MCQ for Spectrophotometric determination of nucleic acid purity

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Question 1: Which of the following are important factors to consider the suitability of isolated DNA as an analyte for a given technique?
(i) Amount or concentration of DNA
(ii) Purity of the DNA
(iii) Integrity of the DNA
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  • CORRECT. The amount of DNA obtained must be sufficient for all analyses, and DNA must also be available at a workable concentration. Moreover, purity is important as the isolated DNA should be as free as possible from all contaminants that may inhibit subsequent analyses. Also, integrity means high molecular weight (HMW) DNA (ranging from 50 to 200 kbp) can be a requirement for some types of analyses, whereas in other cases a degree of DNA degradation can be tolerated.
Question 2: Which of the following ratio is used to calculate the purity of nucleic acid?
  • CORRECT! Absorbance of DNA at 260nm/ Absorbance of DNA at 280nm is the ratio used to calculate the purity of DNA.
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Question 3: In assessment of DNA purity, what is the desirable ratio of absorbance at 260 nm and 280 nm for pure sample of DNA?
  • INCORRECT! This lower ratio implies the sample is contaminated with protein.
  • INCORRECT! This lower ratio implies the sample is contaminated with protein.
  • INCORRECT! This lower ratio implies the sample is contaminated with protein.
  • CORRECT! A pure sample of DNA should have a ratio of 1.8-2.0. Lower ratio implies the sample is contaminated with protein.
Question 4: Which of the following methods can be used to increase the purity of DNA?
(i) Impurity can be removed from crude DNA extracts by the addition of appropriate nucleases.
(ii) Impurity can be removed by appropriate ion exchange columns.
(iii) Impurity can be removed by addition of chelating agents.
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  • CORRECT. To increase the purity DNA, RNA may be removed from crude DNA extracts by the addition of appropriate nucleases. Other impurities that may act as inhibitors can, in some cases, be removed by appropriate ion exchange columns or the addition of chelating agents.
Question 5: Care must be taken when using the A260:A280 ratio as an assessment of sample purity, as the ratio is also dependent on the pH of the sample. As pH increases, the absorbance at 280nm _______ but the absorbance at 260nm _______, which results in the ratio having elevated values at higher pH.
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  • CORRECT. The A260:A280 ratio as an assessment of sample purity is also dependent on the pH of the sample. As pH increases, the absorbance at 280 nm decreases but the absorbance at 260 nm is unaffected, which results in an elevated value at higher pH.