Genomic sequencing by ligation mediated polymerase chain reaction using direct blotting and non-radioactive detection. 1993

J P Halle, and H Wurst, and C Schmidt
Fakultät für Biologie, Universität Konstanz, Republic of Germany.

Genomic sequencing has become an important tool for analyzing uncloned cellular DNA with regard to the methylation status of cytidines as well as to DNA-protein interactions within cells. The hybridization step of the genomic sequencing procedure requires a very high sensitivity, rendering the method fairly difficult. Using a modified ligation mediated polymerase chain reaction procedure (LMPCR) and a sensitive non-radioactive detection method, we have developed a procedure avoiding the high amounts of radioactivity formerly needed for detection of chemically cleaved genomic DNA. The detection limit of our method of genomic sequencing is less than 1 microgram mammalian DNA, which is much better than the detection limit of the original genomic sequencing method and comparable with the detection limit of radioactive detection after the LMPCR procedure. In addition to the advantages of the non-radioactive detection technique we simplified the blotting step of the genomic sequencing procedure by using the direct blotting electrophoresis method. The method was applied to a region 5' to the human c-myc promoter of HeLa cells and was able to verify the sequence obtained by other authors and to specify the methylation status of five CpG-pairs within this sequence.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009838 Oligodeoxyribonucleotides A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties. Oligodeoxynucleotide,Oligodeoxyribonucleotide,Oligodeoxynucleotides
D003562 Cytidine A pyrimidine nucleoside that is composed of the base CYTOSINE linked to the five-carbon sugar D-RIBOSE. Cytosine Ribonucleoside,Cytosine Riboside,Ribonucleoside, Cytosine,Riboside, Cytosine
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004268 DNA-Binding Proteins Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases. DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
D004273 DNA, Neoplasm DNA present in neoplastic tissue. Neoplasm DNA
D006367 HeLa Cells The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays. Cell, HeLa,Cells, HeLa,HeLa Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015247 Deoxyribonuclease HindIII One of the Type II site-specific deoxyribonucleases (EC 3.1.21.4). It recognizes and cleaves the sequence A/AGCTT at the slash. HindIII is from Haemophilus influenzae R(d). Numerous isoschizomers have been identified. EC 3.1.21.-. DNA Restriction Enzyme HindIII,Deoxyribonuclease BstFI,Deoxyribonuclease EcoVIII,Endonuclease HindIII,B Pertussis Restriction Enzyme I,BpeI Endonuclease,Endodeoxyribonuclease BpeI,Endonuclease Asp52I,Endonuclease BbrI,Endonuclease BpeI,Endonuclease BstFI,Endonuclease Cfr32I,Endonuclease ChuI,Endonuclease Eco65I,Endonuclease Eco98I,Endonuclease EcoVIII,Endonuclease Hin1076III,Endonuclease Hin173I,Endonuclease HinJCII,Endonuclease HinbIII,Endonuclease HinfII,Endonuclease HsuI,Endonuclease LlaCI,Endonuclease MkiI,LlaCI, Endonuclease

Related Publications

J P Halle, and H Wurst, and C Schmidt
January 1993, Methods in molecular biology (Clifton, N.J.),
J P Halle, and H Wurst, and C Schmidt
April 1991, Current protocols in molecular biology,
J P Halle, and H Wurst, and C Schmidt
May 1990, Nucleic acids research,
J P Halle, and H Wurst, and C Schmidt
August 1996, Clinical and diagnostic virology,
J P Halle, and H Wurst, and C Schmidt
May 1992, Zhonghua Minguo wei sheng wu ji mian yi xue za zhi = Chinese journal of microbiology and immunology,
J P Halle, and H Wurst, and C Schmidt
February 1993, Nucleic acids research,
J P Halle, and H Wurst, and C Schmidt
January 1993, Methods in enzymology,
J P Halle, and H Wurst, and C Schmidt
January 1993, Methods in enzymology,
J P Halle, and H Wurst, and C Schmidt
January 2017, Methods in molecular biology (Clifton, N.J.),
J P Halle, and H Wurst, and C Schmidt
December 1994, The Journal of antimicrobial chemotherapy,
Copied contents to your clipboard!