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Lecture 4 Nucleic acid hybridization Assays

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Title: Lecture 4 Nucleic acid hybridization Assays


1
Lecture 4 Nucleic acid hybridization Assays
  • Chapter 5
  • Strachen and Reid 2nd Ed.

2
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.

3
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

4
Types of probes
5
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.

6
Nick translation
Why do they call it that?
Random priming
Which is better?
7
Two types of end labeling
8
Synthesis of RNA probes.
Why are RNA probes useful?
9
Isotopic and nonisotopic labeling
  • 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.

10
The half-life and energy of emission of typical
isotopes
11
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.

12
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

13
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.

14
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

15
Fluorescence microscopy
Common Fluorophores
16
Indirect Nonisotopic Labeling
17
Structure and digoxigenin and biotin
18
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

19
Principles of nucleic acid hybridization
20
Stringency
  • Strand length
  • The longer the probe the more stable the duplex
  • Base Composition
  • The GC base pairs are more stable than AT
  • Chemical environment
  • The concentration of Na ions stablize
  • Chemical denaturants (formamide or urea)
    destablize hydrogen bonds.

21
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.

22
Denaturation and hyperchromic shift and Tm
23
Equations for calculating the Tm of an oligo
24
The identification of specific sequences in a
complex mixture.
25
Dot blot or slot blot
26
Southern Blot
27
Northern Blot
28
Mutation detection by RFLP
29
Assay of RFLP (restriction site polymorphism)
This has a variety applications including VNTR
RFLPs and DNA fingerprinting.
30
Detection of gene deletions by restriction mapping
31
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

32
Tissue In situ hybridization
33
Nucleic acid hybridization and microarray
technology
Colony hybridization
34
Gridded clone hybridization
Clones can be identified by screening gridded
microarrays. The positive clones can be picked
from predetermined co-ordinates from microtiter
plates.
35
Construction of DNA and oligo microarrays
36
(No Transcript)
37
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

38
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

39
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.
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