An EcoRI restriction fragment length polymorphism (RFLP) in the human c-erb A locus

Summary An EcoR1 restriction fragment length polymorphism (RFLP) was detected in the 3 end of the locus of the c-erb-A proto-oncogene. The frequency of the rarer allele was around 3.0% in a normal population of 107 unrelated individuals. This frequency did not significantly differ in DNA samples from patients with breast tumors or acute leukemias.     

Multiplex-terminal restriction fragment length polymorphism

A novel method called "multiplex-terminal restriction fragment length polymorphism (M-TRFLP)" has been recently developed which can be used for simultaneous analysis of the community composition of two or more microbial taxa (up to four). This method can also be used for microbial diagnostic purposes. For M-TRFLP analysis, primers specific to different target genes are used for multiplex-PCR, with one primer for each target being labeled with a unique fluorescent dye at its 5' end. Restriction digestion of the amplified products followed by fragment size analysis on a DNA sequencer produces profiles for targeted genes, which can be distinguished from each other by the color of the terminal fragments imparted by the unique fluorescent dye used for primer labeling. In contrast to current protocols, M-TRFLP allows multiple communities or multiple targets (genes) data to be obtained in just one reaction and therefore saves time, cost and labor. This protocol can be completed in 5-8 h.     

An NcoI restriction fragment length polymorphism at the human steroid sulphatase gene locus

Summary The DNA diagnosis of X-linked recessive ichthyosis vulgaris (incidence: approx. 1 in 5000 males) can be complicated by the absence of restriction fragment length polymorphisms (RFLPs) in the STS (steroid sulphatase) gene. An RFLP sequence in NcoI-digested genomic DNA is reported, which it is hoped may prove helpful in diagnosis.     

Mapping of a restriction fragment length polymorphism within the human aldolase B gene

Summary Peripheral blood DNA was hybridized to the full-length cDNA and the cloned structural gene of human aldolase B. With PvuII endonuclease a restriction fragment length polymorphism was detected that was present in the heterozygous state in about 21% of the individuals tested. A map of the human aldolase gene was constructed for the two groups of individuals found to produce different fragments after PvuII digestion. This allowed the localization of the polymorphic site within the gene, which was found to be due to the loss of a PvuII site in the last intron upstream from the 3 end. This polymorphism may be used as a genetic marker to study individuals affected by hereditary fructose intolerance.     

Renin locus restriction fragment length polymorphism

Summary The use of two genomic EcoRI fragments as probes is discussed.     

A new restriction fragment length polymorphism in the haptoglobin gene region

Summary Direct gene analysis of the haptoglobin gene region was carried out by Southern blotting using an Hp cDNA as probe. Two types of polymorphism were observed: one due to intragenic duplication, is characterized by a constant fragment length difference of 1700bp observed with several enzymes and by complete correspondence with the protein molecular weight polymorphism; the second type, due to point mutation, was represented by two additional restriction sites for Eco RI and Pst I, with a frequency comparable to that of other genes. These two mutations segregated together in families, suggesting that the recently described Hp related gene is closely linked to the Hp gene. Moreover, they were completely associated with each other. The evolutionary significance of this finding is discussed.     

An additional restriction fragment length polymorphism at the thyroglobulin gene.

The study was aimed at the screening of human chromosomal DNA for restriction fragment length polymorphism (RFLP) at the human thyroglobulin (hTg) gene locus. The RFLP screening was performed in a typical way. As hybridization probes were used 5 Pst I fragments of hTg cDNA of the total length 5.1 kb pairs cloned in pBR 322. One not described polymorphism was found by using the probe hTg 10, (nucleotides from position 4830 to 5810 in the 3' flanking region of hTg). Restriction enzyme Msp I identified a single two allele polymorphism: A1: 3.5 kb and A2: 2.5 kb. Of 32 unrelated healthy individuals two were homozygous for 3.5 kb, one was homozygous 2.5 kb and 29 were heterozygous for both 3.5 kb. and 2.5 kb. Thus, the frequencies of the 3.5 and 2.5 kb Msp I alleles were 0.52 and 0.48 respectively.     

Amplified restriction fragment length polymorphism in parasite genetics

The amplified restriction fragment length polymorphism (AFLP) technique is a relatively new method for the analysis of polymorphism that has not yet been widely used in parasitology. In this article, Dan Masiga, Andy Tait and Mike Turner provide a brief introduction to AFLP and illustrate how it can be used in the investigation of marker inheritance in genetic crosses and in the analysis of polymorphism of field populations. They also briefly highlight the strengths and weaknesses of AFLP in comparison with other methods for detecting polymorphism and conclude that AFLP is a very useful addition to the range of techniques available.     

BamHI restriction fragment length polymorphism (RFLP) at the human GST3 gene locus

A new restriction fragment length polymorphism (RFLP) detected for the human glutathione S-transferase-pi (GST3) gene with the restriction endonuclease,BamHI (GGATCC) is described. Because of the association of GST isozymes with certain human diseases, the data on involvement of different GST loci, their chromosomal location and information on RFLPs are of potential diagnostic value.     

Novel restriction fragment length polymorphism of the growth hormone gene in inbred rats

A novel restriction fragment length polymorphism in inbred rats was detected by Southern blot analysis with rat growth hormone cDNA as a probe. Four alleles, characterized by PstI fragments of 1.2, 1.1, 0.9, and 0.7 kb, respectively, were detected in 27 strains examined. The same distribution of polymorphisms was observed on digestion of DNAs of these strains with three other enzymes, PvuII, HindIII, and BamHI. Moreover, the same differences in length of allelic restriction fragments were obtained with these restriction enzymes as with PstI. These findings suggested that the polymorphism was caused by insertion or deletion of variable DNA segments in the second intron of the growth hormone gene. Linkage analyses using backcross progeny provided no evidence for close linkage between the restriction fragment length polymorphism locus and 10 other loci examined.     

HLA-DR genotyping by restriction fragment length polymorphism analyses

We have established unique restriction fragment length polymorphism (RFLP) patterns characteristic of homozygous typing cells (HTCs) for HLA-DR-1 through HLA-DR-8 haplotypes. These RFLP patterns were found to segregate in family members and correlate 100% with HLA-DR antibody phenotyping. The RFLP patterns were used to type chronic myelocytic leukemic cells which have a Philadelphia translocation from 23 randomly selected Caucasoid patients. The results show an alternative method for the determination of the HLA-DR types without using live cells and to study disease association with the HLA-DR region.     

Use of restriction fragment length polymorphism to detect dihydropteroate synthase gene mutations in strains of Pneumocystis jirovecii isolated in Poland

Point mutation of the dihydropteroate synthase gene causes Pneumocystis jirovecii resistance to sulfa drugs. The RFLP method was used to search for DHPS polymorphism in P. jirovecii strains isolated in Poland. Positive results of DHPS gene fragment amplification using nested PCR were achieved in 25 out of the 30 examined Pneumocystis DNA samples. Two (8%) of the 25 isolates had point mutation at codon 55 (Thr55Ala). The mutation-positive strains were obtained from HIV positive (1/15) and HIV negative (1/10) patients. The observed DHPS gene polymorphism may point to the appearance of P. jirovecii strains resistant to sulfa drugs in Poland.     

Evaluation of restriction fragment length polymorphism in Cucumis melo

Summary The objectives of this study were to assess the degree of restriction fragment length polymorphism (RFLP) in Cucumis melo and to determine interrelationships among cultivated varieties. Initial screening of a genomic PstI library revealed that approximately 40% of the clones were repetitive. A total of 162 unique and low-copy sequence clones were hybridized to seven diverse accesions of C. melo and a C. sativus cultivar Pacer to evaluate RFLP variation. Of these, 130 probes (80%) detected a polymorphism between C. melo accessions and C. sativus, and the majority were polymorphic with more than one enzyme digest. In contrast, only 53 probes (33%) were useful in differentiating at least one of the seven accessions. Of those, only 9% were informative with more than one enzyme digest. This indicates that within C. melo, the differences among accessions are due to infrequent base substitutions, whereas between the two species, differences are mainly due to genome rearrangements such as insertions and deletions or numerous base substitutions. Of the informative probes, 34 were used in analyzing 44 C. melo lines to establish a data base of RFLP hybridization patterns. Percent similarity based on RFLP profiles was computed among lines and analyzed by principal component analysis, to visualize relationships among lines. There were clear demarcations among, but not within, muskmelon and honeydew groups.     

A search for restriction fragment length polymorphism on the human Y chromosome

Summary A systematic search for restriction fragment length polymorphisms (RFLPs) on the human Y chromosome was performed. DNA samples from 16–34 individuals were screened with five restriction enzymes and 12 Y-chromosomal probes, 3 of which detect lowly repetitive sequences and 9 of which are apparently single copy in genomic DNA. None of the single-copy probes revealed any variation. The repetitive sequence probe p21A1 (DYZ?) revealed a TaqI RFLP with q = 0.05. The frequency of fixed point mutations in Y-chromosomal DNA outside the pseudoautosomal region is probably less than 1 in 18000 bp.     

Rapid DNA purification for restriction fragment length polymorphism analysis

This paper describes a method for isolation of DNA from blood samples involving a rapid chemical disintegration of proteins with 8 M urea and with a minimum of exposure to phenol. The DNA is further desalted and purified on Sephadex G-25 prepacked disposable columns. DNA isolated in this way was pure enough to be immediately cleaved by restriction enzymes.     

Nuclear-encoded subunits of human cytochrome c oxidase: SstI restriction fragment length polymorphism

A DNA polymorphism of the nuclear-encoded subunit Va of the human cytochrome c oxidase (COX), a mitochondrial respiratory enzyme, is reported. No polymorphism was detected in genes for the subunits IV and Vb of the same enzyme.