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