Using ion-exchange chromatography to purify DNA from a cell extract - Is DNA more negatively charged then RNA?

Question

When applying this method we have a glass or plastic column of resin which is positively charged. Then we pour cell extract into the column in order to capture the negatively charged particles which include DNA. After that we gradually add salt which neutralizes the molecules. But it does this gradually so strongly attached DNA molecules fall last while RNA and proteins fall first. The question is why does DNA have a greater negative charge than RNA ?

Answer 1

DNA is has more negative charge (and thus a stronger interaction with the positively charged resin) because DNA is double stranded while RNA is largely single stranded. So even if you had the same amount of DNA and RNA in a sample, DNA will have 2x more negative charges.

Answer 2

The question essentially transforms into: why does DNA have a stronger ion pair attraction to the diethylaminoethyl group of the ion exchange resin?

This principle is not explained in the Qiagen literature, but must be deduced by chemistry reasoning. It's NOT a matter of size. It IS all about what that extra -OH group does in the RNA, that the -H group on the corresponding DNA does not do. And that is, affect the ionization behavior of the phosphate group attached adjacent. The phosphate has (formally) one doubly-bonded O, and one singly-bonded O (in addition to the two O's which link to adjacent bases, that's a common feature to both DNA and RNA). But remember, these are hybidized bonds -- not permanently =O and -O respectively. So when the DNA has a non-polar C-H on the base adjacent, that makes that nearest-by P-O bond more towards P=O and less towards P-O(-) . That is, two non-polar groups are happier adjacent than are one non-polar and one polar. Then, the P-O bond on the *other* side of the P, the one pointing towards the resin, has to"take up the slack" and become *more* like P-O(-) . Hence, it attracts the resin (+) charged group more strongly. Since this happens at many sites along the chain of the RNA or DNA, the increase in attractive interaction going from RNA to DNA is profound, and requires much higher salt concentration to get in and break the DNA away.

Answer 3

There are two reason why DNA falls slowly, with one reason being related to the other. One reason is due to how much more mass DNA has compared to proteins and RNA. Because there is more mass associated with DNA, it has more negatively charged atoms than protein and RNA.