Detect Restriction Fragment Length Polymorphisms Research Articles

The Duffy blood group is linked to the alpha-spectrin locus in a large pedigree with autosomal dominant inheritance of Charcot-Marie-Tooth disease type 1.

The alpha-spectrin locus (SPTA) on chromosome 1 maps to 1q22-q25 and alpha-spectrin specific probes detect restriction fragment length polymorphisms (RFLPs) with the endonucleases MspI and PvuII. The Duffy blood group (FY) has been mapped to the 1p21-q23 region. We found positive linkage between the alpha-spectrin and the Duffy loci with a maximal Lod score of 3.81 at theta = 0.0 using the computer program MLINK. This indicates that both loci are very closely linked and probably localized to 1q22-q23.

EFFECTIVE STRATEGY FOR PRENATAL PREDICTION OF DUCHENNE AND BECKER MUSCULAR DYSTROPHY

Deletions in the gene sequence for Duchenne (DMD) and Becker (BMD) muscular dystrophy were detected in affected males with four cDNA probes, Cf 56a, Cf 23a, Ca1A, and Cf 27. Most of the deletions were seen with only one of the probes. Cf 23a detected all BMD deletions seen with Cf 56a and some that were not. The same markers also detected restriction fragment length polymorphisms for those cases where deletions were not evident. The probes were also used successfully for prenatal diagnosis in two families each with two DMD affected males. In DMD families successive application of probes Cf 56a, Ca1A, and Cf 27 will give a 70% chance of detecting the mutation. BMD families should first be screened with the Cf 23a probe.

Molecular analysis distinguishes two HLA-DR3-bearing major histocompatibility complex extended haplotypes.

We detected restriction fragment length polymorphisms that distinguish the extended haplotype HLA-B8,DR3,SCO1 from HLA-B18,DR3,F1C3O at the DR beta and DQ beta loci with five of seven restriction endonucleases used. One set of restriction fragments was always found on HLA-B8,DR3,SCO1 and associated with DRw52a, while the other was present on HLA-B18, DR3,F1C3O and correlated with DRw52b (the gene encoding the subtype of DRw52 associated with the BO1 or LB-Q1 antigen). Furthermore, using a full-length DQ beta gene probe, we found division in the DQw2 haplotype, in which DQw2a always associated with HLA-B8, DR3,SCO1, while DQw2b always occurred with HLA-B18,DR3,F1C3O. Our evidence thus indicates that serologically defined HLA-DR3, HLA-DRw52, and HLA-DQw2 are each produced by two structurally very different sets of genes, one set occurring in HLA-B8, DR3,SCO1, and the other in HLA-B18,DR3,F1C3O.

A rapid and efficient screening method for DNA restriction fragment length polymorphisms.

Genomic loci displaying DNA sequence polymorphisms represent useful landmarks on the genetic linkage map. We describe an integrated experimental approach to facilitate the detection of DNA restriction fragment length polymorphisms (RFLP) with useful allelic frequencies at loci covered by cloned DNA sequences. The essential feature of the screening method presented is the pooling of DNA from unrelated individuals for Southern blot hybridization analyses using non-repetitive DNA sequences identified in and preparatively isolated from genomic lambda phage clones. This procedure results in the detection of RFLP with maximal values of heterozygosity while counterselecting for RFLP with unfavourable allelic frequencies. The described experimental protocol should therefore facilitate the identification and characterization of polymorphic loci with frequent heterozygosity.

Germline mosaicism and Duchenne muscular dystrophy mutations.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disease with an incidence of approximately 1 in 3,500 newborn boys. The DMD locus has a high mutation frequency: one third of the cases is thought to result from a new mutation. Linkage studies using probes to detect restriction fragment length polymorphisms and DNA deletion studies have greatly improved DMD carrier detection and prenatal diagnosis. Here we report on two families in which a pERT87 (DXS164) deletion was transmitted to more than one offspring by women who showed no evidence for the mutation in their own somatic (white blood) cells. We also show that the deletion in both siblings in one of the families is identical, indicating that the deletion must have occurred during mitosis in early germline proliferation, leading to a germline mosaicism. This phenomenon may turn out to be a major factor contributing to the induction of DMD mutations, and has important implications for the counselling of DMD families.

Chromosomal Localization of the Mouse Homolog of the Huntington's Disease Linked G8 (D4S10) Marker

The human DNA probe G8 defines D4S10, a polymorphic locus tightly linked to the Huntington's disease gene on human chromosome 4. A subclone of G8, pSC33, showed significant cross-hybridization to discrete restriction fragments in total genomic mouse DNA. The probe detected restriction fragment length polymorphisms (RFLPs) with the enzymes Taq I and Msp I, permitting chromosomal localization of the mouse G8 homolog by linkage analysis using three sets of recombinant inbred mouse strains: BXH, BXD, and AKXD. The mouse locus was mapped to the central region of chromosome 11 at 1 centiMorgan from the SPARC gene, a locus whose human counterpart is on human chromosome 5.

Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.)

As a first step in developing a detailed genetic map of lettuce (Lactuca sativa L.), 156 cDNA and 123 genomic DNA clones of lettuce were compared for their efficiency to detect restriction fragment length polymorphism (RFLP) between four lines of lettuce. Polymorphism was detected 2.5 times more frequently with cDNA probes than random genomic probes. Less polymorphism was detected with cDNA clones homologous to single copy than with cDNA clones homologous to multiple copy DNA sequences. A lower percentage of polymorphism was detected with genomic DNA clones homologous to repetitive sequences than with other types of probes. Digests with each of nine restriction endonucleases were compared; increased polymorphism was not correlated with the presence of a CpG dimer in the recognition sequence of the restriction endonuclease. Digests with enzymes recognizing four base pairs, however, displayed RFLPs less frequently. The six pairwise comparisons of the four lettuce lines showed different frequencies of polymorphism which only approximately corresponded to genetic distances obtained from previous isozyme analyses.

Rapid RFLP screening procedure identifies new polymorphisms at albumin and alcohol dehydrogenase loci.

A rapid screening procedure for restriction fragment length polymorphisms (RFLPs) is reported. DNA from ten individuals is pooled and compared to DNA isolated from a cell line containing a single chromosome 4. This single chromosome-containing line is an obligate hemizygote for chromosome 4 RFLPs so that only one band corresponding to a single allele will appear on a Southern blot. In the pooled DNA sample lane bands corresponding to both alleles will be seen. The technique allows for efficient detection of RFLPs with easier use of large numbers of enzymes. It provides estimates of allele frequencies and disequilibrium. New RFLPs for albumin and alcohol dehydrogenase detected with this technique are described.

Pathovars of Xanthomonas campestris Are Distinguishable by Restriction Fragment-Length Polymorphism

Cloned deoxyribonucleic acid (DNA) fragments, derived from a cosmid library of a Florida isolate of Xanthomonas campestris pv. citri 3041 were used to detect restriction fragment-length polymorphism among 93 strains of X. campestris, which make up 26 pathovars. DNA clones were radiolabeled and hybridized against Southern blot transfers of genomic DNAs of different strains of X. campestris digested with restriction endonucleases. Autoradiographs for DNA clones probed against genomic DNA revealed hybridization profiles which appeared to be highly conserved and unique for each pathovar tested. As a species, the population structure of X. campestris appeared basically clonal. Variability appeared to depend on the DNA probe used; some probes represented DNA loci which were highly variable, and some represented DNA loci which were highly conserved at the species level. By using more than one DNA probe, or by digesting the genomic DNAs with different restriction endonucleases, we were able to differentiate all of the strains of X. campestris described as belonging to a given pathovar. Differences among the pathovars were also confirmed by pathogenicity experiments on plants. Some phytopathogenic strains isolated from plants not previously described as susceptible to X. campestris could be grouped as being related to previously described pathovars.

Molecular genetics of human chromosome 21.

Chromosome 21 is the smallest autosome, comprising only about 1.9% of human DNA, but represents one of the most intensively studied regions of the genome. Much of the interest in chromosome 21 can be attributed to its association with Down's syndrome, a genetic disorder that afflicts one in every 700 to 1000 newborns. Although only 17 genes have been assigned to chromosome 21, a very large number of cloned DNA segments of unknown function have been isolated and regionally mapped. The majority of these segments detect restriction fragment length polymorphisms (RFLPs) and therefore represent useful genetic markers. Continued molecular genetic investigation of chromosome 21 will be central to elucidating molecular events leading to meiotic non-disjunction and consequent trisomy, the contribution of specific genes to the pathology of Down's syndrome, and the possible role of chromosome 21 in Alzheimer's disease and other as yet unmapped genetic defects.

Isolation and characterization of DNA probes from the short arm of the human X chromosome that detect restriction fragment length polymorphisms.

We have isolated 30 X chromosome specific probes from a flow-sorted library enriched for the human X chromosome. Hybridization to somatic cell hybrids containing different regions of the X chromosome localized nine of these probes to Xp. After testing 185 probe-enzyme combinations, three of the Xp probes were found to detect restriction fragment length polymorphisms.

Identification of 28 DNA fragments that detect RFLPs in 13 distinct physical regions of the short arm of chromosome 5.

A series of 175 lambda phage carrying human inserts isolated from a library that is specific for the short arm of human chromosome 5 (5p) have been regionally mapped on 5p using a deletion mapping panel of 16 human-hamster cell hybrids, each of which contains a chromosome 5 with a different deletion in the short arm. Seventy-five single copy DNA fragments were screened with 12 restriction enzymes for their ability to detect restriction fragment length polymorphisms (RFLPs). Twenty-eight of these DNA fragments, which are located in 13 distinct physical regions of 5p, were found to detect RFLPs. These DNA markers make it possible to construct a linkage map that will span the entire length of 5p and will allow the relationship between genetic and physical distance for this region of the genome to be examined at a high level of resolution.

Restriction fragment length polymorphism (RFLP) of the human insulin receptor gene in Japanese: its possible usefulness as a genetic marker.

Restriction fragment length polymorphism of the human insulin receptor gene was analyzed with a 4.2 Kb cDNA probe in Japanese normal subjects and Type 2 (non-insulin-dependent) diabetic patients. Restriction endonuclease Rsa I digestion showed polymorphism of the human insulin receptor gene, with a band at 6.7 Kb, 6.2 Kb or 3.6 Kb. The frequency of the 6.7 Kb band was less than that in Caucasians. Furthermore, 15% of all the Japanese subjects examined lacked a 3.6 Kb band, which is commonly found in Caucasians. We have also detected restriction fragment length polymorphism in the human insulin receptor gene by Pvu II or Stu I digestion. Although no significant association of restriction fragment length polymorphism with Type 2 diabetes was found in the present study, our results suggest that the restriction fragment length polymorphism in the human insulin receptor gene varies among ethnic groups, and that the restriction fragment length polymorphism linked to the human insulin receptor gene might be a useful marker for the linkage study of the genes located close to the human insulin receptor gene on chromosome 19.

Isolation of DNA clones revealing restriction fragment length polymorphisms in the human genome.

A recombinant human DNA library was constructed using pUC 18 as the cloning vector. Plasmid DNA isolated from a small scale culture was used as the hybridization probe; many recombinant clones could be easily tested for their ability to detect restriction fragment length polymorphisms (RFLPs). Forty-five arbitrary single copy DNA fragments were isolated from this library and five clones revealed RFLPs. Probe OS-5 had three alleles and probe OS-7, which detected insertion/deletion polymorphisms, had several alleles.

A polymorphic locus on the long arm of chromosome 20 defined by two probes from a single cosmid.

Two probes from the random human cosmid c1-37 detect restriction fragment length polymorphisms in humans. The loci revealed by these probes are in linkage equilibrium and constitute a compound polymorphic locus with a polymorphism information content of 0.54. A somatic cell hybrid panel has been used to map the probes to chromosome 20; in situ hybridization studies confirm this localization and indicate that the locus is on 20q13. This is the first polymorphic locus to be assigned to the long arm of chromosome 20.

Molecular analysis of the rat MHC. I. Delineation of the major regions in the MHC and in the grc.

Southern blot analysis with liver DNA from a unique series of recombinant (R10, R11, R16, R18, R21, and R22), congenic (Y0.1U.grc+, Y0.1U.grc+/Y0.1L.grc, and Y0.1L.grc) and inbred rats has been performed to examine the restriction fragment length polymorphisms of class I genes. After digestion with Xba I or Eco RI, the genomic DNA was resolved on agarose gels, was transferred to nitrocellulose membranes, and was hybridized with murine H-2 cDNA probes. Eighteen to 25 bands of varying intensities could be clearly resolved in any given strain. Analysis of these hybridization patterns detected restriction fragment length polymorphisms that permitted the assignment of 17 specific fragments to regions within the major histocompatibility complex: RT1.A, RT1.B/D, and the RT1.E-grc-T1 alpha region. Fragments have been identified that are specific for grc, grc+, and RT1.E, and mark the junction sites between these loci. In addition, several markers identify the region around the sites of recombination in some strains. The hybridization pattern of the R18 recombinant had a unique band that specified a point of recombination within the grc. The recombinant R11 presented a unique restriction pattern unrelated to either of the parental strains or other related strains. This result suggests that R11 arose from a recombination event(s) undetected by conventional serologic methods.

Prospects for applications of genetic engineering in pig breeding

First, they can be used as gene-specific probes to identify homologous sequences in cellular DNA or chromosomes by molecular hybridization and to detect restriction fragment length polymorphisms in these homologous sequences. Thus, it can be expected that numerous polymorphic genetic markers will soon be localized at random sites on pig chromosomes. These markers will in turn be used to assign potentially important genes to precise chromosomal regions and predict genotypes at the corresponding loci. Eventually, this could lead to the isolation and cloning of some of these genes. Next, functional genes can be introduced into cultured mammalian cells or into fertilized eggs, providing the opportunity to investigate their importance in pig’s main physiological traits and possibly to produce improved transgenic pigs.

Mapping of the mouse X chromosome using random genomic probes and an interspecific mouse cross.

Two libraries enriched in murine X chromosome material have been constructed in the lambda vector NM 1149 from flow-sorted chromosomes. Inserts of unique genomic sequence DNA were purified and their X chromosome specificity characterised by hybridisation to a panel of somatic cell hybrid lines. Of the first five such X chromosome-specific probes characterised, all detect restriction fragment length polymorphisms (RFLPs) between inbred mouse laboratory strains such as C57BL/6 and BALB/c and the SPE/Pas mouse strain established from a wild Mus spretus mouse, when their DNAs are digested with the restriction enzyme TaqI. Taking advantage of these RFLPs, all five probes have been localised on the X chromosome using an interspecific backcross between the B6CBARI and SPE/Pas mouse strains segregating the X chromosome markers hypoxanthine phosphoribosyl transferase (Hprt) and Tabby (Ta). Three of the probes map to the region between the centromere and Hprt, and two distal to Ta. Since such X-specific sequence probes detect RFLPs between M. spretus and M. musculus domesticus DNAs with high frequency, a large panel of well localised probes should soon be available for studies of biological problems associated with the X chromosome which can best be approached using the murine species.

Restriction fragment length polymorphism detected by human salivary amylase cDNA.

The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7 kb and 6.5 kb fragment alleles, are 0.55 and 0.45, respectively.

Isolation of probes detecting restriction fragment length polymorphisms from X chromosome-specific libraries: potential use for diagnosis of Duchenne muscular dystrophy.

We have isolated 23 human X chromosome-specific DNA fragments from lambda libraries, prepared from flow-sorted X chromosomes. To increase diagnostic potential for X-linked genetic disorders, including Duchenne muscular dystrophy (DMD), the fragments were tested for restriction fragment length polymorphisms (RFLPs) with six restriction enzymes. All fragments were regionally mapped to segments of the X chromosome with a panel of somatic cell hybrids and with human cell lines carrying unbalanced chromosomal abnormalities. Two of the isolated probes detected a high frequency RFLP. One, 754, maps between Xp11.3 and Xp21 and detects a PstI polymorphism with an allele frequency of 0.38. The other, 782, maps between Xp22.2 and Xp22.3 and reveals an EcoRI polymorphism with an allele frequency of 0.40. According to a pilot linkage study of families at risk for Duchenne muscular dystrophy, 754 gives a maximum Lod score of 7.6 at a recombination fraction of 0.03. Probe 782 lies telomeric to DMD with a maximum Lod score of 2.2 at a recombination fraction of 0.17. Using our X-chromosomal probes and a set of autosomal probes, isolated and examined in an identical way, we found a significantly lower RFLP frequency for the X chromosome as compared to the autosomes.