Topic 5 Flashcards
What is the principal of nucleic acid hybridization?
Heat the double stranded DNA to denature it, then cool it allowing it to renature or anneal.
What does the association of two complementary strands depend on?
Depends on the Tm because it takes more energy to break a higher number of H-bonds.
So what does the Tm depend on?
DNA length, base composition (G-C content b/c 3 H-bonds), and the chemical environment (Na+ increases renaturation by increases Tm thus keeping DNA as double stranded).
How can you adjust the stringency of renaturation of DNA?
You can adjust the Tm. Higher temperatures increase the stringency.
What are some other ways to adjust the stringency of renaturation?
Decreasing the Na+ ions concentration, increasing urea and formamide concentration and increasing the temperatures temperatures increase the stringency as well.
Describe a typical nucleic acid hybridization assay.
You start first with a DNA mix that has been cut into fragments using restriction enzymes. Next you design a probe based on what type of DNA you’re targeting and add it to the mix (in excess). We then use nucleic acid hybridization to check for the target gene. You heat the mix to make it single stranded, then cool it, allowing the target gene to form a hybrid with the DNA probe. Following the hybridization, you wash the the DNA mix and image it using radiography or fluorescence microscopy depending on how you labeled your probe.
What is the point in adjusting stringency?
It can be adjusted according to your research goal. It can be adjusted to bind to DNA sequences that are exactly complimentary, have one nucleotide difference, etc.
What are the two targets that the probe can bind to?
DNA or RNA
What are the different sources of probes?
You can use fragments of cDNA or genomic DNA (can use RNA, but it is difficult because it is degraded easily). Then you do PCR to amplify your cDNA or genomic DNA template. Another way is having a company synthesize oligonucleotides.
What is the most common way to label DNA probes?
Radioisotope labeling.
What are the three major ways to label probes using radioisotopes?
Nick translation, random priming, and end labeling.
What is the most common form of phosphate?
P-30
What is another form of phosphate? What is the significance of this?
P-32: DNA has many phosphates because of the phosphodiester bonds. P-30 and P-32 have the same chemical properties, therefore P-32 can be used as a radioisotope because it gives off beta rays which can be seen in radiography.
What is Nick translation?
First, you use the enzyme DNase 1, which cuts the phosphodiester bond. This creates a “nick” (bases aren’t connected by a phosphodiester bond). Next, DNA Polymerase 1 is added from E.Coli which adds radioisotope labeled P (P-Alpha-32)
