Download C. Nucleic acid hybridization assays using cloned target DNA, and

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 nonisotope 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.