4.2 LEC Flashcards
What was the classical method used to label probes in Southern analysis, involving a radioactive element?
Radioactive labeling with 32P
What are two common nonradioactive labels used today in medical laboratories?
Biotin and digoxigenin
What method adds labeled nucleotides to the ends of probes?
End labeling
What method incorporates labeled nucleotides into single-stranded breaks using DNA polymerase?
Nick translation
What method uses oligomers of random sequences to prime synthesis of new strands incorporating labeled nucleotides?
Random priming
What type of probes are transcribed from cloned or amplified DNA and labeled during synthesis?
RNA probes
What is the nucleotide triphosphate commonly used for labeling DNA or RNA probes?
UTP or CTP
Three methods of probing
End labelling
Nick translation
Random priming
What is critical for determining the specificity of hybridization results?
Probe Design
What is a probe with internal complementary sequences likely to form?
Secondary Structure
What type of probes provide greater specificity in Southern blotting due to resistance to mutations?
Long Probes (500 to 5,000 bp)
What property of a probe can interfere with binding efficiency due to strong folding?
High GC Content
What kind of probe is sensitive to single-base-pair changes and useful for mutation analysis?
Short Probes (less than 500 bp)
What offers lower background noise in hybridization procedures?
Longer Probes
What is a risk when using shorter probes in Southern blotting?
Higher Background Noise
