Subject(s)
DNA-Directed DNA Polymerase/genetics , Endopeptidases/genetics , Genome, Viral , Luteovirus/genetics , Poaceae/virology , Amino Acid Sequence , Base Sequence , DNA, Complementary , Luteovirus/classification , Luteovirus/enzymology , Luteovirus/ultrastructure , Microscopy, Electron , Molecular Sequence Data , Nucleic Acid Conformation , Phylogeny , Promoter Regions, Genetic , RNA, Viral/chemistry , RNA, Viral/genetics , Sequence Homology, Amino AcidABSTRACT
New systems are proposed for the PCR analysis of HindIII polymorphic sites in the gamma A and gamma G globin genes and of TaqI polymorphic site in the human factor IX gene of blood population. DNA fragments amplified according to the systems described contain constant restriction site of the appropriate endonuclease, in addition to the polymorphic one, which significantly improves the reliability of the RELP analysis. The systems proposed are highly specific and may be used for DNA diagnosis of beta-thalassemia and haemophilia B.
Subject(s)
Factor IX/genetics , Globins/genetics , Polymorphism, Restriction Fragment Length , Base Sequence , Deoxyribonuclease HindIII , Deoxyribonucleases, Type II Site-Specific , Genetic Markers , Humans , Hydrolysis , Molecular Sequence Data , Polymerase Chain Reaction , Restriction MappingABSTRACT
Using oligonucleotide probes, sequences containing the Mbcr locus involved in chromosome translocation t(9:22) were cloned form the library of human genes in the Charon 4A vector. The recombinant clone lambda BCR 1.1 obtained contained Mbcr sequences, but the 3' region of the Mbcr locus in lambda BCR 1.1 clone was strongly altered. Subcloning of a fragment of the altered region and blot hybridization analysis using it as a DNA probe revealed recombination in the 3' region of the Mbcr locus in clone lambda BCR 1.1 which resulted in insertion of unknown sequences into the region. A modified system is suggested for chromosome 22 breakpoint identification using restriction analysis of genome DNA with four restriction endonucleases and one 5'-DNA probe.
Subject(s)
Chromosomes, Human, Pair 22 , Chromosomes, Human, Pair 9 , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Translocation, Genetic , Base Sequence , Cloning, Molecular , Humans , Molecular Sequence Data , Oligonucleotide Probes , Recombination, Genetic , Restriction MappingABSTRACT
The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7. The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.
Subject(s)
Adenovirus E1A Proteins/genetics , Adenoviruses, Simian/metabolism , Bacteriophage M13/genetics , DNA, Single-Stranded/isolation & purification , DNA, Viral/metabolism , Oligonucleotides/metabolism , Base Sequence , Hydrolysis , Molecular Sequence Data , Restriction MappingABSTRACT
Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.
Subject(s)
DNA, Single-Stranded/genetics , Mutagenesis, Site-Directed , Oncogenes , Adenoviruses, Simian , Alkylating Agents , Animals , Cell Line, Transformed , Cell Transformation, Viral , DNA, Single-Stranded/metabolism , Nucleic Acid Hybridization , Phenotype , Polymerase Chain Reaction , RodentiaABSTRACT
A new variant of the PCR test system is discussed which allows one to detect Bcl I and Hind III polymorphic sites of FVIII gene. It can be used for rapid and effective diagnosis of hemophilia A, especially, in combination with the blot-hybridization technique that detects other polymorphic variants of FVIII gene. The method proposed is highly accurate, reliable and simple. It allows one to analyze submicrogram quantities of DNA without using radiolabeled probes. The whole procedure takes several hours. The variant discussed can, possibly, be the part of the general scheme of hemophilia diagnosis completely based on the effective PCR test.