RANELLE JANINE L.

ASI
BIO 120 S-5L
EXERCISE 9. Genomic DNA Isolation & Exercise 10. Polymerase Chain Reaction (PCR)
Cell and molecular biology is a science based on the various systems of a cell resulting to its
regulation, maintenance and function. Many of these systems involve genetic information hence the
study of the DNA is an essential part of this field. To able to analyze DNA, it must first be isolated and
purified from its natural environment filled with biological molecules and compounds that cause
physical or chemical interference in an experimental set-up. Several protocols have been established
to efficiently extract DNA, one of which, the CTAB method, was performed and studied in this exercise.
(De la Parte and Dita, 2014)
As in all biological studies, the analysis of DNA requires samples in high enough amounts. A
method called Polymerase Chain Reaction (PCR), which is significantly dependent on the size
distribution and quality of the extraction of DNA, has been developed to produce exponential copies of
or simply, to amplifyDNA. PCR follows basic elements of natural DNA synthesis and replication
processes and uses them in a more simplified manner easily duplicated in the laboratory such as
denaturation by heating and exposure of DNA to polymerases in a reaction mix. The introduction of
PCR has revolutionized and accelerated the field of molecular biology and has expanded the
applications of DNA analysis to various biological and medical procedures (McPherson and Moller,
2006).
In the CTAB method for DNA extraction, the buffer used to suspend the DNA contains the
following: (1) hexadecyl trimethyl ammonium bromide (CTAB, from which the method is named after),
a detergent which disrupts fluid membranes, (2) EDTA, a chelating agent that acts on magnesium ions
required for DNase activity, (3) Beta-mercaptoethanol, which helps in denaturing proteins by breaking
disulfide bonds and removing tannins and polyphenols present in the crude extract, (4) PVP, which
removes phenolic compounds in plant DNA extracts, and (5) Tris-HCl at pH 8 and NaCl, which aids in
precipitation by neutralizing the negative charges on the DNA. (Gaikwad, n. d.) In this experminent,
PVP and Beta-mercaptoethanol was not used as the chemicals are too dangerous for undergraduate
students to handle.
A classic method for DNA purification is phenol-chloroform extraction in which the nucleic acid
solution is extracted by successively washing with a volume of chloroform:isoamyl alcohol. The phenol
removes protein contaminants by denaturing them as they accumulate in the organic phase during
intermittent centrifugation. DNA is non-soluble in alcohol and is hence precipitated upon addition of
100% isopropanol or ethanol. The DNA solution is finally washed using 70% of the alcohol used to
remove salt and alcohol remnants. (Gaikwad, n. d.)
The quality of extracted DNA can be enhanced by following some additional steps: grinding of
leaf samples in liquid nitrogen to lyophilise them as dry tissue can be efficiently disrupted while DNA is
unhydrated, taking care to only collect the aqueous phase of the solution after addition of
chloroform:isoamyl as it contains all the DNA and the less noticable interphase underneath it contains
contaminants, and the use of fresh sterile tips and tubes for every steps to minimize re-entry of
previous contaminants in the solution. (Zidani et. al, 2005)
In the PCR method, a reaction mix is prepared using the following reactants: (1) PCR buffer
containing the dipolar Tris-HCl which optimizes the pH in the mix for the action of DNA polymerases,
KCl, which assists in primer-template annealing and most importantly, MgCl 2, which affects the
specificity and efficiency of the reaction as it is a cofactor required for the action of Taq DNA
polymerase, (2) dNTP mix, a stock solution containing equimolar concentrations of the four types of
dNTPs to be used as the building blocks of new DNA strands to be primed by the polymerases, (3)
sense and antisense primers, which are complementary to the ends of the two strands of the DNA
target to signal where DNA priming should begin, (4) Taq polymerase, a thermostable DNA polymerase

able to withstand high temperatures that the mix will be subjected to during the main steps, and (5)
genomic DNA from which the target DNA fragment to amplified will be replicated from (McPherson &
Moller, 2006). Sterile nanopure water was used as a blank for comparison with the reactions happening
in the tube containing genomic DNA.
PCR proceeds in three main steps: denaturation, annealing and extension or synthesis.
Denaturation is carried on by heating the double-stranded DNA at 94C to separate the
complementary strands that will serve as template in further cyclings. Pre-denaturation is sometimes
done at the same temperature to ensure complete separation of strands. Annealing then occurs upon
rapid cooling of the solution, allowing oligonucleotide primers to hybridize to the template. In this
phase, however, the single strands of the template are too long and complex to be able to completely
reanneal spontaneously. The gene fragment to be amplified will completely form double-stranded
fragments upon further cycling of this step and the extension step. The extension step involves
heating of the reannealed DNA to 72C, the temperature at which the thermostable DNA polymerase in
the mix will operate most efficiently in synthesizing new DNA strands.

Gel Electrophoresis exposes the molecular sizes of different DNA fragments as the lightest or
shortest fragments travel fastest down the gel and the heaviest or largest fragments travel most
slowly and are left near the top part of the gel. In this run, samples A-F show almost identical bands,
indicating that all six samples are the same DNA. Two bands are found in each well which implies that
each sample has two differently sized DNA fragments. The higher bands are most likely genomic DNA
and the lower, larger bands are DNA in similarly sized fragments in higher concentrations. (Magdeldin,
2012)

The result of spectrophotometry using BioTek Gen5 indicates raw absorbance values, meaning
they were read without subtracting the blank values, absorbance corrected to 1 cm pathlength and
subtracted with blank values, the 260/280 ratio used to identify the type of sample, and calculated
DNA concentration to quantify the amount of the DNA present in the sample. (Brescia & Banks, 2012)
Nucleic acids absorb maximally at 260 nm, the report indicated values on the 260 column to
be higher than readings at other wavelengths, hence initially confirming the sample as nucleic acid.

According to Brescia and Banks (2012), the 260/280 ratio for pure DNA is 1.8 to which all of the values
in the column are near, further confirming the identities of all the samples in the wells to be nucleic
acids. Elevated ratios usually indicate presence of RNA, while ratios below 1.8 signal presence of
contaminating protein or phenol; hence most of the samples were not pure. Concentrations of dsDNA
are computed by multiplying the 260 value by the samples dilution factor then by dsDNAs correction
factor, 50. The results show high concentration of DNA in the samples as dilution was kept to a
minimum during preparation.

In this sample run, the band in the second lane indicates that it is longer than 1,500 kbp as it is
genomic DNA that was not fragmented. However, the DNA from the third to the seventh lane travelled
down the gel as they were smaller due to fragmentation during amplification. The bands on the five
lanes also travelled different distances indicating that fragment lengths vary as the DNA polymerase
replicated (and eventually fragmented) the DNA strands at different loci where the primer attached on
each species genome. The site where the primer attached for Species A was around 700 kb from the
tip of the segment, and hence resulted to a fragment that is 700kbp long. Species Bs fragment size
was about 550 kbp, Species C and Ds are the same at around 300 kbp (hence, they have same gene
loci), and Species Es was about 250 kbp.
Negative controls in gel electrophoresis serve the purpose of indicating the presence of
contaminants in the reaction mix used in the PCR or in the gel itself. In this run, there is no indication
of any contaminants in the samples.

REFERENCES:
De la Parte, E. M. & Dita, M. (2014) Basic Aspects of Molecular Biology and DNA Extraction [Lecture
slides]. Retrieved from
http://www.fao.org/fileadmin/templates/agphome/documents/Pests_Pesticides/caribbeantr4/10Molecula
rBiology.pdf
Gaikwad, A. B. (n. d.) DNA extraction: Comparison of Methodologies. Retrieved from
http://www.nbprgr.ernet.in
McPherson, M. J. & Moller, G. S. (2006) PCR (2nd ed.) New York, NY: Taylor & Frnacis Group
Schleif, R. (1993) Genetics and Molecular Biology (2nd ed) London: The Johns Hopkins University Press.

Zidani, S., Ferchichi, A. and Chaieb, M. (2005) Genomic DNA extraction method from pearl millet
(Pennisetum glaucum) leaves. African Journal of Biotechnology Vol. 4. Retrieved from
http://www.academicjournals.org/AJB
Brescia, P. J. & Banks, P. (2012). BioTek Tech Note: DNA Quantification using Gen5TM. Winooski, VT:
BioTek Instruments, Inc.
Magdeldin, S. (ed.) (2012) Gel Electrophoresis: Principles and Basics. Rejika, Croatia: InTech Books and
Journals.