In a certain species of ciliated protist, there is genomic DNA segment that is o
ID: 222531 • Letter: I
Question
In a certain species of ciliated protist, there is genomic DNA segment that is occasionally removed. This deletion correlates with cellular mating. It has been proposed that this genetically controlled process is a type of recombination, specifically, a site-specific recombination where the DNA on either end of the nucleic acid segment joins together and the deleted DNA yields a circular DNA reaction product. Suggest how the researcher might use PCR to detect the presence of the circular form of the deleted DNA in an extract collected from the protest and briefly explain the procedure. What method can the researcher employ to quantitate the relative copy numbers of the target sequence? Explain the biochemistry and chemistry that allows for this quantification.Explanation / Answer
a). Circular and linear both types of DNA can serve as templates for PCR, the resulting products will always be linear DNA . But the in vitro procedure of ligation-during-amplification (LDA) for selective amplification of closed circular DNA by using specific primers for the particular circular DNA can be done to amplify the circular DNA specifically in the closed circular form. In LDA a thermostable DNA ligase should be used in a PCR reaction that uses a closed circular DNA as template. After a primer is fully extended on the circular template, the ligase closes the gap to form a double-stranded DNA. Following thermal denaturation, the two circular DNA strands serve as templates for the next round of extension and ligation.
b) to quantify the copy number of target sequnce can be quntify by spectrophotometer quntification method or by UV flurosence tagging of the DNA.
Spectrophotometric analysis of DNA is based on the principles that nucleic acids absorb UV light at 260 nm. In the case of DNA should be treated or exposed to ultraviolet light at 260 nm of wavelength and a photo-detector measures the light that passes through the sample. Th elight which is absorbed by the sample wil give the optical density as the concentration of DNA in your sample. More OD means the more DNA concentration. It depends on the principle of BEER LAMBERT law and be calclulated by the following method
Optical Density= Log (Intensity of Incident Light / Intensity of Transmitted Light)