ABSTRACT
We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.
Subject(s)
DNA Ligases/chemistry , Polymorphism, Single Nucleotide , Tumor Suppressor Protein p53/genetics , DNA Probes , Genes, p53 , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Tumor Suppressor Protein p53/chemistryABSTRACT
Identification of chromosome rearrangements is of importance for exact diagnosis, risk assessment, and therapy in blood malignancies. A new method was proposed for rapid and accurate identification of leukemia forms caused by chromosome rearrangements involving MLL (11q23). The method combines reverse transcription-multiplex PCR and hybridization with an oligonucleotide microarray. The microarray was designed to detect the five most common MLL rearrangements: t(4;11) MLL/AF4, t(9;11) MLL/AF9, t(11;19) MLL/ELL, t(11;19) MLL/ENL, and dup(11) MLL/MLL. With clinical specimens, the method was shown to efficiently identify the chromosome translocations in leukemia patients.
Subject(s)
DNA-Binding Proteins/genetics , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Proto-Oncogenes/genetics , Transcription Factors/genetics , Translocation, Genetic , Base Sequence , Cell Line , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 4 , DNA Primers , Histone-Lysine N-Methyltransferase , Humans , Myeloid-Lymphoid Leukemia ProteinSubject(s)
Calcitonin/genetics , Escherichia coli/genetics , Galactosidases/genetics , Gene Expression Regulation , beta-Galactosidase/genetics , Amino Acid Sequence , Calcitonin/biosynthesis , Escherichia coli/metabolism , Genes, Bacterial , Genetic Techniques , Humans , Plasmids , Recombinant Proteins , beta-Galactosidase/biosynthesisSubject(s)
Escherichia coli/metabolism , Genetic Engineering , Growth Hormone/biosynthesis , Animals , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Growth Hormone/analysis , Growth Hormone/isolation & purification , Growth Plate/drug effects , Humans , Hypophysectomy , Immunodiffusion , RatsABSTRACT
The chromatographic profiles of isoacceptor tRNA's for 9 amino acids from the loach embryos at two early developmental stages were compared by the method of chromatography on sepharose column in the decreasing gradient of ammonium sulphate concentration to elucidate the possible role of tRNA in the control of early embryogenesis processes. Certain differences both in the proportions of some isoacceptor peaks and their relative positions on the chromatographic profile were found for 8 aminoacyl tRNA. Methionyl-tRNA was represented by one constant peak.