Lecture 4 Nucleic acid hybridization Assays

1
Lecture 4Nucleic 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.