Answer to Question #343533 in Genetics for AMOS

Question

1. You are a Geneticist employed by Thato Biotech Pty Ltd in a criminal case, which could not be solved due to the integrity of DNA used as evidence. You are requested to ascertain the integrity of DNA evidence. You decided to resuspend the purified DNA in a volume of 100uL of double-distilled nuclease-free sterile water. You then diluted 10uL of the DNA sample to a total volume of 100uL with water quality for spectrophotometric analyses. On measuring the absorbance of this diluted sample at 230, 260, and 280nm she obtained the following readings:

A280nm= 0.494; A260nm = 0.364; A230nm= 0.191

a) What is the DNA concentration of the 100uL aliquot, in 3 significant figures

b) How much total DNA was purified by the procedure?

c) Is this DNA pure? If yes, how do you know? if not, what is the most likely contaminant?

d) if this DNA was contaminated with salts what would you expect in the absorbance readings?

e) If DNA was contaminated with RNA, how would you purify your DNA?

Answer 1

a) In order to calculate the DNA concentration in µg/mL, we use the following formula:

• C(dsDNA) = A260nm*dilution factor*50;

where:

• A260nm — the absorbance value of the DNA sample at a wavelength of 260 nm;
• dilution factor — the value of how many times the sample was diluted, in this case it is 10;
• 50 — the value of the DNA concentration in µg/mL, at which the A260nm is 1.

• C(dsDNA) = 0.364*10*50 = 182 µg/mL.

b) Based on the calculated DNA concentration, as well as the known volume of the DNA sample, we calculate the mass of DNA that was purified:

• m(dsDNA) = 182 µg/mL*0.1mL=18.2 µg.

c) To find out if the DNA is pure, we calculate the quality of the DNA sample as the following ratios:

• A260nm/A280nm, which shows the quality of the DNA sample in terms of protein content;
• A260nm/A230nm, which indicates the quality of the DNA sample in terms of salts and organic compounds such as phenol.

An indicator of high quality DNA is the value of the ratio A260nm/A280nm from 1.7 to 2, as well as the value of the ratio A260nm/A230nm from 2 to 2.2. Values less than the lowest value in each range indicate that the DNA is contaminated.

• A260nm/A280nm = 0.364/0.494 = 0.7.

The calculated value is almost 2 times less than the required minimum of 1.7, which means that the DNA is very heavily contaminated with proteins.

• A260nm/A230nm = 0.364/0.191 = 1.9.

The calculated value is also slightly less than the required minimum of 2, which means that there is some contamination of the DNA with salts and organics.

Thus, the main contaminants are proteins, as well as, to some extent, salts and organic substances.

d) The DNA actually turned out to be slightly contaminated with salts, since the A230nm is less than 2 times inferior to the A260nm. At the same time, DNA would be considered pure with respect to salts if the boundary values of A230nm were as follows:

• A260nm/2 = 0.364/2 = 0.182;
• A260nm/2.2 = 0.364/2.2 = 0.165.

The experimentally obtained value of A230nm, equal to 0.191, does not fall within the outlined boundaries. Therefore, DNA is considered to be contaminated with salts.

e) If a DNA sample is contaminated with RNA molecules, the first can be isolated using the phenol-chloroform DNA extraction technique, which uses RNases and low pH, in which the DNA is precipitated and the RNA remains in the aqueous phase.