Nucleic Acid Hybridization: Principles & Applications 1. Preparation of nucleic acid probes: - DNA: from cell-based cloning or by PCR.

Probe is double stranded. Labeling by DNA polymerase-based DNA strand synthesis. - RNA: by transcription from DNA cloned in an expression vector. Probe is single stranded. Labeling by run-off transcription. - Oligonucleotide: by chemical synthesis. Probe is single stranded. Labeling is by end labeling.

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 DNA and RNA probes could be labeled in vitro by one of two methods: - Strand synthesis. By using DNA or RNA as a template to generate a labeled DNA strand. DNA or RNA polymerase are used and one of the four dNTPs in the reaction usually has a labeled group e.g. 32P- dCTP. DNA could be labeled by nick-translation, random primed labeling, or PCR-mediated labeling. RNA probes are labeled by in vitro transcription. - End-labeling: Used in labeling single strand probes by adding one (kinase end-labeling) or very few (fill-in end-labeling) labeled groups at the 5 end.

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 Isotopic labeling is detected by exposure to X-ray film (autoradiography). Non-isotopic labeling includes: - Direct labeling using modified nucleotides containing a fluorophore (a chemical group that when exposed to light of certain wavelengths will fluoresce). - Indirect labeling using a reporter molecule attached to a nucleotide precursor (a spacer of 11-16 side C chain is used to distant the reporter from the nucleotide). An affinity molecule binds very strongly to the reporter molecule. Affinity molecules could be detected by a conjugated marker molecule. Two widely used methods are biotinstreptavidin (detected by fluorophores) and digoxigenin.

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2. Principles of nucleic acid hybridization (NAH): NAH is used to identify how close DNA molecules are. Factors to consider when performing a NAH assay between a probe and a target molecule are: - strand length - base composition - chemical environment monovalent cations stabilize the duplex while polar molecules such as formamide and urea are chemical denaturants. - melting temperature - hybridization stringency temperature and salt concentration (high NaCl conc. and low temp. is low stringency while low NaCl and high temperature is high stringency).

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3. NAH assays: Dot-blot hybridization used with allele-specific oligonucleotides ASO probes where the probes are labeled and hybridized to immobilized target genomic DNA. In reverse blot hybridization, the ASO probes are not labellled and are immobilized on a membrane then hybridized to the labeled target DNA (genomic DNA).

Dot Blot

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Direct detection of pathogenic point mutations by restriction mapping

VNTR based RFLP

FISH

PCR

Allele-specific PCR amplification (ARMS test)

 DNA microarray technology here the filters (membranes) have been replaced by a microscopic slide chemically-treated (e.g. nitrocellulose coated microscopic glass slides). Two type of microarrays depending on how the nucleic acid samples were generated and delivered to the microarray:

 DNA microarray technology has very important applications in biomedical research and diagnostic approaches. Two main principal applications are: 1. Expression screening: here RNA expression levels are monitored by using using cDNA microarrays or gene-specific oligonucleotide microarrays. 2. DNA variation screening: Oligonucleotide microarrays are used. Will be used for assaying for mutations in known human disease genes (diagnostic). Will also be used to identify and catalog human single nucleotide polymorphism (SNP) markers.

SSCP

DGGE