Chapter 5 Nucleic Acid Hybridization Assays A. Preparation of nucleic acid probes: 1. Labeling DNA & RNA - Nick Translation - Random primed DNA labeling - End-labeling of DNA - End labeling of RNA

2. Non-isotopic DNA labeling - Direct nonisotopic labeling by using fluorophores - Indirect DNA labeling where a reporter molecule is incorporated into the probe DNA. The reporter group can be easily detected by its very high affinity to a ligand. The latter can be visually detected by its attachment to marker such as a fluorescent dye that can be detected by in a fluorimetric assay. Alternatively, a marker can be an enzyme such as alkaline phosphatase which can be detected by an enzyme assay that is assayed colorimetrically.

B. Using labeled probes in hybridization reaction for screening tests: 1. Dot blot hybridization: A rapid method that employs allele-specific oligonucleotide (ASO) probes to distinguish between alleles that differ in a single point mutations. Example is the screen for individuals with the sickle cell mutation.

2. Southern blot hybridization: Target DNA (genomic DNA) is digested with the appropriate restriction enzyme, the resulting DNA fragments are separated by agarose gel electrophoresis, the DNA fragments in the gel are then denatured (made single strands), and finally blotted onto a nylon or nitrocellulose membrane. The labeled probe (whether by isotope or non- isotope labeling methods) is first denatured (made single strand) and then hybridized in solution to the nylon membrane containing the template DNA. After hybridization, the location of the fragments in the blotted template DNA that hybridized to the labeled probe by autoradiography (or any other appropriate method).

3. Northern blot hybridization: RNA is extracted from several different tissue, denatured and electrophoresed in an agarose gels. The gel is blotted onto a nylon membrane and hybridized in solution to a labeled DNA probe (gene or fragment of a gene). The tissue in which the gene is expressed will show a signal band upon detection while the tissue that does not express the gene will not show any signal indicating lack of hybridization.

Applications of Southern blot hybridization: different ways to detect Restriction Fragment Length Polymorphism RFLP a) Direct detection of pathogenic point mutations by restriction mapping. Example the sickle cell mutation destroys an Mst II site and generates a disease-specific RFLP.

b) detection of conventional RFLPs mutations are detected in non-disease coding genes or in non-coding regions by using type II restriction endonucleases type. This could identify alleles based on restriction site polymorphism (RSP) and leads to RFLP’s.

c) VNTR-based RFLPs and DNA fingerprinting Due to their wide distribution anf high degree of polymorphism, using hypervariable minisatellite VNTR as a probe results in a highly variable banding pattern that distinguishes between any two individuals (excluding identical twins). d) Detection of gene deletion by restriction mapping Hetrozygotes (carriers) will display a 2:1 ratio of signal intensity for the two alleles (the full length and the shorter deleted allele). Homozygotes for the deletion will show no band for the deleted gene.

4. In situ hybridization: a) Chromosome in situ hybridization The signal obtained identifies a specific chromosome band and thus the map location of a gene in a single step. This technique is known as fluorescence in situ hybridization (FISH)

b) Tissue in situ hybridization single stranded riboprobes (cRNA) are hybridized against RNA in tissue sections. When using isotopes the localized of the hybridization signal is localized using dark-field microscopy.

C. Nucleic acid hybridization assays using cloned target DNA, and microarray hybridization technology: 1. Colony blot and plaque lift are methods for screening separated bacterial colonies or plaques following bacteriophage infection of bacteria

2. Gridded high density arrays of transformed cell clones or DNA clones have greatly increased the efficiency of DNA library screening 3. DNA microarray “DNA chips” technology DNA clones delivered by microspotting onto the surface of a microscopic slide Expression screening by using cDNA microaarays or gene specific oligonucleotide microarrays Screening of DNA variation in disease genes as in breast cancer susceptibility gene BRCA1. Also identifying and cataloging human single nucleotide polymorphism (SNP) markers.