Nucleic acid UV absorption & purity ratios (260/280 and 260/230)

We can use the absorption at a single wavelength, UV260, to calculate the concentration of nucleic acids, but you can learn a lot more also looking at the whole spectrum. Because molecules have overlapping spectra (e.g. both DNA & RNA absorb light of 260nm wavelength) you look to ratios. A couple key ratios for when you’re analyzing nucleic acid (DNA or RNA) quality are 260/230 & 260/280. 
Longer blog post : bit.ly/dnauvbeer
In terms of biomolecules, at 260 - dominant absorbers are DNA & RNA & at 280 - dominant absorber is protein 
 
260/280: tells you about protein “contamination” - I put contamination in quotes because if you’re analyzing protein, it’s the DNA that’d be the contaminant! 
 
260/230: tells you about “contamination” from proteins and/or things like phenol or salts that are left over from the purification process 
 
But why? Where does that absorption come from? 
 
DNA & RNA only have 4 letters each, and all of them absorb 260nm light, but it’s their unique part that does the absorbing - adenine, guanine, cytosine and thymine/uracil bases. These are aromatic rings (aromatic rings are rings where there’s some communal electron sharing that stabilize them - more here: bit.ly/phenylal.... This electron delocalization through resonance reduces the cost to move (to promote an electron), so aromatic rings are often present in things like dyes. 
 
Resonance is also involved in why proteins absorb light. Proteins peak at 280 & 230. The parts of proteins that absorb at 280 are aromatic rings (sound familiar?) Only 3 of the 20 common amino acids have these - Tryptophan (Trp) & Tyrosine (Tyr) are the major contributors - Trp the most so - Phenylalanine has a ring too, but it doesn’t absorb here as much. Cysteine crosslinks can also absorb, where applicable - and different proteins have different numbers of all these. So different proteins absorb 280nm light differently, which is reflected by different extinction coefficients. Proteins also absorb at 230nm and that absorbance is from the generic backbone part - corresponds to absorbance by the peptide bonds linking the letters. These peptide bonds also have resonance, but not as much as rings do, and they absorb ~190-230nm. More on using UV to measure protein concentration here: bit.ly/proteinm...  
 
DNA & RNA bases all absorb at 260, but to different extents. If you measure the 260/280 ratios for each nucleotide separately you get: Guanine: 1.15; Adenine: 4.50; Cytosine: 1.51; Uracil: 4.00; Thymine: 1.47 
 
one thing you might notice is that uracil (U) which is in RNA but not DNA has a much higher 260/280 than its DNA counterpart, thymine (T) - as a result, pure RNA has a higher 260/280 ratio than pure DNA 
 
If you take the weighted average of the different bases into account, pure RNA should have an A260/A280 ratio of ~2 - it absorbs ~ 2 times more 260nm light than 280nm light) & pure double-stranded DNA, which only absorbs ~1.8 times more at 260 vs 280, should have an A260/A280 ratio of ~1.8 
 
Because DNA absorbs so strongly at UV260, where protein doesn’t, it’s relatively easy to see if you have DNA in your protein prep, but it’s harder to tell if you have protein in your DNA prep - 260 will dominate the 260/280 ratio 
 
You also want to look at the 260/230 values. For pure nucleic acids, these are usually ~2.0-2.2  (you want greater than 1.8)
 
So far, the “contaminants” we’ve discussed are biomolecules that co-purified with the molecule you purified, but contaminants can also come from chemicals you used in the purification process. For example, phenol & chaotropic salts like guanidine are often used when purifying nucleic acids, such as with phenol-chloroform or Trizol extractions (more here: bit.ly/2Xj4Zyc ) - Also carbs such as glycogen you might have used as a coprecipitant to help with pelleting.
 
These absorb strongly at 230nm, which (in addition to the fact that proteins also absorb there) is why a low 260/230 ratio could be concerning if you’re looking at a nucleic acid prep. 
 
Now, as promised, let’s get discuss how we can convert absorbance to concentration using Beer’s Law. We can characterize how much a molecule absorbs light at any wavelength (we usually choose its “favorite” - peak absorption) using its extinction coefficient. 
 
The equation is: A = εcl 
 
A = absorbance 
 
ε = extinction coefficient (aka molar absorptivity coefficient) - specific for particular molecule & particular wavelength; units of L mol⁻¹cm⁻¹ 
 
c = concentration (in mol/L) - this is molarity - a mole is just a chemist’s “baker’s dozen” - it’s Avogadro’s number (6.022 x 10²³) of something - solute molecules or donuts, it’s just a number bit.ly/c1v1equa...
 
l = path length (in cm) 
 
rearrange that a bit and you get  
 
c= A/εl 
 
finished in comments

[@insanity.defeated]

I just did 6 midi prep today, and found this so helpful 😍

[@thebumblingbiochemist]

Great to hear!

[@mytoeisgreen]

Good video as usual! I would daresay though, that for quantification purposes if you have access to a Qubit, you should use it over UV-VIS whenever possible, especially in cases where a more stringent purification like PAGE gel excision is not undertaken. I've found that PCRs and T7s invariably leave behind free dNTPs/NTPs with spin column clean ups like the popular Qiagen kits and even with ethanol precipitations sometimes, and the free nucleotides will cause a erroneously higher reading on nanodrop.

[@thebumblingbiochemist]

Great points! Thanks for raising them!

[@thebumblingbiochemist]

Even though the base composition will affect the exact extinction coefficient (the measure of how well a specific something absorbs a specific wavelength - the thing you stick into Beer’s law to convert absorbance to concentration, you can still estimate extinction coefficients for “generic” DNA & RNA 
 
dsDNA: (0.02 μg/mL)^-cm 
 
ssDNA: (0.027 μg/mL)^-cm 
 
ssRNA: (0.025 μg/mL)^-cm 
 
To make these easier to use, there’s a shortcut. “Standard Coefficients” which assume a 1cm pathlength (if you’re using a different one, then divide the A by that. bit.ly/3dg5yDi  
 
Basically, since  
 
c= A/εl, then c = A/l * 1/ε 
 
if l = 1cm, then this is c = A * 1/ε 
 
So if we calculate 1/ε, then we can just multiply that by A to get the concentration. So, instead of remembering 0.02, 0.027, & 0.025, remember 
 
dsDNA: 1/0.02= 50μg/mL(ng/μL) -> A260 of 1.0 corresponds to 50μg/mL(ng/μL) of pure dsDNA. A260 of 2.0 corresponds to 100μg/mL(ng/μL) of pure dsDNA. etc…  
 
ssDNA: 33μg/mL -> A260 of 1.0 corresponds to 33μg/mL(ng/μL)of pure dsDNA. A260 of 2.0 corresponds to 66uμg/mL(ng/μL) of pure ssDNA. etc…  
 
note: 1/0.027 is actually ~37μg/uL so I honestly don’t know what’s going on here, but most sources say 33?!  
 
RNA: 40μg/mL (this one makes since again since 1/0.025=40) -> A260 of 1.0 corresponds to 40μg/mL(ng/μL) of pure dsDNA. A260 of 2.0 corresponds to 80μg/mL(ng/μL) of pure ssRNA. etc…  
 
From these values you can see that strandedness matters. When DNA is double stranded, its bases (the parts that absorb the light) are “hidden” & “busy” binding to the base on the other strand. But with single stranded DNA, the bases are out in the open and free to absorb. As a result you get a hyperchromic shift -> single-stranded DNA (ssDNA) absorbs more UV light than double-stranded DNA (dsDNA) & free nucleotides absorb more strongly than either of those. 
 
final note: You also want to look at the absorbance at 320nm. This tells you about the turbidity of your sample - if your sample is “cloudy” - suspended particles in the solution can scatter the light, preventing it from reaching all your molecules. So this gets adjusted for. So for DNA, you can get concentration using this equation: concentration (ug/mL) = (A260 reading - A320 reading) x dilution factor x 50ug/mL 
 
 references:
QIAGEN Newsletter March 15, 2010 www.qiagen.com/us/~/media/1ea8aec3bfa24543a28fcaea25986514.ashx
Thermo T042‐TECHNICAL BULLETIN, Assessment of Nucleic Acid Purity medicine.yale.edu/keck/dna/protocols/tube/t042-nanodrop-spectrophotometers-nucleic-acid-purity-ratios_407666_284_7035_v1.pdf
Thermo T042‐TECHNICAL BULLETIN, 260/280 and 260/230 Ratios, www.uvm.edu/~vgn/microarray/documents/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
more about how nano drops work: blog: bit.ly/nanodrop_science ; TH-cam: th-cam.com/video/Yrn8AO2qfhk/w-d-xo.html
for more details on nucleic acid precipitation: bit.ly/na_precipitation ; TH-cam: th-cam.com/video/RE6Sd-xHoks/w-d-xo.html  
more about co-precipitants like glycol blue: TH-cam: th-cam.com/video/SSLQcIvz6wQ/w-d-xo.html  
more about RNA extraction: blog form:  bit.ly/trizolRNAextraction ; TH-cam: th-cam.com/video/zpJEYXYls2E/w-d-xo.html   
more about spin column purification: bit.ly/spincolumns
  
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@wendelun]

Thanks for the video about UV absorption. I’ve been thinking a lot about this recently. I’ve got identical purified protein samples that are showing different absorptions at 280nm. I think I’ve narrowed it down to oxidation of the tryptophan residues. The sample with the lower absorption also has less tryptophan, and so it will absorb less. Your video helped me better conceptualize it. Thanks.

[@thebumblingbiochemist]

Glad to hear!

[@sukantshetty2146]

any primary source of reference for these ratios?

[@thebumblingbiochemist]

QIAGEN Newsletter March 15, 2010 www.qiagen.com/us/~/media/1ea8aec3bfa24543a28fcaea25986514.ashx
Thermo T042‐TECHNICAL BULLETIN, Assessment of Nucleic Acid Purity medicine.yale.edu/keck/dna/protocols/tube/t042-nanodrop-spectrophotometers-nucleic-acid-purity-ratios_407666_284_7035_v1.pdf
Thermo T042‐TECHNICAL BULLETIN, 260/280 and 260/230 Ratios, www.uvm.edu/~vgn/microarray/documents/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf

[@sukantshetty2146]

Thank you