Nucleic acid hybridization assays using cloned DNA probes
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Nucleic acid hybridization assays using cloned DNA probes to screen uncloned nucleic acid populations
Numerous applications in molecular genetics involve taking an individual DNA clone and using it as a hybridization probe to screen for the presence of related sequences within a complex target of uncloned DNA or RNA. Sometimes the assay is restricted to simply checking for presence or absence of sequences related to the probe. In other cases, useful information can be obtained regarding the size of the complementary sequences, their subchromosomal location or their locations within specific tissues or groups of cells.
5.3.1. Dot-blot hybridization is a rapid screening method which often employs allele-specific oligonucleotide (ASO) probes to discriminate between alleles differing at a single nucleotide positionThe general procedure of dot-blotting involves taking an aqueous solution of target DNA, for example total human genomic DNA, and simply spotting it on to a nitrocellulose or nylon membrane then allowing it to dry. The variant technique of slot-blotting involves pipetting the DNA through an individual slot in a suitable template. In both methods the target DNA sequences are denatured, either by previously exposing to heat, or by exposure of the filter containing them to alkali. The denatured target DNA sequences now immobilized on the membrane are exposed to a solution containing single-stranded labeled probe sequences. After allowing sufficient time for probe-target heteroduplex formation, the probe solution is decanted, and the membrane is washed to remove excess probe that may have become nonspecifically bound to the filter. It is then dried and exposed to an autoradiographic film.
5.3.2. Southern and Northern blot hybridizations detect target DNA and RNA fragments that have been size-fractionated by gel electrophoresisSouthern blot hybridization
Northern blot hybridization
5.3.3. Southern blot hybridization permits restriction mapping and assay of RFLPs and moderately small scale mutations
Southern blot hybridization has been used extensively in molecular genetic studies as a means of genomic restriction mapping: a labeled DNA probe from one genome can be used to infer the structure of related sequences in the same or different genomes. Because the genomic DNA samples are fractionated by separation of restriction fragments according to size, mutations which alter a restriction site, and significantly large insertions or deletions occurring between neighboring restriction sites, can be typed. Such mutations will result in altered restriction fragment lengths, that is restriction fragment length polymorphisms (RFLPs) .
Direct detection of pathogenic point mutations by restriction mappingDetection of conventional RFLPs
VNTR-based RFLPs and DNA fingerprinting
DNA probes can also be used to monitor VNTR polymorphisms where alleles differ by a variable number of tandem repeats. To do this, genomic DNA samples are digested with a restriction endonuclease which recognizes well-conserved restriction sites flanking a specific VNTR locus. The resulting restriction fragments are separated according to size on agarose gels, transferred to a suitable membrane and hybridized with a probe representing a unique sequence from the corresponding locus. The resulting pattern of locus-specific RFLPs does not reflect RSP: instead, the differences in sizes of the restriction fragments represent integral numbers of the tandemly repeated unit.
Although the term VNTR could, in theory, encompass a wide range of repeat lengths, in practice the term is usually reserved for moderately large arrays of a repeat unit which is typically in the 5 64 bp region (so-called hypervariable minisatellite DNA, distinguishing it from simple tandem repeat polymorphism (where the repeat unit length is from 1 to 4 bp; i.e. microsatellite DNA) and tandem repeat polymorphism associated with very large arrays of satellite DNA.
DNA fingerprinting (see Sections 17.4.2 17.4.4 ).
Detection of gene deletions by restriction mappingCertain diseases are associated with a high frequency of deletion of all or part of a gene. If a partial restriction map has been established for the gene under investigation, deletions can be screened by Southern blot hybridization using an appropriate intragenic DNA probe. If the deletion is a small one, for example a few hundred base pairs, it is often apparent as a consistent reduction in size of normal restriction fragments in the gene. An individual who is homozygous for this mutation, or is a heterozygote with one normal allele and another with a small deletion, can easily be identified by detecting the aberrant size restriction fragments.
5.3.4. In situ hybridization usually involves hybridizing a nucleic acid probe to the denatured DNA of a chromosome preparation or the RNA of a tissue section fixed on a glass slideChromosome in situ hybridization
Tissue in situ hybridization