Direct analysis of single nucleotide variation in human DNA and RNA using in situ dot hybridization.

Using oligonucleotide hybridization, single and multiple nucleotide differences between alleles were detected directly in genomic DNA without electrophoretic separation. The DNA was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. An oligonucleotide complementary to a unique sequence region of the human major histocompatibility complex gene HLA-B27 only hybridized with genomic DNA from an HLA-B27-positive individual. Two other oligonucleotides complementary to the normal human beta-globin gene (beta A) and to the sickle cell globin gene (beta S) were synthesized. Using competition hybridization conditions which included the presence of a 10-fold molar excess of unlabeled oligonucleotide complementary to the other beta-globin allele, DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) hybridized to the beta A probe exclusively, whereas DNA from individuals homozygous for the sickle cell globin gene (beta S beta S) hybridized only with the probe for the sickle cell gene. As expected, DNA from heterozygous individuals bound to both probes. Similar results were obtained with total human RNA immobilized in in situ dots. Possible applications of this methodology include genetic disease diagnosis, population carrier screening, HLA "DNA" typing, and DNA and RNA sequence polymorphism analysis.

A method for binding specificity analysis of anti-DNA autoantibodies in SLE.

A pattern of differential binding between an NZB/NZW mouse-derived monoclonal anti-ssDNA antibody, V'D2, and restriction fragments of plasmid pBR322 DNA was shown by electrophoresis of the fragments through a denaturing agarose gel followed by their transfer onto nitrocellulose membrane and subsequent reaction of the immobilized DNA with the antibody and 125I-protein A. The antibody showed preferential binding to a 328 base pair Alu I + Hinf I fragment (denoted FD) (AT content, 60%), compared with the other fragments (AT contents, 40-56%). In dot blot assays the antibody bound only to poly(dT) and poly(dA,dT), failing to bind to other synthetic deoxyribopolynucleotides even at the highest concentration tested (300 ng). In competition experiments, the ability of unlabeled DNA to inhibit binding of V'D2 to FD increased with AT content of the DNA. It is concluded that V'D2 has preference for AT-rich DNA. In addition, poly(dA,dT) inhibited binding to a greater extent than either poly(dA) or poly(dT), indicating that base sequence may be important in defining the antigenic determinant. The method, appropriately modified, may be applicable to a wide range of natural nucleic acids and monoclonal antibodies, allowing detection and isolation of specific DNA fragments for detailed studies of antigenic determinants.

Discrimination among the human beta A, beta S, and beta C-globin genes using allele-specific oligonucleotide hybridization probes.

Synthetic nonadecanucleotides complementary to the human beta A-, beta S-, or beta C-globin sequences were used as hybridization probes to screen human genomic DNA samples for these genes. The oligonucleotides were 32P-labeled and used as probes to genotype restriction endonuclease digests of human genomic DNA. The data obtained show that hybridization with oligonucleotide probes, unlike restriction fragment length polymorphism (RFLP) analysis or direct restriction enzyme digestion, can be used to directly distinguish among the three alleles of beta-globin, beta A, beta S, and beta C, when present either in one (heterozygous) or two copies.

Prenatal diagnosis of beta-thalassemia. Detection of a single nucleotide mutation in DNA.

We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of beta-thalassemia due to a single nucleotide mutation. The beta 0 thalassemia we tested is produced by a nonsense mutation and is commonly found in Sardinia and other parts of the Mediterranean. In this DNA lesion, the glutamine codon CAG at the beta 39 position is mutated to TAG, which results in a stop codon and premature termination of the beta-globin chain. We synthesized two oligonucleotides: one homologous to the normal beta A gene and the other to the beta 0 thalassemia gene at the beta 39 location. The oligonucleotides were labeled with 32P and used as hybridization probes for normal and thalassemic DNA. The beta A probe hybridized only to the normal DNA, and the beta-thalassemia probe only to thalassemic DNA, thus providing a technique for direct demonstration of the mutation. The method is sensitive enough to be applied directly to DNA that is isolated from uncultured cells obtained from only 20 ml of amniotic fluid as early as the 16th gestational week.

Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides.

Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.

Isolation of a non-histone chromosomal high mobility group protein from mouse liver nuclei by hydrophobic chromatography.

A histone-binding polypeptide of 25,000 Mr (J2) in the saline-EDTA wash of mouse liver nuclei was purified to homogeneity in three steps: 1) ammonium sulfate fractionation, 2) hydrophobic chromatography using omega-amino butyl agarose, and 3) DEAE-cellulose chromatography. The isolation conditions were mild, and avoided extremes of pH and use of chaotropic reagents. The purified polypeptide was similar, although not identical, to calf thymus HMG-1 (high mobility group protein). The molecular weight, migration in two-dimensional polyacrylamide gels, and the amino acid sequence of the first 9 residues of the NH2-terminal region were identical for the two proteins. The amino acid analysis, however, indicated the mouse liver polypeptide was more acidic, with a lower content of lysine and higher contents of serine, glutamic acid, and aspartic acid. The mouse liver and calf thymus proteins differ in extraction from nuclei and solubility at neutral pH. The J2 polypeptide reacted with affinity-purified HMG-1 antibody by complement fixation, but always at a lower level than the homologous antigen purified with chaotropic reagents. We conclude the J2 polypeptide is an HMG-1-like protein. The intrinsic hydrophobicity of HMG-1 may be important in its functional interaction with DNA and histones.

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography.

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.