Question

Which statement is false regarding DNA/RNA isolation?

a) Remove protein: proteinase K, pronase, phenol/CHCl3

b) Concentrate with EtOH + salt

c) DNA: DNAase denatured by heat (650C), EDTA

d) RNA: RNAase – very unstable, need to inhibit RNAases (cellular or extraneous)

Answer 1

Nucleic acid (DNA or RNA) is the genetic material of all organisms. It is DNA in majority of organisms. For cutting the DNA with restriction enzymes it needs to be pure and free from other macromolecules. Because the DNA is covered by the membranes, it has to break the cell open to release DNA and other macromolecules like RNA, proteins, polysaccharides and lipids. It is obtained by treating the cells with certain enzymes like lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). The genes that are present on long molecules of DNA interwind with proteins like histones. Proteins are removed by treating with Proteinase K and pronase., which breaks the peptide bonds and inactivate endogenous DNases and RNases in the cell lysate. The degraded proteins and other contaminating substances are then separated with organic solvents containing phenol or chloroform. RNA is an unstable molecule and is susceptible for degradation. RNA is unstable due to the presence of RNases, so it is isolated by using strong denaturants like DNase that inhibit endogenous RNases and intact the RNA. DNase is an endonuclease that specifically cleave and degrade DNA. It can able to digest single and double stranded DNA into single bases or oligonucleotides and finds application in RNA isolation, reverse transcription preparation, etc. DNase removes any sort of DNA contamination from RNA samples by denaturing DNA, subjected to a temperature of 65^oC followed by the adddition of EDTA which prevent RNA denaturation.

After the removal of degraded proteins and other macromolecules, the resulting nucleic acid is precipitated with the addition of concentrate ethanol and salt like NaCl.

So the correct answer is (c).