Hybrdization
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Hybrdization

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Hybrdization Hybrdization Document Transcript

  • NUCLEIC ACID HYBERDIZATION: Nucleic acid hybridization is a technique in which single-stranded nucleic acids, the DNA's and RNA's, are allowed to interact. This will result to occurrence of complexes called hybrids. These hybrids are being formed by molecules with similar, complementary sequences Through nucleic acid hybridization, the degree of sequence identity between nucleic acids can be determined and specific sequences detected in them. The hybridization can be carried out in solution or with one component immobilized on a gel or, most commonly, on nitrocellulose paper. Hybrids are detected by various means: visualization in the electron microscope; by radioactively labelling one component and removing non-complexed DNA; or by washing or digestion with an enzyme that attacks single-stranded nucleic acids and finally estimating the radioactivity bound. Hybridizations are done in all combinations: DNA-DNA (DNA can be rendered single-stranded by heat denaturation), DNA-RNA or RNA-RNA. METHOD OF HYBERDIZATION: i. ii. In situ hybridization fluorescent in situ hybridization in situ hybridization molecular hybridization used to analyze prepared cells or histologic sections in situ in order to analyze the intracellular or intrachromosomal distribution, transcription, or other characteristics of specific nucleic acids. molecular hybridization formation of a partially or wholly complementary nucleic acid duplex by association of single strands, in order to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands.
  • Fluorescent in situ hybridization (FISH): a genetic MAPPING technique using fluorescent tags for analysis of chromosomal aberrations and genetic abnormalities. Called also chromosome painting. Fluorescent In Situ Hybridization A method for locating a segment of DNA on a chromosome. The DNA is labeled with a fluorescent dye and hybridized to a cytological preparation of chromosomes that has been denatured to allow nucleic acid hybridization between chromosomal DNA and the probe. The site of hybridization is determined by fluorescent microscopy. FISH is a hybrid of 3 technologies: cytogenetics, fluorescence microscopy, and DNA hybridization, which is used to determine cell ploidy and detect chromosome segments by evaluating interphase—non-dividing—nuclei; in FISH, fluoresceinated chromosome probes are used for cytologic analysis and cytogenetic studies, and to detect intratumoral heterogeneity. In genetics, FISH provides a physical mapping approach to detect hybridization of probes with metaphase chromosomes and with the less-condensed somatic interphase chromatin DNA probes may be applied to cell preparations on a slide; if the complementary DNA sequence is present, it binds to DNA and can be detected by light microscopy; FISH labels probes nonradioactively either directly with fluorochromes, or indirectly with biotin and fluorochrome-labeled avidin, with digoxeginin and fluorochrome-labeled anti-digoxeginin, or others; the use of multiple band-pass filters allows simultaneous viewing of numerous probes for different chromosomal sequences labeled with different fluorochromes; FISH is useful in cytogenetic studies, where probes for particular chromosomes—e.g., chromosomes 13, 18, 21—or chromosomal regions—e.g., ABL and BCR genes in the Philadelphia translocation—can be used for the prenatal diagnosis of common aneuploidies or to detect early stages of lymphoproliferative disorders; FISH is as sensitive as other analytical techniques—e.g., conventional cytology and flow cytometry, used to diagnose transitional cell carcinoma of the urinary bladder Pros FISH is simpler, less labor-intensive, and time-consuming—48 hours—than classic cytogenetics—karyotyping—2-3 weeks Cons Only one question can be asked at a time, i.e., rather than asking ‘global issues’—e.g., what is the genetic composition of a population of cells HYBERDIZATION PROCESS  Done by blotting process ,immobilization of nucleic acid on solid support (nylon or nitrocellulose membrane)  Blotted nucleic acid are targets in hybridization experiment  All DNA must be single stranded (at high temperature or with NAOH  Complementary DNAs find each other and anneal  Blot is visualized by labeling DNA
  • Main blotting procedures are: – – – – – Southern blot: DNA digested by a restriction enzyme then separated on an electrophoresis gel Northern blot: use RNA on the gel instead In situ hybridization: probing a chromosomes or tissue Colony hybridization: detection of clones Microarrays Applications a) Isolation and quantification of specific nucleic acid sequences b) Intracellular localization: presence and absence of a particular gene and its copy number in the genome of an organism c) Degree of similarity between chromosomal gene and the probe sequence. d) Presence and absence of recognition sites for particular restriction endonucleases in the gene. e) Expression and regulation of a particular gene. f) Diagnosis of infectious and inherited diseases. Mutation Mutation is a change in the genetic material. This means changes to the DNA or to the chromosomes which carry the DNA. All such changes are heritable (can be passed on to the next generation) unless they have lethal effects. DNA mutations When DNA is copied mistakes are sometimes made – these are called mutations. There are four main types of mutations:     Deletion, where one or more bases are left out. Insertion, where one or more extra base is put in. Substitution, where one or more bases are substituted for another base in the sequence. Duplication, where whole genes are duplicated. IRTRODUCING OF MUTATION IN DNA View slide
  • is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation., or as a result of exposure to mutagens.this process is also called mutagenesis . Mutagenesis laboratory technique Mutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, or strains of organism. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the functioning of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with interesting properties, or enhanced or novel functions that may be of commercial use. Mutants strains may also be produced that have practical application or allow the molecular basis of particular cell function to be investigated. Early methods of mutagenesis produces entirely random mutations, later methods of mutagenesis however may produce site-specific mutation. Types of mutagenesis       Directed mutagenesis Site-directed mutagenesis PCR mutagenesis Insertional mutagenesis Signature tagged mutagenesis Transposon mutagenesis Site-directed mutagenesis Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering. With decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis. View slide
  • Basic mechanism of Mutagenesis The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion. The single-strand primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected The original method using single-primer extension was inefficient due to a low yield of mutants. The resulting mixture contains both the original un-mutated template as well as the mutant strand, producing a mixed population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of mismatch repair system that favors the methylated template DNA, resulting in fewer mutants. Many approaches have since been developed to improve the efficiency of mutagenesis. Advantages of Mutagenesis By this process we can get benefits in plants and animal by following way • Plants:     • More disease-resistant Larger yields More transportable More nutritious Animals:  Make proteins for medicinal purposes  Make organs for transplant to humans