Probe based technology Flashcards
What is a probe in nucleic acid techniques?
A single stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome.
Probes contain a label for detection, such as a fluorophore or radioactive marker.
What is the purpose of labeling probes?
Allows for detection of the probe’s binding to its complementary sequence.
Common labels include fluorophores and radioactive isotopes like P-32.
What are the external exposure hazards of P-32?
High energy beta particles that can produce x-rays and present a hazard if exposed to the eyes or skin.
P-32 is a radioactive isotope used in probe labeling.
What is the difference between external and internal exposures to radiation?
External exposure occurs when radiation penetrates the body; internal exposure occurs when a radiative substance is inhaled, ingested, or absorbed.
Internal exposure can also happen through breaks in the skin.
Name three techniques that commonly use probes.
- Southern Blot
- Fluorescence in situ hybridization (FISH)
- Microarray
Despite the popularity of sequencing, newer techniques still utilize probes.
What is a Southern Blot?
A method used to detect specific sequences in a sample of nucleic acid.
Named after its inventor, Edward Southern.
What are the initial steps in a Southern Blot procedure?
- Quantitate and normalize DNA
- Digest DNA with a restriction enzyme
- Separate DNA by gel electrophoresis
This results in equal intensity bands on the gel.
What role do restriction enzymes play in Southern Blotting?
They cut DNA at specific sequences to create fragments for analysis.
A frequent cutter is preferred for Southern Blot analysis.
How is DNA transferred from gel to membrane in Southern Blotting?
By incubating the gel in a basic solution to denature DNA and using capillary action to transfer it onto a nylon membrane.
Nylon is positively charged and attracts negatively charged nucleic acids.
What is the purpose of stringency washes in Southern Blotting?
To remove unbound probe and determine the strength of hybridization conditions.
Stringency is influenced by salt concentration and temperature.
What is the visualization method used for 32P labeled probes?
Autoradiography.
Involves exposing X-ray film to radioactive emissions from the probe.
What is the main purpose of Northern Blotting?
To detect specific RNA sequences in a sample.
Unlike Southern Blotting, RNA does not need to be digested because it is already short.
What is Fluorescence in situ hybridization (FISH)?
A diagnostic technique that helps to identify chromosomal abnormalities.
FISH has a resolution of 1-5kb and can detect various chromosomal changes.
What is the significance of growing cells in culture for FISH?
To propagate more samples and examine chromosomes in metaphase.
Cells such as blood, bone marrow, and tissue can be grown for analysis.
What is the purpose of a counterstain in FISH?
To visualize the nucleus during fluorescence examination.
DAPI is commonly used as a counterstain.
Fill in the blank: The hybridization buffer in FISH contains _______.
Formamide, salmon sperm DNA, SSC, dextran sulfate, phosphate buffer.
These components help stabilize hybridization and block non-specific binding.
What control types are necessary in FISH?
- Internal control
- Negative control
- Positive control
These controls ensure the probe’s effectiveness and validate the scoring method.
