Lecture 2 - Southern Blot & Hybirdization Flashcards
what is the purpose of the Southern blot and hybridization technique?
To detect a specific DNA molecule(s) among a complex mixture of DNA molecules
Why is it called Southern blot and hybridization?
Because Edward Southern developed the technique.
How do you denature double-stranded DNA?
With heat (>95degC) or chemicals (eg. formamide or NaOH)
How does renaturation of single-stranded DNA occur?
by cooling or removal of chemicals
What can the radioactively-labelled hybridization probe be?
either DNA or RNA
What are the characterizations of the Southern blot?
1) Size-fractionation of a complex mixture of DNA molecules by agarose gel-electrophoresis.
2) Denaturation of the double-stranded DNA by NaOH (alkali) to break the hydrogen bond between bases
3) Transfer of the single-stranded DNA molecules to a rigid support, a nitrocellulose filter.
What are the steps for filter hybridization?
- high molecular mass DNA to low molecular mass DNA fragments
- single-stranded DNA becomes immoblized to the nitrocellulose filter
- radioactively labelled hybridization probe anneals to target DNA
- wash off probe not hybridized to the target DNA
- overlay filter with X-ray film for autoradiography
What creates the photographic emulsion in autoradiogrphy?
- Silver halide (bromide, chloride, iodide) crystals suspended in gelatin on a plastic film
What is the chemical process of autoradiogrphy?
Photons or radiation cause silver metallic speck to form. During development, entire crystal is converted into metallic silver that is visible on film
What does autoradiography do?
Allows radioactive emissions to activate silver grains in the film. Develops X-ray film
In an autoradiogram, all size markers show because they are radioactive; in lanes 2, 3 and so on, only the bands hybridized with what are visible?
hybridized with probe
How does the Southern blot and hybridization technique diagnosis sickle-cell anemia?
It detects the Dde I fragments on a Southern blot by probing with beginning of beta-globin gene
What is amniocentesis?
A procedure for obtaining fetal cells for genetic testing
What DNA probe is used to diagnosis Thalassemias?
Globin genes
What DNA probe is used to diagnosis hemophilia?
Factor VIII gene
What DNA probe is used to diagnosis dwarfism?
Growth hormone gene
What DNA probe is used to diagnosis diabetes?
Insulin gene
What DNA probe is used to diagnosis collagen disorders?
collagen gene
What DNA probe is used to diagnosis some cancers?
Oncogenes
What DNA probe id used to diagnosis Muscular Dystrophy?
MD gene
What DNA probe is used to diagnosis Huntington disease?
Huntingtin gene
What DNA probe is used to diagnosis cholesterol, metabolism and heart disease?
Low density lipoprotein receptor
What DNA probe is used to diagnosis Leukemia and Lymphomas?
Immunoglobulin genes
What DNA probe is used to diagnosis emphysema?
Alpha-antitrypsin gene
What DNA probe is used to diagnosis immune deficiencies?
T-cell receptor gene
What DNA probe is used to diagnosis cystic fibrosis?
CF gene
What are the four types of hybridization probes and where do they come from?
1) Messenger RNA that are abundant in particular cells, e.g., globin mRNAs make up 80% of mRNA in red blood cells
2) Heterologous probes, i.e., genes already cloned from related organisms
3) Synthetic DNA probes. Can use reverse genetics to design a DNA probe based on the protein sequence coded by the gene.
4) Anonymous DNA sequences that recognize Restriction Fragment Length Polymorphisms (RFLP)
What is the frequency of recombination or crossing over dependent on?
The distance between the gene and the RFLP
