotype and the vanA gene was located on plasmid DNA. These data suggest that cross-infection with a single strain of vancomycin-resistant E. faecalis occurred in most instances. In contrast, 23 vancomycin-resistant E. faecium isolates showed greater heterogeneity, comprising 8 ribotypes, suggesting that multiple strains were present in the unit. Twenty-one of these 23 isolates harbored a 24-MDa plasmid which hybridized with the vanA probe, implying that interstrain dissemination of a vancomycin resistance plasmid may have occurred among E. faecium isolates in the renal unit.

Structure, organization, and chromosomal mapping of the human macrophage scavenger receptor gene.

Macrophage scavenger receptors (MSR) mediate the binding, internalization, and processing of a wide range of negatively charged macromolecules. Functional MSR are trimers of two C-terminally different subunits that contain six functional domains. We have cloned an 80-kilobase human MSR gene and localized it to band p22 on chromosome 8 by fluorescent in situ hybridization and by genetic linkage using three common restriction fragment length polymorphisms. The human MSR gene consists of 11 exons, and two types of mRNAs are generated by alternative splicing from exon 8 to either exon 9 (type II) or to exons 10 and 11 (type I). The promoter has a 23-base pair inverted repeat with homology to the T cell element. Exon 1 encodes the 5'-untranslated region followed by a 12-kilobase intron which separates the transcription initiation and the translation initiation sites. Exon 2 encodes a cytoplasmic domain, exon 3, a transmembrane domain, exons 4 and 5, an alpha-helical coiled-coil, and exons 6-8, a collagen-like domain. The position of the gap in the coiled coil structure corresponds to the junction of exons 4 and 5. These results show that the human MSR gene consists of a mosaic of exons that encodes the functional domains. Furthermore, the specific arrangement of exons played a role in determining the structural characteristics of functional domains.

Genetic diversity of penicillin-resistant Neisseria meningitidis.

The genetic relatedness of 42 penicillin-resistant Neisseria meningitidis isolates obtained during a 2-year period from a single hospital was studied by multilocus enzyme electrophoresis and by restriction fragment length polymorphism (RFLP) analysis of penicillin-binding protein (PBP) 2 genes. The PBP 2 genes of 7 susceptible strains gave identical RFLP profiles. Sixteen different PBP 2 RFLP profiles were found among the 42 resistant strains, but 4 were found in greater than 1 resistant isolate. Multilocus enzyme electrophoresis revealed a high level of genetic diversity. Four clusters of resistant strains could be distinguished at a genetic distance of 0.75. Resistant strains with the most common PBP 2 RFLP profile were restricted to one of these clusters and may be derived from a common ancestral strain. However, resistant strains with the 3 other common RFLP profiles were distributed in two or more of the clusters.

Restriction fragment length polymorphism analysis of Listeria monocytogenes and its application to epidemiological investigations

The restriction fragment length polymorphisms (RFLPs) of 64 random potentially related strains of Listeria monocytogenes were analysed and compared using a probe comprised of two L. monocytogenes chromosome fragments cloned into a α vector. Twelve RFLP types were defined using 14 isolates of clinical origin, 42 food isolates and eight food associated environmental strains. Of the RFLP types, some were common to a particular serovar and source, whereas other were widespread amongst all serovars and sources. One of the two most common RFLP patterns was associated with serovar 1 2 isolates from food or the environment, whereas another dominant pattern was associated most commonly with serovar four isolates from all sources. The potential relationships between epidemologically related strains were examined, with the analysis of types from a suspected listeriosis outbreak, from clinical maternal-foetal cases, and from an ice-cream factory environmental study. Serotyping alone was not a sufficient marker for the comparison of these strains whereas further discrimination of strains was possible with RFLP analysis.

Use of DNA haplotype analysis in diagnosis of familial hypercholesterolaemia in 31 German families.

In the majority of patients, familial hypercholesterolaemia (FH) is caused by different mutations affecting the well defined low density lipoprotein receptor (LDLR) gene. However, 3% of patients in Munich with a clinical diagnosis of FH have a particular mutation in the apolipoprotein B gene causing familial defective apolipoprotein B-100 (FDB). To date none of the LDLR mutations causing FH in German patients has been identified and it is therefore not yet possible to use DNA technology for direct diagnosis. However, indirect molecular diagnosis based on genetic linkage of common restriction fragment length polymorphisms (RFLPs) of the LDLR gene can be used for diagnosis in family studies. Patients with FDB were excluded from this study. Genotypes and haplotypes of four RFLPs (StuI, ApalI 5', PvuII, and NcoI) were determined in a total of 164 independent alleles from 31 pedigrees that included 222 subjects. Allele frequencies and four RFLP haplotype distribution did not differ significantly from those reported in other populations. The applicability of DNA diagnosis in our lipid clinic is comparable with the estimation from calculations on frequencies and heterozygosities of RFLPs, as predicted using these four RFLPs before. On the basis of cosegregation of haplotypes and normo- or hypercholesterolaemia in one or more sibs or offspring, defective and normal LDLR gene alleles could be distinguished in 42 of 58 heterozygous FH patients who were heterozygous for at least one RFLP. In 97 of 134 (72%) children of these 42 subjects, diagnosis of FH could be carried out unambiguously.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular defects in haemophilia B: detection by direct restriction enzyme analysis

The common restriction fragment length polymorphisms (RFLPs) associated with the FIX gene: 5' BamH I, Dde I, BamH I (2), Taq I and 3' Hha I were absent or of low incidence in Southern Chinese and are therefore not useful for linkage analysis. No deletion was detected amongst seven consecutive unrelated haemophilia B patients, but one had an insertion of a 15 kb Pvu II fragment containing exon d. Using an alternate strategy of polymerase chain reaction (PCR) amplification and direct sequencing, the molecular defect in the other six patients was defined. The four novel mutations characterized were: nucleotide (nt) 6410 G----C (Gly12----Ala); nt 31261 delta T (stop codon 31 bp downstream); nt 31260 C----G (Thr380----Ser) and nt 31122 C----A (Ala34----Asp). Two patients had the same mutation at nt 6365, G----A (Arg-4----Gln), identical to one previously described in other ethnic groups, suggesting that this is a hotspot for mutation. Each of the mutations was found to affect an enzyme recognition site and could thus be identified by direct visualization of abnormal restriction fragments in amplified genomic DNA. This allows rapid and accurate DNA diagnosis of haemophilia B in an ethnic group which otherwise shows little or no polymorphism for the common RFLP sites.