Lecture 6 - Chap11 - Nucleic Acid Hybridization-7

Nucleic acid hybridization Assays
Human Molecular Genetics

Chapter 6
Strachen and Reid (3rd Ed.)
Learning Objectives
• Understand how nucleic acid probes are produced.
• Understand the principles of nucleic acid hybridization.
• Understand how cloned DNA can be used to screen uncloned DNA.
• Understand RFLP analysis of uncloned DNA.
• How probes can be used for in situ hybridization
• How microarrays can be used.
Hybridization
• Nucleic acid hybridization is a fundamental tool in molecular genetics.
It takes advantage of the complementary nature of double stranded
DNA or RNA to the DNA or even RNA to RNA.
• Nucleic acid probes are used extensively in many different diagnostic
tests.
• Hybridization is also used in cloning and PCR
Types of probes
RNA and DNA probes
• RNA and DNA probes can be labeled by incorporation of a radioactive
or labeled base.
• Synthesis of new RNA or DNA containing the labeled base.
• End labeling by swapping the terminal phosphate group.
Nick translation Random primed labeling
Which is better?
Two types of end labeling
Synthesis of RNA probes. Why are RNA probes useful?
Isotopic labeling and detection
• Traditionally nucleic acids have been labeled with radionuclides. The
isotopes used were 32P, 33P, 35S, and 3H. The reason these isotopes are
used is they can be detected using film by autoradiography. Each
isotope has its advantages. Some have high emmission intensities
while others are lower. Some have short half-lives while others are
longer. 3H was used for chromosome in situ hybridization, while 32P,
33P, 35S, were and are used for DNA sequencing.
The half-life and energy of emission of typical isotopes
Principles of autoradiography
• This is the principle of localizing and recording a radiolabeled
compound within a solid sample. This involves the production of an
image on a photographic emulsion. The silver halide crystals in a
gelatinous phase are exposed to beta or gamma particles. The Ag
ions are converted to Ag atoms and then are developed to produce
visible image. The undeveloped Ag are removed in the fixation
process. A sample can be exposed to X-ray film and the exposed
atoms turn black giving an image.
Indirect autoradiography
• Some beta particles 3H and 35S are not that suitable for direct
detection due to the low intensity of their emission. The use of
Scintillator or Fluor can help detect these weaker signals.
• Some beta particles 32P are too strong and pass thru film so the use of
intensifying screen can be used. A solid inorganic scintillator are used
behind the film to capture high intensity emission
Nonisotopic labeling and detection
• Direct nonisotopic labeling
• The use of a nucleotides containing at fluoro-phore.
• Indirect nonisotopic labeling
• Chemical coupling of a modified reporter molecule. The reporter molecule
can bind with high affinity to another ligand.
Indirect labeling
• Biotin-streptavidin
• Biotin is a naturally occuring vitamin which binds with high affinity (10-14).
Highest known interaction in biology.
• Digoxigenin
• A plant steroid which has a very specific antibody
Fluorescence microscopy
Common Fluorophores
Indirect
Nonisotopic
Labeling
Structure and digoxigenin and biotin
Principles of hybridization
• The addition of a probe to a complex mixture of target DNA. The mix
is incubated under conditions that promote the formation of
hydrogen bonds between complementary strands.
• Factors that affect hybridization characteristics
• Strand Length
• Base Composition
• Chemical environment
Principles of nucleic acid hybridization
Stringency
• Strand length
• The longer the probe the more stable the duplex
• Base Composition
• The % G:C base pairs are more stable than A:T
• Chemical environment
• The concentration of Na+ ions stabilize
• Chemical denaturants (formamide or urea) destablize
hydrogen bonds.
Reassociation Kinetics
• When double stranded DNA is denatured by heat the speed at which
the strands form double stranded DNA is due to the starting
concentration of DNA. If there is a high concentration of
complementary DNA then the time required will be reduced.
Reassociation Kinetics is the speed at which complementary single
strands form duplexes. Two parameters is Concentration (Co) and
time (t) in sec. (Cot) This dictates that single copy genes hybridize
more slowly than multicopy sequences. Therefore give weaker
signals on a southern.
Denaturation and hyperchromic shift and Tm
Equations for calculating the Tm of an oligo
The identification of specific sequences in a complex mixture.
Dot blot or slot blot
Southern Blot
Southern Blot
Northern Blot
Mutation detection by RFLP
Assay of RFLP (restriction site polymorphism)
This has a variety applications including VNTR (variable number tandem repeat)
RFLPs and DNA fingerprinting.
In situ hybridization
• Chromosome in situ hybridization
• Metaphase or protometaphase chromosomes are probed with labeled DNA .
The DNA can be labeled with a fluorochrome (FISH).
• Tissue in situ hybridization
• Sliced or whole mounted preparations can be probed with RNA probes to
detect mRNA expression
Tissue In situ hybridization
Nucleic acid hybridization and microarray technology
Colony hybridization
Summary I
• Hybridization is due to complementarity of DNA strands.
• DNA can be labeled various ways
• Isotopic and non isotopic
• Hybridization can detect identical or similar sequences.
• Governed by Cot
Summary II
• A variety of techniques utilize hybridization of DNA or RNA probes
• ASO
• Southern Blot, RFLP, VNTRs, Mutation detection, deletion detection
• Northern Blot, tissue specific expression
• In situ hybridization
• Chromosome location and integrity
• Tissue specific expression
Summary III
• Colony hybridization can be used to identify specific clones. Once you
have one clone you can find others that hybridize to it.
• Screening of gridded clones . One can identify genomic clones
homologous to a cDNA or identify cDNA expressed in a cell line.
• Microarrays; minaturize hybridization analysis. Can be used in many
ways to analyze gene expression in various cell types, in response to
various stimuli.
Identifying protein binding sites on a DNA
molecule
• Gel retardation of DNA-protein complexes
Figure 7-33
Figure 7-34
Modification interference assays can identify nucleotides that actually form attachments
DMS attaches methyl groups to quinine nucleotide
This modification is carried out under limited conditions, so an
average of just one nucleotide per DNA fragment is modified
A chemical cleaves DNA at methylated nucleotides

Chromatin immunoprecipitation can detect protein association with DNA in the cell
Formaldehyde for cross-linking

Genomic DNA is sonicated into small fragments
Identifying and studying the translation
product of a cloned gene
• Hybrid-release translation (HRT)
• Hybrid-arrest translation (HART)
All translated proteins are labeled
Remove the unbound
molecules, the hybridized
mRNA is recovered and
translated in a cell-free
system
Entire sample is translated

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Nucleic acid hybridization Assays

Human Molecular Genetics

A chemical cleaves DNA at methylated nucleotides

Formaldehyde for cross-linking

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