Chemical cleavage of DNA duplexes with single base mismatches as a basis for detection of random point mutations

The most promising approaches to detection of random point mutations are based on chemical cleavage of mismatches and other noncomplementarities. To demonstrate the specificity of this method, a model system was obtained for the first time as sets of 50-mer imperfect DNA duplexes containg all variants of mismatched and unpaired internal residues located in an invariant context and flanked by either A · T or G · C base pairs. Chemical cleavage of DNA duplexes immobilized on magnetic beads via the biotin-streptavidin interaction was accomplished using potassium permanganate or hydroxylamine, which are sensitive to the secondary DNA structure and react with thymine and cytosine, respectively. The reactivity of different mismatches was connected with the local duplex structure and depended on their type, orientation, and flanking nucleotides. The use of potassium permanganate and hydroxylamine to modify a heteroduplex mixture makes it possible to unambiguously detect a mismatch and, based on the type of reagent and the size of the cleavage products, to suppose the type and position of the mismatch and the flanking nucleotides. The model system can be used to evaluate the sensitivity of a chemical cleavage method and to control false-positive and false-negative results when different protocols are applied to the detection of DNA point mutations.     

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

Electron microscope analysis of mouse and rabbit globin and immunoglobulin gene sequences

Mouse and rabbit globin and immunoglobulin gene sequences, which had been synthesized in vitro from eukaryotic mRNAs and inserted into plasmids, have been examined in the electron microscope. The size of the inserted β rabbit and α and β mouse globin DNA sequences has been estimated as 620 base pairs while the size of the inserted α rabbit globin DNA sequences was found to be about 490 base pairs. Heteroduplex analysis has revealed no structural abnormalities at the insertion sites of the chimeric plasmids except in the case of a plasmid containing an immunoglobulin light chain gene sequence of about 830 bases, in which a 3 kb deletion adjacent to the insertion site was observed.     

Insertion sequence IS2 associated with int-constitutive mutants of bacteriophage lambda.

We have examined mutations in bacteriophage lambda called int-c, which confer elevated constitutive expression on the int gene for prophage integration. One class of mutations, which map between the b538 and bio386 endpoints, does not appear to be associated with any major chromosomal modification, whereas the second class has the IS2 insertion sequence in orientation II within the region between gene int and the b538 endpoint, All int-c mutations are within gene xis, with the possible exception of int-c548, which might be located between int and xis. The present data are most consistent with the following notion: (1) the point mutations of class one inactivate the tI terminator signal of the pI-tI leader RNA for gene int and thus render int expression independent of the antiterminating action of the cII and cIII products, and (2) the second class of int-c mutants is constitutive for Int because the IS2 insertion, when strategically located between int and tI, provides a new constitutive promoter for int transciption.     

Identification and characterization of new BoLA-DRB3 alleles by heteroduplex analysis and direct sequencing

A sample of 52 mixed-breed dairy cattle (Holstein Friesian and Jersey) and 51 beef cattle (Hereford) from south-east Queensland was studied. The second exon of BoLA-DRB3 was amplified by polymerase chain reaction (PCR), and polymorphisms were detected by heteroduplex analysis. A large number of different heteroduplex patterns indicated extensive sequence polymorphism. Direct sequencing of PCR products from 17 homozygotes and cloning and sequencing of PCR product from two heterozygotes resulted in the identification and characterization of four novel alleles. The previously described allele BoLA-DRB3 * 2A is characterized by an amino acid deletion at position 65. We have identified three animals that are homozygous for this amino acid deletion, indicating that the deletion is unlikely to result in loss of function. Two of these animals had allele BoLA-DRB3 * 2A, and one had a novel allele with codon 65 deleted but differing from BoLA-DRB3 * 2A at a number of other amino acid positions. In conclusion, heteroduplex analysis allows rapid discrimination between homozygotes and heterozygotes, and enables rapid identification of new BoLA-DRB3 alleles.     

Characterization of a cloned, moderately repeated sequence from Balbiani ring 2 in Chironomus tentans

pCtBR2-1 is a recombinant plasmid with a 750-bp insert of Chironomus tentans genomic DNA. When pCtBR2-1 was hybridized in situ to salivary gland polytene chromosomes, it hybridized exclusively to Balbiani ring 2 (BR2), a giant chromosomal puff. It was also shown that the insert contained four tandemly repeated sequences that were delineated by HinfI sites which occurred every 190 bp. The purified insert reassociated to C. tentans DNA with a C0t1/2 = 0.48 indicating that the sequence was moderately repeated within the genome. Hybridization of radioactive pCtBR2-1 to nitrocellulose blots containing partial HinfI digests of genomic DNA revealed that the 190-bp repeats were organized into one or more blocks of 11 to 12 copies in tandem. Hybridization of the recombinant plasmid to limit digests of genomic DNA also demonstrated that repeated sequences in BR2 were not homogeneous. As much as 70% of BR2 appeared to be represented by a 26-kb HhaI-resistant core, while the remaining 30% may have HhaI sites at 190-bp intervals, similar to pCtBR2-1.