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Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are perhaps the most important molecules in cell biology, responsible for the storage and reading of genetic information that underpins all life.
3:30 4:21 So cytosine and thyine are the pyrimidines. So theyre the One Ring bases adenine and guanine areMoreSo cytosine and thyine are the pyrimidines. So theyre the One Ring bases adenine and guanine are the two ringed bases. And now you have a labeled DNA.
DNA and RNA labeling techniques Oligonucleotides can be labeled at either the 3 or the 5 end. Using polynucleotide kinase and ATP-gamma-32P, the 5 end is labeled. Using terminal transferase and deoxynucleotide triphosphate labeled on the alpha phosphate, the 3 end is labeled.
It is done either by random priming or nick translation using PCR amplification techniques or by in vitro transcription using labeled dNTPs or NTPs. For protein interaction-based applications, such as pull-downs or gel-shift assays, its required to generate end-labeled probes. This can be done by PCR using primers.
Two methods are commonly used for 3-end labeling RNA: T4 RNA ligase with 3,5[5-32P]pCp (1,2), and poly(A) polymerase with [-32P]cordycepin 5-triphosphate (CoTP or 3-deoxy-ATP) (3,4). Labeling with T4 RNA ligase requires high concentrations of pCp and enzyme, and is less efficient with long RNAs (3).
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