Chapter Six 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.

06_01.jpg

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. 32 P- 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.

06_02.jpg

06_02_2.jpg

06_03.jpg

06_04.jpg

06_04_2.jpg

Isotopic labeling is detected by exposure to X-ray film (autoradiography) and also by counting the dpm of the labeled molecule using a scintillation counter. 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 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 biotin- streptavidin (detected by fluorophores) and digoxigenin.

06_05.jpg

06_05_2.jpg

06_06.jpg

06_07.jpg

06_07_2.jpg

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).

06_08.jpg

06_09.jpg

06_10.jpg

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).

06_11.jpg

Southern and Northern blot hybridizatoions are used to hybridize a labeled probe to fractionated and immobilized DNA (Southern) or RNA (Northern).

06_12.jpg

06_12_2.jpg

06_13.jpg

3. Pulsed field gel electrophoresis (PFGE) used in conjugation with rare-cutter restriction endonucleases that recognize CpG islands in genomic DNA of vertebrates. CpG islands occur at low frequency in human (or other vertebrate) DNA which results in few recognition sites and a small number of large fragment (Mb range).

06_14.jpg

In situ hybridization: a probe is hybridized to a chromosome preparation (on a microscopic slide) or to RNA of a tissue fixed on a slide

4.Hybridization assays using cloned target DNA (libraries) and microarrays. Colony hybridization and plaque lift hybridization.

06_16.jpg

Gridded high density arrays of transformed cell clones or DNA clones – now performed by robotic gridding devices. Membranes filters could be copied and distributed to a lrage number of laboratories.

06_17.jpg

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:

- microarrays of pre-synthesized nucleic acids – individual DNA clones or oligonucleotides are spotted at individual locations (x, y coordinates of a miniaturized grid) using a robote.

06_18.jpg

- micrarrays of oligonucleotides synthesized in situ – here DNA chips are constructed by taking advantage of photolithography and the chemistry of oligonucleotide synthesis. The probe is a set of unlabeled nucleic acids fixed to the microarray. The target DNA (e.g. genomic DNA) is labeled with a fluorophore and allowed to mix with the micorarray to form heteroduplexes. After washing the microarray of excess hybridization solution, a laser scanner is used to acquire an image of the fluorophores (Cy3, green excitation; Cy5 red excitation) to produce a ratio image. Digiatl imaging software is used to analyze a signal emitted by each spot on the microarray.

06_18_2.jpg

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.