BABS 2413 MOLECULAR BIOLOGY

Name: Chong Khai En

Student ID: 15WAU08702
Programme: RBS 2- Group 1
Date: 12-10-2015
Title: Lab 1- Spectrophotometry of DNA and RNA.
Objective:
1. To determine the absorbance of different concentration of DNA solution.
2. To study the thermal denaturation and annealing of DNA.
3. To measure the concentration and purity of DNA samples using spectrophotometer.
Introduction:
DNA is a polymer. The monomer units of DNA are nucleotides, and the polymer is known as
a "polynucleotide." Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing
base attached to the sugar, and a phosphate group. There are four different types of nucleotides found
in DNA, differing only in the nitrogenous base. The four nucleotides are given one letter
abbreviations as shorthand for the four bases. A is for adenine, G is for guanine, C is for cytosine and
T is for thymine.
A spectrophotometer is used to measure the amount of light that a sample absorbs. The
instrument operates by passing beam of light through a sample and measuring the intensity of light
reaching a detector. There is a chance the analyte will absorb the photon when a photon meets
analyte molecule. Therefore, this absorption reduced the number of photons in the beam of light and
reducing the intensity of the light beam.
The absorbance of isolated nucleotides single stranded DNA is greater than that of doublestranded DNA. With the absorption of double stranded DNA being approximately 40% less than
would be exhibited by a mixture of free nucleotides of the same concentration. This difference is due
to the structural properties of the nucleic acid and is called the hypochromic effect. This is due to the
hydrophobic and dipole-dipole interactions between the electrons of the individual bases causes their
stacking in the parallel array of the double helix. In single stranded DNA, the bases have a tendency
to stack on top of one another but this is not applied in double stranded DNA.
Wavelength of 260nm is used to estimate concentration of DNA in the sample. This is
because DNA absorbs light most strongly at 260nm. On the other hand, wavelength of 280nm is used
an as indicator of protein contamination since tyrosine and tryptophan residues absorb strongly at
this wavelength. Absorbance generated at 280nm is used in the ratio A260:A280 which estimates the
DNA purity. Furthermore, high absorbance at wavelength 230nm can be indicative of carry-over of
either guanidium salts phenol (used in phenol/chloroform extractions) that known to absorb strongly
at 230nm.

Methods:
DNA had prepared by lab assistant. TE buffer was used to dissolve DNA sample in a beaker.
Students were divided to four groups to carry out different parts of serial dilution for 500, 100, 50,
10g ml-1 of sample DNA. After dilution, each different concentration of DNA was placed into
Nanodrip to measure absorbance at 260nm, A260. The results were recorded down in Table 1. 500g
ml-1 of DNA had chosen by our group and 0.5ml of DNA solution was transferred into each of three
test tubes-- one test tube (Tube A) was maintained at room temperature, another remaining two test
tubes (Tube B and Tube C) were placed in 90oC water bath for 15 minutes. After the incubation
period, the tubes were removed. Tube B was quick cooled in an ice bath while Tube C was slow
cooled to room temperature over a period of about half an hour. The absorbance of DNA solution
was measured and recorded at wavelength (nm) of 200, 260 and 300 on each of the three tubes in
Table 2.
Results:
(i) Absorption spectrum for 10g ml-1 DNA.

(ii) Absorption spectrum for 50g ml-1 DNA.

(iii) Absorption spectrum for 100g ml-1 DNA.

(iv) Absorption spectrum for 500g ml-1 DNA.

Table 1: Table of DNA concentration and absorbance.

Tube
1
2
3
4

DNA concentration (g ml-1)

10
50
100
500

A260
0.093
0.504
0.949
4.821

Graph 1: Graph of absorbance against DNA concentration.

Graph of Absorbance(A260) against DNA Concentration

6.00
5.00
4.00

Absorbance (A260)

3.00
2.00
1.00
0.00
0

50

100

150

200

250

300

350

400

450

500

DNA concentration (g ml-1)

Table 2: DNA absorbance of different wavelength and different condition.

DNA Absorbance
Wavelength
Room temperature
Quick cool after 90oC
Slow cool after 90oC
(nm)
(Tube A)
(Tube B)
(Tube C)
200
0.993
1.000
1.909
260
3.535
3.505
3.570
300
0.176
0.162
0.160
Graph 2: Graph of DNA absorbance against wavelength.

Graph of Absorbance against Wavelength

4.000
3.500
3.000
2.500
2.000

Room temperature
Absorbance

Quick cool after 90oC

Slow cool after 90oC

1.500
1.000
0.500
0.000
200

210

220

230

240

250

260

270

280

290

300

Wavelength (nm)

Discussion:
DNA concentrations can be accurately measured by UV absorbance spectrometry. From
Graph 1, we can clearly see that the amount of UV radiation absorbed by a solution of DNA is
directly proportional to the concentration of DNA sample, usually absorbance is measured at 260 nm.
This is because 260nm is the wavelength at which an absorbance of 1.0 corresponds to 50 g of
double-stranded DNA per ml. From Graph 1, we know that the absorbance increases with the
increase of DNA concentration. High concentration of DNA contains more nitrogenous bases,
therefore higher absorbance.
The absorption of single stranded DNA is higher than the absorbance of double stranded
DNA. This is known as a hyperchromic effect. The hydrogen bonding between the paired bases in
the double helix limits the resonance behavior of the aromatic ring of the bases. Thus, the UV
absorbance of double stranded DNA decreases, which known as hypochromic effect. It results from
hydrophobic and dipole-dipole interactions between the electron systems of the individual bases
made possible by their stacking in the parallel array of the double helix. In single stranded DNA, the
bases have a tendency to stack on top of one another but this is maximized in double stranded DNA.
It is these interactions and stacking arrangement that stabilise the helical structure of DNA rather
than hydrogen bonding which determines the specificity of base pairing. Both native and denatured
DNA is capable of absorbing UV light at a wavelength of 260nm due to the aromaticity of
nitrogenous bases. Therefore, single stranded DNA has higher absorbance than double stranded DNA
at the same concentration. From the results obtained, we can see that denatured DNA absorbs more
ultraviolet light than regular double helix DNA. This is because denaturation of DNA disrupts
stacking of nitrogenous bases in native DNA which interferes with UV absorption, allowing for more
absorbance by the bases.

When the DNA in test tube B and C was heated, the energy of the heat pulls the two strands
of DNA apart. This process is called denaturation. The denaturation of the ends of the molecule, and
of more mobile AT-rich internal regions, will destabilize adjacent regions of helix, leading to a
progressive melting of the whole structure at a temperature corresponding to the midpoint of the
smooth transition, known as melting temperature. The melting temperature depends on both the
length of the DNA, and the nucleotide sequence composition. Higher guanine (G) and cytosine (C)
content has higher melting temperature. This is because the triple hydrogen bonds between G and C
need more energy to disrupt than the double bonds between Adenine (A) and Thymine (T).
The separated two strands still have the same nucleotide sequences, so they are
complementary. When the tubes were cooled, the random molecular motion will reform the double
stranded DNA. This process is called annealing. Only the complementary strands will come together
after identifying specific strands of DNA in mixture. The thermal denaturation of DNA may be
reversed by cooling the solution. The rate of cooling can affect the outcome. Rapid cooling allows
only the formation of local regions of double stranded DNA, formed by the base pairing or annealing
of short regions of complementarity within or between DNA strands, thus decrease in A260 is rather
small. There is not enough time for nitrogenous bases to reform. In this case, the bases form random
recombination with nearby bases and the single stranded DNA does not form the double helix
structure properly and orderly. The extinction of the solution at room temperature is higher than the
original DNA solution before heating, and thus higher absorbance. If the DNA solution is cooled
slowly, there is enough time for bases on the single stranded to reform the base pairs. The two
threads recombine and the cooling curve is supposed to be superimposed on the melting curve. This
annealing of regions of complementarity between different nucleic acid stands is known as
hybridization.
The purity of a DNA solution can be determined using a comparison of the optical density
values of the solution at various wavelengths. The ratio of the readings at 260 nm and 280 nm:
A260/A280; provides an estimation of the DNA purity with respect to contaminants that absorb UV
light, such as proteins or RNA. Ratio of more than 1.5 was considered adequate while between 1.7
and 2.0generally represents a high-quality DNA sample. For pure DNA, the observed A260/A280
ratio will be near to the value of 1.8. For Tube A, the A260/A280 ratio is 1.54, A260/A280 ratio for
Tube B is 1.52, and A260/A280 ratio for Tube C is 1.55. A260/A280 ratios below 1.8 often indicate
the presence of a contaminating protein or phenol, the purity of DNA obtained was considered very
low possibly. Alternatively, protein or phenol contamination is indicated by A230/A260 ratios greater
than 0.5. Therefore, we can say that all three DNA solutions have protein or phenol contamination.
There are some precautions to improve the purity of DNA solution. Gloves must be worn
throughout the experiment to prevent contamination. The apparatus and glassware were cleaned
thoroughly before handling DNA sample. On the other hand, talking, sneezing, and coughing were
avoided over the DNA sample to prevent contamination.

Conclusion:

The absorbance of isolated nucleotides single stranded DNA is greater than that of doublestranded DNA. This difference is due to the structural properties of the nucleic acid and is called the
hypochromic effect. The strands of DNA can be seperated by heat, this is known as denaturation. The
thermal denaturation of DNA may be reversed by cooling the DNA. The random molecular motion
will reform the double stranded DNA. This process is called annealing. High quality, intact pure
DNA is required for many applications; therefore care must be taken to ensure reliable and accurate
results. The purity of a solution of DNA can be determined using a comparison of the optical density
values of the solution at A260/A280 ratio, pure DNA will be near to value 1.8.

References:
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2. Chapter 5, An Introduction to DNA: Spectrophotometry, Degradation, and the 'Frankengel'
Experiment, written by William Clark and Kimberley Christopher, viewed on 12th October 2015.
Available at:
http://www.ableweb.org/volumes/vol-22/5-clark.pdf
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http://www.scribd.com/doc/24147687/Quantification-of-DNA#scribd
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http://www.answers.com/Q/Why_denaturation_of_DNA_is_followed_by_rapid_cooling
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http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/pg2.html
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