Wright 2 - Southern Blot and Hybridization 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
When is the Southern blot and hybridization technique used?
When the DNA molecules being studied are complex genomic DNA from an eukaryotic organism, as compared to simple prokaryotic DNA, which can be assayed by gel electrophoresis
What must be done to DNA before it is hybridized?
It must be denatured with heat of over 95 degrees or with chemicals such as formamide or NaOH
Is it true that that radioactively labelled hybridization probe can be either DNA or RNA?
True. Can be both, is attached during renaturation (cooling or removal of chemicals)
What are the steps of the Southern blot?
- Cut DNA with restriction enzymes
- Run it through agarose gel with electrophoresis
- Denature DNA
- Transfer to nitrocellulose (ssDNA binds) with capillary action and buffer
- Label with 32P-label DNA (hybridization probe)
- Wash off probe not annealed to target DNA
- Overlay filter with x-ray film for autoradiography
- Radioactive emissions activate silver grains in the film and develop x-ray
Where is the Southern blot and hybridization method commonly used?
In diagnosis of genetic disease
What is the mutation responsible for sickle-cell anemia?
A mutation in the beta-globin gene that codes for an essential polypeptide in the O2/CO2 carrier protein, hemoglobin.
Single nucleotide substitution from A to T in the 6th codon of the gene replaces glutamine for valine.
How can sickle-cell anemia be detected using Southern blot and hybridization?
By using a restriction enzyme (DdeI), which cuts in normal genes but not in the mutant Beta-globin gene.
There will be two prob lines for normal homozygotes, three for heterozygotes and only one for sickle-cell anemia genes.
What is amniocentesis? What type of sample is taken?
A procedure for obtaining fetal cells for genetic testing.
Amniotic fluid is taken, which contains fetal cells which are cultured and then analyzed by chemical, DNA and chromosomal analysis.
What is chorionic villus sampling (CVS)? What advantage does this have over amniocentesis?
It is sampling from the chorion of the placenta, with ultrasound and a catheter (more invasive technique).
Advantages are that culturing is not required and the CVS can be performed earlier in pregnancy.
What are heterologous probes?
Genes that are already cloned from related organisms.
How are synthetic probes made?
By using reverse genetics to design a DNA probe based on the protein sequence coded by the gene.
What are Restriction Fragment Length Polymorphisms?
Refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites. Can be observed with probes to see if a person is a carrier of a gene.
