RAPID HUMAN GENOMIC DNA (gDNA) EXTRACTION FROM

WHOLE BLOOD FOR PCR ANALYSIS

Abstract

Deoxyribonucleic acid (DNA) extraction was first performed in 1869 and since then it has substantially advanced.
DNA extraction no doubt is one of the most vivacious technique or step required to perform many important
applications in the field of not just molecular biology but whole of the scientific disciplines. DNA can be obtain
from many sources like blood, bone, skin, hair, urine etc. but whole blood is one of the major sources to extract
DNA, although there are many protocols to extract DNA from the nucleus but these methods vary from manual to
more refined and rapid techniques including automatic DNA extraction technique. Based on the comprehensive
range of existing options, it would be idyllic to determine those that perform best in terms of cost-effectiveness and
time efficiency. We have reviewed DNA extraction history and the most commonly used methods for DNA
extraction from whole blood samples and experimented one of the most ideal methodology among all techniques.

Keywords: DNA, DNA Extraction, Gel Eelectrophoresis, Lab Protocols

Introduction:

Friedrich Miescher is known to be the pioneer to extract DNA in 1861, bizarre advances have been accomplished in
the field of DNA treatment since then. At the moment nucleic acids can be extracted from any type of biological
substantial such as tissues, cells and viruses. Furthermore, growing data of human genome is tiling the approach to
an active employment of pharmacogenomics and genetic-based analytical tests in medicine (Yuan, 2012)

Modern research improvements in genomic syndromes have imposed the pool of hefty sums of good quality DNA
that needs to be attained from diverse sample sources. DNA typing is presently the most indorsed method for the
particular identification of human bodily fluid stains found at crime scenes (Griffiths, 2014) . In a varied variety of
genetic studies, the commonly used method is to obtain genomic DNA from nucleated cells of peripheral blood; as a
result of the invasiveness of this approach, it might be challenging to acquire samples from the study subjects.

Fresh blood is one of the several existing sources to obtain genomic DNA (gDNA), and it has been broadly used in
amenities around the world (Bag, 2016). Therefore, I will emphasize DNA extraction protocols expending whole
blood samples. Concerns regarding collection, storage, and manual handling of human whole blood specimens is
also included in the scope of this publication and is mentioned briefly in the context. Sample handling is one of the
most crucial and sensitive step for the initiation of the process as it could potentially impact on the performance and
success of any DNA extraction technique chosen.

Initial development of DNA extraction techniques

In 1869, Friedrich Miescher experimented on leukocytes that he collected from the samples on renewed surgical
bandages and conducted experiments to disinfect and sort protein residues in these cells. During his experiments he
recognized an unusual substance in the nuclei, which he called “nuclein”. He then established two etiquettes to
separate cells’ nuclei from cytoplasm and to segregate this novel compound, nowadays known as DNA, which
contrasted from proteins and other cellular constituents. This scientific verdict, along with the isolation protocols
used, was published in1871 in collaboration with his mentor, Felix Hoppe-Seyler. (Griffiths, 2014)
Nonetheless, it was only in 1958 that Meselson and Stahl settled a monotonous laboratory technique for DNA
extraction. They performed DNA extraction from bacterial mockups of Escherichia coli using a salt density ascent
centrifugation protocol (Ghatak, 2013). Since then, DNA extraction practices have been altered to perform
extractions on many different types of
biological sources.

DNA extraction approaches follow some communal processes designed to accomplish effective disruption of cells,
denaturation of nucleoprotein complexes, inactivation of nucleases and other enzymes, removal of biological and
chemical contaminants, and finally DNA precipitation. Utmost of them follow analogous basic steps and consist of
the use of organic and nonorganic reagents and centrifugation methods. Finally, they have established into a
diversity of automated procedures and commercially available kits.

Many procedures have been circulated concerning DNA isolation from blood. Several of these distributed etiquettes
applied enzymes and organic solvents for extracting high quality DNA, bereft of PCR inhibitors, while others
counting salting out technique targeted toward sophisticated DNA yields. Therefore some protocols are lavish and
time consuming while others compromised with the DNA quality.

Main types of DNA extraction methods from human whole blood samples

The below mentioned table demonstrates the chief categories and subsections of DNA extraction procedures from
whole blood samples that are mostly used in research facilities globally. Laboratory reagents frequently used for
each phase of the nucleic acid extraction protocol are encompassed in this table permissible to highlight
correspondences and variances between them.
Materials and protocols for DNA extraction:

The first and foremost responsibility of a researcher working in lab is to clean up the extraction point where the
procedure is going to performed both before and after.

The technician should wear synthetic gloves, lab coat, glasses, mask and complete all the personal protective
equipment (PPE) requirements. After properly dressed up, he /she should clean the area with alcohol swabs or 20%
ethyl-alcohol spray. Afterwards check all the necessary and basic instruments needed for DNA extraction , these
include , Pipettes, tips , eppendroff, test tubes , falcon tubes, centrifuge , mini centrifuge , vortex , water and dry
baths, kingfisher purifier, spectrophotometer, incubator , cold storage. (Guha, 2017)

If all the instruments are complete start the DNA extraction procedure in a closed chamber to avoid any
contaminations. Discard the gloves after extraction and don’t reuse pipette tips in any step. Take almost 5ml of
blood in EDTA tube and store it under -70 degree centigrade for short period if you want to startt the process early
but for long period i.e above 7 hours keep the blood refrigerated at -20 degree centigrade.

DNA Extraction from Blood Sample

White Blood Cells (WBCs) from whole blood was segregated by lysing the red blood cells (RBCs) by using a
buffer (ammonium bicarbonate and ammonium chloride; Himedia) with minimal lysing effect on lymphocytes.
Three tomes of RBC lysis buffer was added to blood sample and assorted by vortexing and capsizing scrupulously
for 5 min and centrifuged (Eppendorf 5415R) at 40,000 g for 05 min. The supernatant was habitually castoff,
leaving behind 1 ml to preclude loss of cells. To the pellet, 3000 µl RBC lysis buffer was pipetted, and vortexed,
inverted, and centrifuged, steps were repeated two to three times until a vibrant supernatant and a clean white pellet
were obtained. After the final wash, the supernatant was discarded completely, and the pellet was resuspended in
500 l PBS, followed by addition of 400 l cell lysis buffer (10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl, 10%
SDS, pH 7.5) and 10 l proteinase K (10 mg/ml stock; Himedia). The sample was vortexed to dissolve the pellet
completely and incubated for 2 h at 56°C in a water bath (CW-30G; Jeio Tech) for lysis. An equivalent tome of
phenol (equilibrated with Tris, pH 8) was then added to the tube and assorted well by inverting for almost 1 min.
The tube was centrifuged at 10,000 g (at 4°C) for 10 min, and the aqueous upper layer was transferred to a fresh
tube containing equal volumes (1:1) of phenol and chloroform:isoamyl alcohol (24:1). The tube was mixed by
inverting for 1 min and centrifuged for 10 min at 10,000 g (at 4°C). The supernatant was then transferred to a fresh
tube, and 10 l of 10 mg/ml RNase A (Fermentas, Thermo Scientific) was added. The sample was incubated at 37°C
for 30 min before an equal volume of chloroform: isoamyl alcohol (24:1) was added and mixed by inverting the tube
for 1 min and centrifuging at 10,000 g (at 4°C) for 10 min. The supernatant was transferred to a fresh tube, and
twice the volume of absolute alcohol (Merck) was added and inverted gently a few times and chilled at 20°C, then
was centrifuged at 40,000 g at (4°C) for 05 min. The supernatant was discarded, 250 l 70% ethanol was added, and
the pellet was tapped gently, followed by centrifugation at 10,000 rpm for 10 min and decanting the supernatant
gently. (Ghatak, 2013) The pellet was air-dried in a laminar air flow, and the dried pellet was resuspended in 50 l
nuclease-free water or 1 TE buffer and frozen at 20°C or 80°C for storage.

Concentration and Purity Determination

A quantitative spectrophotometric assay of DNA was performed using a Cary 60 UV-visible spectrophotometer
(Agilent Technologies, Santa Clara, CA, USA). Absorbance was measured at wavelengths of 260 and 280 nm. The
absorbance quotient (OD 260/OD 280) provides an estimate of DNA purity. An absorbance quotient value of 1.8
ratio (R) 2.0 was considered to be good, purified DNA (Griffiths, 2014). A ratio of 1.8 is indicative of protein
contamination, where as a ratio of 2.0 indicates RNA contamination.

Gel Electrophoresis examination:

2% agarose gel was prepared by adding 10x TE Buffer , after setting up the gel in electrophoresis chamber ,
samples were loaded and after completion , following results were observed under Gel Doc.

Figure 2 the obtained bands indicates that the DNA is uncontaminated

and accurately extracted.

Agarose gel electrophoresis showed that the molecular weight of genomic DNA extracted was more than 10
kilobases with uniform brightness, suggesting that the extracted DNA was integrated and that the extraction effects
were stable.

CONCLUSION:
The protocol mentioned in this study may prove to be efficient in yielding considerable amount of genomic DNA
from both fresh human blood sample. Furthermore, the elimination of time consuming steps such as enzymatic
incubation (for Proteinase K and RNAase) and avoiding the use of toxic organic solvents such as Phenol made the
protocol time-saving and economical without affecting the quality of the DNA samples which could be reliable
enough for applications in advanced molecular biological techniques.
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