Attempt to calculate the allele frequency distribution of a TaqI RFLP detected by the highly polymorphic probe YNH24

Summary To improve the analysis of parentage testing with the additional technique of DNA polymorphisms, the usefulness of probe YNH24 was studied. The allele frequency distribution of restriction fragments detected by probe YNH24 on TaqI-digested genomic DNA from 100 unrelated individuals was determined. For this purpose, the size of the fragments was calculated by making use of HindIII-digested lambda DNA as an internal marker and of a digitizing tablet coupled to a computer. The size of the fragments ranged from 2.53 kb to 5.89kb. The mean standard deviation was 0.05kb. The differences between the fragment sizes appeared to be smaller than the standard deviation. For this reason, it was not possible to calculate the allele frequency distribution of this highly polymorphic genetic system.     

Restriction fragment length polymorphisms detected by anonymous DNA probes mapped to defined intervals of human chromosome 16

Summary Three anonymous DNA probes ACH207, ACH224, and ACH202, isolated from a flow-purified chromosome 16 library and mapped to defined intervals of human chromosome 16, detected restriction fragment length polymorphisms (RFLPs). The RFLPs were of simple two allele types. The ACH207 (D16S4) probe detected a TaqI and an MspI RFLP with polymorphism information content (PIC) values of 0.30 and 0.27; the ACH224 (D16S5) probe detected and RsaI RFLP, PIC value of 0.34; and the ACH202 (D16S14) probe detected an XbaI RFLP, PIC value of 0.22.     

The potential use of DNA probes to identify and type strains within the Mycobacterium tuberculosis complex

DNA was extracted and purified from 11 strains of Mycobacterium bovis isolated from cattle in Ireland. After digestion with restriction endonuclease PvuII and electrophoresis on an agarose gel, the separated DNA fragments were transferred to a nylon membrane and sequentially hybridized with three DNA probes derived from BCG.
None of the three probes detected restriction fragment length polymorphism (RFLP) within the 11 M. bovis strains, indicating a very close genetic relationship. One probe, pBCG12, detected RFLPs between the M. bovis strains and a reference PvuII digest of DNA from M. tuberculosis R37Rv, confirming that M. bovis and M. tuberculosis are closely related though genetically distinct.     

Mitochondrial DNA variation in the grasshopper Sinipta dalmani: application of long-PCR to the development of a homologous probe.

RFLP analysis of mtDNA in natural populations is a valuable tool for phylogeographic and population genetic studies. The amplification of long DNA fragments using universal primers may contribute to the development of novel homologous probes in species for which no previous genomic information is available. Here we report how we obtained the complete mtDNA genome of Sinipta dalmani (Orthoptera) in 2 fragments (7 and 9 kb) using primers of conserved regions. The specificity of the PCR reactions was ultimately confirmed by several lines of evidence. These fragments were used as a probe for a mtDNA RFLP study in S. dalmani that analyzed the pattern of haplotype distribution and nucleotide diversity within and among chromosomally differentiated natural populations. Our results suggest that the restriction in gene flow detected at the molecular level may explain the chromosome differentiation detected previously and the maintenance of chromosome polymorphism in some areas of S. dalmani geographic distribution.     

A polymorphic DNA marker genetically linked to congenital malformations

Unusual restriction fragments were detected by DNA blot hybridization with PCNA (DNA polymerase-delta auxiliary protein) probe in one of seven cases of congenital malformations. Chromosomal in situ hybridization localized PCNA gene to region q31-35 of human chromosome 2. To discover the locus more closely associated with congenital malformations, a cloned DNA segment which has been mapped to chromosomal region 2q33-36 was tested for restriction fragment length polymorphisms (RFLPs) in these patients. The 2q33-36 probe hybridized with 2.1-kb, 1.9-kb and 1.7-kb fragments in ten normal control samples. In seven cases of congenital malformations examined, however, the band of 2.1 kb is absent in six cases and the band of 1.7 kb in one case. These results indicate that the locus closely linked to congenital malformations is present in the proximity of PCNA locus.     

Characterization and regional mapping of new anonymous chromosome 20-specific DNA markers isolated from a flow-sorted DNA library

Employing the flow-sorted chromosome 20-specific DNA library LL20NS01, we isolated seven novel unique poly- and monomorphic DNA markers specific to human chromosome 20. Initially, 201 phage clones were analyzed regarding insert size and repetitivity. By testing 14 single- and low-copy number clones for their ability to detect RFLPs, three polymorphisms were revealed by two probes, pFMS22-1.4 [D20S22] and pFMS76 [D20S23]. Seven of twenty probes (35%) were assigned to chromosome 20 using a somatic cell hybrid DNA panel. Five of them were regionally mapped by in situ hybridization. Three DNA markers, pFMS51 [D20S29], pFMS76 [D20S23], and pFMS106 [D20S30], were assigned to 20p11.2-p12, and two markers, pFMS22-1.4 [D20S22] and pFMS135 [D20S31], to 20q12-q13.3. Our new chromosome 20-specific DNA markers should be useful for the molecular characterization of this rather underpopulated human chromosome.     

Localization and linkage of three polymorphic DNA sequences on human chromosome 20

The D20S6 locus has been sublocalized by in situ hybridization using the pD3H12 probe to human chromosome band 20p12 and the D20S4 locus using the pMS1-27 probe to 20q13.2. A rare new restriction fragment length polymorphism detected in MspI-digested DNA by the pMSI-27 probe is reported. Linkage studies in nine families have shown that the D20S6 locus is linked to D20S5 (formerly mapped to 20p12 by in situ hybridization) with a maximum likelihood estimate of 0.07 for the recombination frequency (lod score = 9.07) and a confidence interval of 0.02 to 0.14. Estimated recombination frequencies were similar in males and females. Using both two- and multipoint analyses, linkage of D20S4 with the D20S5 and D20S6 loci was excluded and the suggested order for the three loci on chromosome 20 is D20S5-D20S6-centromere-D20S4. D20S5 and D20S6 are very useful markers for linkage studies because of their close proximity and reasonably good polymorphic information content values.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

DNA polymorphism in the 5′ flanking region of the human carbonic anhydrase II gene on chromosome 8

Summary A restriction-fragment-length polymorphism (RFLP) is described which is associated with the human carbonic anhydrase II gene (CA2) that codes for one of the three genetically distinct carbonic anhydrase isozymes, CA I, CA II, and CA III. The isolated DNA was cleaved with several restriction enzymes and subjected to Southern blot hybridization analysis using a DNA probe containing the 5 end of the human CA II gene. A two allele RFLP which was detected with the restriction endonuclease, Taq I, is expressed phenotypically on Southern blots as either a 5.4 kilobase (kb) fragment or as 4.0 and 1.4 kb fragments. These fragments result from the presence or absence of a Taq I recognition site in the 5 flanking region approximately 1.0kb from the initiation codon of the CA II gene. Segregation analysis showed that the alleles are inherited in a Mendelian fashion, with a frequency of 50%.     

Haplotypes of the human immunoglobulin lambda IGLV7S1 and IGLV1S1 genes defined by restriction-site polymorphism and insertion/deletion of a 6-kb DNA fragment.

Haplotypes were defined in the human immunoglobulin lambda locus by using three probes--V lambda VII, V lambda A, and V lambda I--hybridized to BamHI, KpnI, EcoRI, and HindIII digests. Four KpnI alleles were described. Two of them, 13 kb and 16 kb, detected with both the V lambda VII and V lambda A probes, were correlated with 4.6-kb and 10.5-kb KpnI fragments, respectively, which hybridize to the V lambda I probe. The two others (17 kb and 24 kb) were detected with the three probes V lambda VII, V lambda A, and V lambda I. Moreover, we show that two of those haplotypes may reflect an insertion of 6 kb between V lambda A and V lambda 1S1. Familial studies were performed to demonstrate the Mendelian inheritance. Our results demonstrate the absence of association between the C lambda alleles and V lambda haplotypes.     

Cloning and characteristics of DNA sequences amplified in Djungarian hamster cells with multidrug resistance

A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.     

Molecular cytogenetics of alpha satellite DNA from chromosome 12: fluorescence in situ hybridization and description of DNA and array length polymorphisms.

A 340-bp EcoRI fragment of alpha satellite DNA from human chromosome 12 has been isolated and used in molecular cytogenetic and genetic studies. The clone, pSP12-1, detects tandemly repeated 1.4-kb repeat units at the centromeric region of chromosome 12. By fluorescence in situ hybridization, biotinylated pSP12-1 is highly specific for chromosome 12 and has been used to confirm an i(12p) in a case of Pallister-Killian syndrome, both in metaphase spreads and in interphase nuclei. A dominant DNA polymorphism for the centromeric D12Z3 locus is detected with the enzyme TaqI. In addition, a high frequency of D12Z3 array length polymorphisms can be detected using pulsed-field gel electrophoresis. The D12Z3 array has been measured by pulsed-field gel electrophoresis to span approximately 2,250-4,300 kb at the centromeric region of chromosome 12.     

Human population genetic studies of five hypervariable DNA loci.

Population genetic studies were performed using DNA probes that recognize five hypervariable loci (D2S44, D14S1, D14S13, D17S79, and DXYS14) in the human genome. DNA from approximately 900 unrelated individuals, subdivided into three ethnic groups (American blacks, Caucasians, and Hispanics) were digested with PstI and were successively hybridized to each DNA probe. The number of distinct DNA fragments identified for each of these regions varies from 30 to more than 80. An allele frequency distribution was determined for each locus and each ethnic group. The results show significant differences, between ethnic groups, in the pattern of distribution as well as in the relative frequency of the most common alleles of D2S44, D14S1, and D14S13 but only small differences in others (i.e., D17S79 and DXYS14). The results presented show that the analysis of these loci can have useful applications in population genetics as well as in identity tests.     

Similar-sized daughter-strand gaps are produced in the leading and lagging strands of DNA in UV-irradiated E. coli uvrA cells.

The nascent DNA synthesized after UV irradiation contained discontinuity, i.e., daughter-strand gaps. The sizes of these gaps produced in the leading and lagging strands of UV-irradiated Escherichia coli cells were determined by using strand-specific DNA probes. The DNA isolated from irradiated uvrA delta(lac-pro) cells was hybridized with the 32P-labeled single-stranded DNA probes. After digestion with S1 nuclease, the sizes of the bound radioactive DNA fragments were determined by electrophoresis in an alkaline agarose gel. It was found that the average size of gaps produced in the leading strand was about 0.12 kb, whereas those produced in the lagging strand was slightly smaller than 0.12 kb. No gaps larger than 0.5 kb were detected.     

A highly polymorphic locus in human DNA revealed by probes from cosmid 1-5 maps to chromosome 2q35----37.

The highly polymorphic locus D2S3 is revealed by three single-copy probes from cosmid C1-5. These probes, 1-30, 1-32, and 2-96, collectively reveal seven restriction fragment length polymorphisms. Fifty-three of 56 unrelated individuals (93%) were heterozygous at one or more of the seven loci, making the compound locus a very useful marker for gene mapping. Chromosomal assignment of D2S3 was obtained using a panel of human X hamster and human X mouse somatic cell hybrids. Molecular hybridization of EcoRI-digested DNA from these cell lines with the DNA inserts from subclones 1-30, 1-32, and 2-96 showed that all three probes mapped to the long arm of chromosome 2. Additionally, in situ hybridization of [3H]-labeled probe 2-96 to metaphase chromosome preparations allowed more precise assignment of the locus to the region 2q35----37.     

Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome.

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.     

Regional localization of chromosome 3-specific DNA fragments by using a hybrid cell deletion mapping panel.

A series of human chromosome 3-specific DNA fragments isolated and characterized from a lamda phage genomic library were regionally localized on human chromosome 3. This was accomplished using filter hybridization blot analysis of a human chromosome 3 hybrid cell deletion mapping panel. Twenty-three new anonymous DNA fragments were assigned to one of four physical regions of chromosome 3. Seventeen DNA fragments were mapped to the long arm of chromosome 3, including one DNA fragment that demonstrated a restriction fragment length polymorphism (RFLP). Five DNA fragments were assigned to 3p14.2----pter, including one highly polymorphic fragment sublocalized at 3p25----pter by in situ hybridization. This DNA fragment is the second reported distal 3p polymorphic probe. One DNA fragment was localized to 3p14----p14.2. In addition, three fragments previously assigned to chromosome 3 were confirmed. Polymorphic DNA probes DNF15S2 (formerly D1S1) and D3S2 were mapped to 3p14.2----pter. The previous 3p25 in situ localization of the c-raf-1 oncogene was supported by deletion panel mapping. The physical localization of these twenty-three new DNA fragments has more than doubled the number of cloned DNA fragments assigned to chromosome 3. These and future regional assignments of DNA fragment probes will facilitate construction of both a physical and genetic linkage map of chromosome 3. They may also be useful in characterizing the chromosomal and molecular aberrations involved in small-cell lung cancer (SCLC), renal cell carcinoma, other malignancies, and the 3p14.2 common fragile site.     

Long range restriction map of the von Hippel-Lindau gene region on human chromosome 3p

Von Hippel-Lindau disease is a heritable tumour syndrome caused by the loss of the function of a tumour suppressor gene on the short arm of human chromosome 3. The interval RAF1-D3S18 (3p25–3p26) has been identfied by genetic linkage studies to harbour the von Hippel-Lindau gene. We have constructed a long range restriction map of this region and have succeeded in demonstrating the physical linkage of loci D3S726 (DNA probe LIB31-38), D3S18 (c-LIB-1, L162E5), D3S601 (LIB1963) and D3S587 (LIB 12–48). Since multipoint analysis has located D3S601 proximal to D3S726, the physical map should be oriented with D3S726 towards the telomere. The order and distances of probes within the von Hippel-Lindau gene region is as follows: telomere — LIB3138 — (<280 kb) — c-LIB-1 — (overlapping) — L162E5 — (900–1600 kb) — (LIB 19-63, LIB 12–48) — centromere. In tissues that included blood, semen and Epstein-Barrvirus-transformed lymphocytes, we detected a putative CpG island flanking D3S18.     

DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996