last  page PLNT3140 Introductory Cytogenetics
Lecture 8, part 1 of 2
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September  26, 2017
RECOMBINANT DNA (cond.)


Learning checklist:

1. Know the basics of how to label DNA by PCR
2. Know the main steps in Southern and northern hybridization experiments.
3. Understand the principles behind sequenceing of DNA


V. LABELING DNA (cond.)

B. Labeling by PCR

PCR is now often the method of choice for labeling a specific DNA fragment.

Click here for review of PCR
The labeling reaction is identical to a regular PCR amplification, except that labeled nucleotides are included along with unlabeled nucleotides.

Advantages:

C. Non-radioactive detection methods

There are two general approaches to non-radioactive labeling. All of these methods incorporate various nucleotide analogues into DNA in a labeling reaction. In chemiluminescence, an enzyme is conjugated to a molecule such as an antibody that is specific for the nucleotide analog. When the appropriate substrate is added, the enzyme breaks it down, resulting in the emission of a photon of light. In fluorescent detection, nucleotide analogs themselves contain a fluorescent tag, which emits light when illuminated with the appropriate excitation wavelength.


1. Commonly-used nucleotide analogs


Digoxygenin-labeled nucleotides (DIG-dNTPs)

see DIG System basics at
https://www.roche-applied-science.com/sis/lad/lad_docs/The_DIG_System-Labeling_and_Detection_of_Nucleic_Acids.pdf

Biotin-labeled nucleotides

For structure, see http://chemcyte.com/products/CC-6008.htm


Fluorescent nucleotides


2. Detection
 

Chemiluminescent detection is typically done using either DIG-dNTPs or Biotin-dNTPs.

Detection of DIG-labeled nucleotides is done using anti-DIG antibodies conjugated to alkaline phosphatase. When a substrate such as CDP* is added, alkaline phosphatase breaks down the substrate, which emits a photon of light.

Detection of Biotinylated DNA is typically done using streptavidin, a bacterial toxin that has an affinity for biotin, conjugated to horseradish peroxidase. Breakdown of substrate (eg. luminol peroxide from Clontech Inc.) results in release of a photon of light.




Fluorescently-tagged DNA probes have the advantage that no enzymatic reaction is necessary for detection. Dye-conjugated nucleotides are incorporated into DNA during labeling eg. PCR labeling.

For structures of Cy3 and Cy5 see http://en.wikipedia.org/wiki/Cyanine#Cy3_and_Cy5

Advantages to non-radioactive detection

Advantages of radioactive detection

VI. HYBRIDIZATION

OBJECTIVE: To use a labeled DNA fragment to detect another DNA fragment of similar sequence.

WHY?:
  1. You have a gene from one species, and want to use it to detect the same gene in another species.
  2. You have a fragment of a gene, and want to use it to find a clone that has the entire gene.
  3. To measure the levels of RNA in a tissue.


If the probe has the same sequence as the target (on filter) it will base-pair with the target. Otherwise, it will just remain in solution, unduplexed. Hybrid duplexes between the radioactive probe and the target can be visualized in autoradiography.

SCHEMATIC OF HYBRIDIZATION EXPERIMENT

A. Method


The filter is a matrix of fibers, each of which can bind a single-stranded DNA molecule. Labeled probe molecules can form hybrid duplexes with free ends of target DNA.
a. denaturation of probe - double-stranded probe is heated or boiled to denature, and quick-chilled on ice.
b. incubation - Probe is added to a plastic bag, a bottle or tube containing the filter, and incubated, typically overnight at 65C, 5xSSC, 0.2M SDS. For a 10 x 12cm. blot, probe would be added in a volume of ~12ml or less. During incubation, radioactively-labeled probe DNA is free to base-pair with complementary target DNA bound to the filter.
c. washing - Unhybridized probe that has not base paried with filter-bound DNA is removed by doing successive 15min. washses in 6xSSC 0.1%SDS, at 65C, followed by two washses in 2xSSC 0.1%SDS.
 
Filters can be rolled up and incubated in hybridization ovens, similar to that shown below. The oven incubates the tubes at the desired temperature, while tubes are rotated to ensure even flow of the hybridization solution across the surface of the filter.

Displayed by hypertext link to http://www.analytik-jena.de/fileadmin/content/import/Vollimport/webexport/14-03-10_OV_500_448_x_336_01.jpg

4) Example

Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency
Hitoshi Kanno, Taiju Utsugisawa, Shin Aizawa, Tsutomu Koizumi, Ken-ichi Aisaki, Takako Hamada, Hiromi Ogura, Hisaichi Fujii
Haematologica June 2007 92: 731-737; doi:10.3324/haematol.10945

Displayed from http://dsas9a9gxtv2e.cloudfront.net/content/haematol/92/6/731/F2.large.jpg


VIDEO: Example of image acquisition by chemiluminescent imaging.
https://www.thermofisher.com/ca/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/e-gel-electrophoresis-system/e-gel-imager-system/e-gel-software.html

B. Types of blots

TAKE HOME EXERCISE: BLOTTING HYBRIDIZATION
 
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last  page PLNT3140 Introductory Cytogenetics
Lecture 8, part 1 of 2
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