GROUP 1 DECEMBER 9, 2019

BATUANG, MARK RYAN C. 0943L BSMLS 2-J

COLLADO, JEFF MICHAEL B.
AFALLA, MARIA PAOLA S.
ALCANTARA, CIELO M.
AL MASRI, SALMA
ALOS, JESSICA GRACE A.

“An Acidic Chemical Environment is NOT Essential for Better Electrophoretic Mobility of DNA
During Electrophoresis”

Introduction

Electrophoresis is often used to separate and identify components of a mixture of biological

molecules. The rate and direction of movement for a specific molecule in an electrical field depends upon
its size and charge. When placed in a pH gradient, a biological molecule will migrate in an electric field until
it reaches a position within the gradient at which it has no net charge. The pH at this position is referred to
as the isoelectric point or pI for that molecule (Friscia, Turchi, Herfer, 1992)

Drabik and Selberring (2016) stated that the observed migration is also affected by the type
of electrophoresis buffer, especially its ionic strength. The electrical conductance of the gel is dependent
on the presence of various ions, including those present in the sample. Gel polymerization is based on
heating the agarose solution to a temperature higher than 40°C. Polysaccharide is solidified again after
cooling to room temperature.

The agarose gel matrix is negatively charged because of covalently attached sulfate, carboxylate
and/or pyruvate groups. These negatively charged residues are surrounded by positively charged
counterions from the buffer. When an electric field is applied, some of the cations in the diffuse double layer
near the gel fibers migrate toward the cathode, carrying along buffer and solvent molecules. The net result
is a flow of the solvent toward the cathode, called the electroosmotic flow (EOF). DNA molecules are
negatively charged and migrate in the opposite direction toward the anode (Stellwagen, N and Stellwagen
E, 2008)

Data

Electrophoresis is a separation technique that utilizes buffers to keep the gel at a stable ph. By
providing a reservoir for a weak acid and base in the form of buffers, the Ph of the solution will be kept
within a narrow range. This prevents a drastic change in the structure of protein and or nucleic acid that
may lead to improper separation when subjected to significant pH changes
Qualifier

1. Provided that the environment of the DNA during the gel electrophoresis is not within the optimal
pH conditions, extreme Ph affects the structure of all macromolecule, in DNA, at high temperature,
the solution is rich in hydroxide ion, and these negatively-charged ions can pull hydrogen ions off
of molecules like the base pairs in DNA. This process disrupts the hydrogen bonding that holds the
two strands together, causing them to separate. On the other hand, at low pH, the hydrolysis and
depurination happens because of acid-catalyzed SN1 reaction mechanism. Nucleophilic centers
on Guanine and Adenine are N1, N3, N7 and 6 th position in which the acid attacks electrophilicity
on N7 position. Extremely low Ph digest the DNA completely and that is why our stomach pH is
low. DNA should be on its optimum condition to function properly. (Bhatt, 2016). Gel electrophoresis
is a technique commonly used in laboratories to separate charged molecules like DNA, RNA,
proteins, according to their size. Electrophoresis is performed in buffer solutions to reduce pH
changes due to the electric field, which is important because the charge of DNA and RNA depends
on pH. (lee, 2015). Charged molecules move through a gel when an electric current is passed
across it. An electric current is applied across the gel so that one end of the gel has a positive
charge and the other end has a negative charge. The movement of charged molecules (migration)
is towards the opposite charge (European bioinformatics institute, 2016). A molecule with a
negative charge will therefore be pulled towards the positive end. Thus, a change in pH will affect
the charge of DNA will in turn affect the movement of the molecules.

Warrant

1. The optimal pH for DNA is from 7.5-9.5 (Worthington, n.d.). lower pH will cause DNA to hydrolyze
where in the phosphodiester bonds break and the bases break off. Higher pH causes DNA to
denature with the phosphodiester backbone intact (university of Hawaii, ND), thus electrophoretic
mobility will only be efficient if the environment is within optimal pH to prevent denaturation of
DNA. Biological macromolecules evolved to perform their function in specific cellular environment
therefore, they should be adapted to the biophysical characteristics of the corresponding
environment, one of them being the characteristic pH. Many macromolecular properties are pH
dependent, such as activity and stability. pH is a significant factor for DNA-nanoparticles, DNA-
nanoparticles, DNA-metal ions, and DNA binding interactions. Every biological process is pH
dependent reflecting the importance of the local pH on all processes in the cell (Talley et al.,
2010)
2. The electrophoretic mobility is dependent on external factors like electric field strength, viscosity,
gel concentration and temperature, and intrinsic properties of the molecule like charge density,
size and hydrophobicity.
3. The electrophoretic mobility of DNA is not affected by pH because the pH of these buffers is
neutral caused by the Tris-Acetate EDTA, the phosphate backbone of DNA which possesses a
net negative charge will migrates towards the anode

Backing

1. Electrophoretic mobility of solutes can be modified by changing the characteristics of the buffer
solution – pH, ionic strength and composition – by means of the addition of different types of
substances (Garcia et al., 2005). The pH of the electrolytic solution is a key parameter to achieve
a separation by capillary zone electrophoresis since it determines the degree of ionization and,
therefore, the relative mobility of the different analytes. As a consequence, a background
electrolyte with a good buffering capacity at a working pH should be chosen (Garcia et al., 2005)
2. Dextran remains in the sample wells; it does shift toward the negative pole at pH 3 and toward the
positive pole at pH 11. The ct-carbox~(l group has a pK of 2.18. 7 The pK of the a-amine group is
8.95/Since it is linked to the DNP moeity, the R-functional group or the ~-N is not affected by pH.
At pH 3, the et-carboxyl group has dissociated while the a-amino group remains protonated. The
net charge on this molecule is positive and it therefore migrates to the negative pole. At pH 7 and
 pH 11, the isoelectric point (5.56) for DNPlysine has been exceeded. The molecule exhibits a net
negative charge and therefore migrates to the positive pole. Buffers at pH 3 and pH 7 are lower
than the isoelectric point (pH 10.7) for cytochrome c. Under these conditions, the molecule has a
net positive charge and migrates to the negative pole. At pH 11, cytochrome c has a negative
charge and migrates to the positive pole. Blue dextran does not migrate into the gel at any pH. This
reflects the large size of this molecule. At pH 7.0, this molecule appears to carry no charge and it
remains equally distributed within the sample well. The chromophore of blue dextran acquires a
positive charge at pH 3 and a negative charge at pH 11. As described above, this causes the
molecule to become concentrated at the gel interface closest to the oppositely charged pole
(Friscia, Turchi, Herfer, 1992).
3. In the absence of ions and water is substituted for electrophoresis buffer in gel or in the
electrophoresis tank, electrical conductivity is minimal and DNA migrates slowly, if at all.
In buffer of high ionic strength electrical conductance become very efficient and significant
amounts of heat will be generated, even when moderate voltages are applied in the worst case,
the gel melts and the DNA denature.
4. The migration of DNA molecules towards anode occurs mainly due to the naturally occurring
negative charge carried by their sugar phosphate backbone and is primarily size-dependent and
decreases as their length increases and is proportional to the strength of the electric field

Conclusion

The electrophoretic mobility is dependent on external factors like electric field strength, viscosity,
gel concentration and temperature, and intrinsic properties of the molecule like charge density, size and
hydrophobicity. The optimal pH for DNA is from 7.5-9.5 (Worthington, n.d.). lower pH will cause DNA to
hydrolyze where in the phosphodiester bonds break and the bases break off. Higher pH causes DNA to
denature with the phosphodiester backbone intact. In buffer of high ionic strength electrical conductance
become very efficient and significant amounts of heat will be generated, even when moderate voltages
are applied in the worst case, the gel melts and the DNA denature.

References

Bhatt D, 2016, Effect of Acid or Base on Nucleic Acid, Retrieved from

https://www/slideshare.net/DhavalBhatt15/effect-of-acid-or-base-on-nucleic-acid?

Drabik A and Silberring J, 2016, Gel Electrophoresis, Proteomic Profiling and Analytical Chemistry
(Second Edition), Retrieved from https://www.sciencedirect.com/topics/pharmacology-
toxicology-and-pharmaceutical-science/agarose-gel-electrophoresis
Friscia, Turchi, and Herfer, 1992, The Influence of pH on Electrophoretic Mobility, A Laboratory
Investigation, Retrieved from https://onlinelibrary.wiley.com/doi/pdf/10.1016/0307-
4412(92)90020-M

Garcia, MA et al, 2005, Separation modes in capillary electrophoresis, Retrieved from

https://www.sciencedirect.com/science/article/pii
Stellwagen N, and Stellwagen E, 2008, Effect of the matrix on DNA electrophoretic mobility, Retrieved
from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643323/

University of Hawaii, ND, Physical and Chemical Structure of DNA, Retrieved from
https://www2.hawaii.edu/~johnb/micro/micr230_lecture%203.html

Westermeier R, ND, Gel Electrophoresis, doi: 10.1038/npg.els.0005335, Retrieved from

http://people.bu.edu/mfk/restricted566/electrophoresis.pdf
Worthington Biochemical Corporation, ND, DNA Polymerase, Taq, Retrieved from
http://www/worthington-biochem/DNAPTAQ/default.html

Collins, P. (2018). The Purpose of the Buffer in Electrophoresis. Retrieved from.

https://sciencing.com/purpose-buffer-electrophoresis-6613320.html

Yg (2016). What is gel electrophoresis?. Retrieved from. https://www.yourgenome.org/facts/what-is-

gel-electrophoresis