Spectrophotometry Lab # 2 UV Spectrophotometry of DNA, RNA, and Proteins

Purpose
Molecular biologists routinely work with DNA, RNA, and proteins and have devised some simple, fast spectrophotometric assays for these molecules. The purpose of this e ercise is to use the !" absorbance of biological samples to obtain #ualitative and #uantitative information about those samples. $n this spectrophotometry e ercise you will%

Measure the absorbance at &'( nm to #uantify DNA. )alculate A&'(*A&+( ratios to estimate the purity of a DNA preparation. ,perate a spectrophotometer in the !" range.

Brief Bac !round Re"ie#

A$ %he UV Absorbance Spectra of Nuc&eic Acids and Proteins Most biological molecules do not intrinsically absorb light in the visible range, but they do absorb ultraviolet light. -iologists take advantage of !" absorbance to #uickly estimate the concentration and purity of DNA, RNA, and proteins in a sample. $t is possible to #uantify the amount of DNA in a sample by looking at its absorbance at a wavelength of &'(nm or &+(nm .in the !" region/. The !" method described in this e ercise is not highly accurate but it is very widely used since it is easy, #uick, and little DNA is re#uired. 0roteins have two absorbance peaks in the !" region, one between &123&4( nm, where peptide bonds absorb, and another at about &+( nm due to light absorption by aromatic amino acids .tyrosine, tryptophan and phenylalanine/. )ertain of the subunits of nucleic acids .purines/ have an absorbance ma imum slightly below &'( nm while others .pyrimidines/ have a ma imum slightly above &'( nm. Therefore, although it is common to say that the absorbance peak of nucleic acids is &'( nm, in reality, the absorbance ma ima of different fragments of DNA vary somewhat depending on their subunit composition. 5igure 1 shows the !" spectra for DNA, RNA, and proteins. ,bserve that although proteins have little absorbance at &'( nm, both proteins and nucleic acids absorb light at &+( nm. Therefore, if nucleic acids and proteins are mi ed in the same sample, their spectra interfere .overlap/ with one another. Table 1 below summari6es the !" wavelengths relevant to the measurement of nucleic acids and proteins

'()UR* +

'i!ure +$ UV ABS,RBAN-* SP*-%RU. ',R DNA AND PR,%*(NS. A. DNA. b. 0rotein .-7A/. ). The absorbance spectrum for a mi ture of DNA and protein. Distinct DNA and protein peaks cannot be resolved.

TA-89 1 !" M9A7!R9M9NT7 ,5 DNA, RNA AND 0R,T9$N7 :A"989N;T< 7$;N$5$)AN)9 ),MM9NT7 &123&4( nm Minimum absorbance for Measurements are generally nucleic acids not performed at this 0eptide bonds in proteins wavelength because absorb light commonly used buffers and solvents, such as Tris, also absorb at these wavelengths. &'( nm Nucleic acids have 0urines absorbance ma imum absorbance ma imum is slightly below &'(= pyrimidines ma imum. is slightly above &'(. 0urines have a higher molar absorptivity than pyrimidines. Therefore, the absorbance ma imum and absorptivity of a segment of DNA depends on its base composition. 0roteins have little absorbance at this wavelength. &>( nm 0henol absorbs strongly 0henol may be a contaminant in nucleic acid preparations. &+( nm Aromatic amino acids Nucleic acids also have some absorb light absorbance at this wavelength. 4&( nm Neither proteins nor nucleic !sed for background acids absorb correction since neither nucleic acids or proteins absorb at this wavelength. B$ -oncentration .easurements of Nuc&eic Acids and Proteins in a Samp&e Determining concentration in a ?pure@ sample containing only proteins or nucleic acids, involves constructing a standard curve. $t is possible to determine the concentration of nucleic acids or proteins based on their absorbance at a wavelength of &'( nm or &+( nm respectively. A calibration curve using standards of known concentration can be constructed. 5or accurate results, the standard curve should be prepared using the protein of interest or DNA that is similar to that in the sample being measured. The linear range for DNA values is reported to be from about 23

2( Ag*m8. Depending on the protein, !" analysis of proteins at &+( nm has a linear range from about (.1 3 2 mg*m8. The average absorptivity constants for proteins and nucleic acids lead to the following relationships%

(f a samp&e containin! pure doub&e/stranded DNA has an absorbance of + at 201 nm, then it contains appro2imate&y 31 4!5mL of doub&e /stranded DNA$ (f a samp&e containin! pure sin!&e/stranded DNA has an absorbance of + at 201 nm, then it contains appro2imate&y 66 4!5mL of DNA$ (f a samp&e containin! pure RNA has an absorbance of + at 201 nm, then it contains appro2imate&y 71 4!5mL of RNA$ Va&ues for proteins "ary$ A "ery rou!h ru&e is that if a samp&e containin! pure protein has an absorbance of + at 281 nm, then it contains appro2imate&y + m!5mL of protein$

5or e ample, 1 mg*m8 of bovine serum albumin is reported to have an A&+( value of (.>. Antibodies .which are a type of protein/ at a concentration of 1 mg*m8 are reported to have an A&+( between 1.42 and 1.&. ."alues from BAntibodies% A 8aboratory ManualB, by 9. <arlow, and D. 8ane, p. '>4. Academic 0ress, New Cork., 1D++./ -$ *stimation of the Purity of a Nuc&eic Acid Preparation $t is possible to use !" spectrophotometry to estimate the purity of a solution of nucleic acids. This method involves measuring the absorbance of the solution at two wavelengths, usually &'( nm and &+( nm, and calculating the ratio of the two absorbances%

An A&'(*A&+( ratio of &.( is characteristic of pure RNA. An A&'(*A&+( of 1.+ is characteristic of pure DNA. A&'(*A&+( ratio of about (.' is characteristic of pure protein

Therefore, a ratio of 1.+ 3 &.( is desired when purifying nucleic acids. .Note that this method does not actually distinguish DNA and RNA from one another./ A ratio less than 1.> means there is probably a contaminant in the solution, typically either protein or phenol. Table & below summari6es the various !" methods described in this e ercise.

TA-89 & A00R,E$MAT$N; T<9 ),N)9NTRAT$,N AND 0!R$TC ,5 DNA, RNA ,R 0R,T9$N $N A 7AM089 -oncentration of doub&e/stranded DNA 9 :31 4!5mL; < :absorbance at 201 nm; -oncentration of sin!&e/stranded DNA 9 :66 4!5mL; < :absorbance at 201 nm; -oncentration of RNA 9 :71 4!5mL; < :absorbance at 201 nm; Turbidity causes an apparent increase in the absorbance of a sample leading to incorrect readings. To compensate for slight turbidity, a background correction can be used. Proteins and nucleic acids do not absorb at 320 nm. Therefore, if a sample absorbs at 320 nm, the absorbance is due to turbidity. The absorbance at 320 can be subtracted from the readings at 260 nm and 280 nm: -oncentration of doub&e/stranded DNA 9 31 4!5mL :A201 / A621; -oncentration of sin!&e/stranded DNA 9 66 4!5mL :A201 / A621; -oncentration of RNA 9 71 4!5mL :A201 / A621; -oncentration of protein 9 + m!5mL < the absorbance at 281 nm -oncentration of protein 9 +3 m!5mL < the absorbance at 2+3 nm Notes% alues for proteins !ary. The rule that " mg#m$ of protein has an absorbance of " is appro%imate. Tris and other common sol!ents also absorb light at 2"& nm. 'or this reason, 280 nm is far more commonly used for protein measurements. Purity An A2015A281 ratio of 2$1 is characteristic of pure RNA An A2015A281 of +$8 is characteristic of pure DNA An A2015A281 of 1$0 is characteristic of pure protein Notes% ( ratio of ".8 ) 2.0 is desired *hen purifying nucleic acids. This method does not actually distinguish +,( and -,( from one another. ( ratio of less than ".. means there is probably a contaminant in the solution, usually either protein or phenol.

Laboratory Acti"ities
Use =uart> cu"ettes in the UV ran!e$ ?and&e them #ith caution@ 1. Measure the A&'( and the A&+( for each sample. )alculate their A&'(*A&+( ratios. &. )alculate the concentration of the DNA, RNA, and protein samples based on their A&'( values. Record the concentrations of these samples based on what is written on their labels. 4. 0lace results for the class on the board. $nclude% a. b.
c.

A&'( and the A&+( for each sample.

A&'(*A&+( ratios for the samples.

Cour calculation of the concentrations of DNA, RNA, and protein in the samples based on the

A&'( values.

&. 4.

$f your results differ from those of your classmates, try to figure out why. $f all students use the same samples, the results should be the same. Try mi ing some protein with one of the DNA or RNA samples to see what this does to the absorbance values.

SU..ARA ,' R*SUL%S UV SP*-%R,P?,%,.*%RA ,' DNA, RNA AND PR,%*(NS

DNA A&'( A&+( A&'(*A&+( 0urityF )oncentration A&'( A&+( A&'(*A&+( 0urityF )oncentration

RNA

0R,T9$N A&'( A&+( A&'(*A&+( 0urityF )oncentration A&'( A&+( A&'(*A&+( 0urityF )oncentration

M$ET!R9

DiscussionB
1. :hat is the estimated concentration of DNA, RNA and protein in your samples based on their absorbances at &'( nmF The DNA, RNA, and protein solutions should be labeled with their concentration based on how much the preparer weighed out and dissolved. ;enerally we make these at (.1 mg*m8 for the DNA and RNA, and 1 mg*m8 for the protein. )ompare the estimated concentrations based on absorbance to the e pected concentration based on the preparation of the samples. Are they the sameF $f not, assume that the concentrations labeled on the solutions are the correct concentrations. &. These !" assays are very commonly used by molecular biologists. :hyF :hat information is obtained from these simple assaysF 4. :hat are the advantages and disadvantages of these !" methodsF )onsider their accuracy, precision, range, ease of use, cost, and any other factors you think are important.

;roup DNA

DNA GH

RNA A&'(, A&+(

RNA GH

0rotein A&'(, A&+(

Mi A&'(, A&+(

A&'(, A&+( A&'(*A&+(

based on A&'( A&'(*A&+(

based on A&'( A&'(*A&+(

A&'(*A&+(

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