DNA Extractions - the Past, Present and Future Approaches

Nur Munirah Bt Majid, Uda Hashim*, Subash C.B. Gopinath, and

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Biomedical Nano Diagnostics Research Group, Institute of NanoElectronic Engineering (INEE), Universiti Malaysia Perlis

(UniMAP), 01000 Kangar, Perlis, Malaysia.

Correspondence to:

Prof. Uda HashimBiomedical Nano Diagnostics Research Group,

1

Institute of Nano Electronic Engineering (INEE), University Malaysia Perlis (UniMAP), 01000 Kangar, Perlis, Malaysia.Email: uda@unimap.edu.my

Abstract

DNA, RNA and protein are the major macromolecules play important

roles in the functional aspects of every living thing. As stated

by ‘central dogma’ DNA is the primary molecules not only as

building block of human genetics but also towards medical

diagnosis and forensic applications, due to high specificity of

DNA and they varies from one individual to another. DNA

molecules in native condition are in the complex form with other

macromolecules in most of the cases to fulfill functions. This

complex formation makes the necessity of separation (extraction)

of DNA molecules from others before being applied for sensing or

forensic applications. Moreover, as technologies develop, there

is an urge toward method to extract macromolecules that specific

to particular species. The processes of extraction and

purification of DNA used previously were complicated, time-

consuming, labor-intensive, and limited in terms of overall

throughput, but now-a-days there are many specialized and

sophisticated methods that can be used to extract DNA in pure

form. Current technologies should allow a high-throughput of

samples to be extracted for DNA, however the crucial

developmental process of DNA extraction during transitional

stages of developments were not explored properly. In this2

overview, we gleaned the extraction methods demonstrated from

past to present and for a future with their critics.

Key words: DNA, Extraction, Purification, Macromolecule,

Nucleases

1. Introduction

Nucleic acid extraction and purification is one of the

essential steps should follow the workflow of genetic analyses

and considered to be a most crucial method used in molecular

biology. Biological macromolecules including deoxyribonucleic

acid (DNA), can be isolated from biological materials such as

living or conserved tissues, cells, virus particles, plant

material or other samples. Thus, extractions of macromolecules

becoming the basic method that are commonly used in molecular

studies. Further, DNAs should be isolated from other complexed

macromolecules, as DNAs are carrying basic genetic information

for subsequent downstream processes and analytical or preparative

purposes. DNA purification involves two major categories that

are isolation of recombinant DNA such as plasmids or

bacteriophage and the isolation of chromosomal or genomic DNA

from prokaryotic or eukaryotic organisms (Doyle, 1996). A

successful use of available downstream applications will gain

with high-quantity and quality DNA output (Pyzowski & Tan, 2007).

There are three important steps that will lead to successful

nucleic acid extraction, firstly the effective disruption of

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cells or tissue, secondly denaturation of nucleoprotein complexes

using appropriate reagents to inactivate the nucleases, for

example, RNase for RNA removal and DNase for DNA removal and

lastly away from contamination (Wink, 2006). The target nucleic

acid should be free of contaminants including protein,

carbohydrate, lipids, or other factors that will lead to impure

output. So, it is essential to choose a suitable extraction

method, and thus, few considerations have to be made when

evaluating the available options. These may include technical

requirements, time efficiency, cost-effectiveness, as well as

biological specimens to be used and their collection and storage

requirements.

Extraction methods involve in DNA isolation becoming more

effective and efficient as extraction kits being developed which

containing most of the components needed to isolate nucleic acid,

but still additional steps are needed to remove other components

depending on the type of specimen, which are time consuming and

tedious. To overcome these problems, automated systems being

designed for medium-to-large laboratory scales have grown in

demand over recent years. The prime issues with DNA extraction is

to increase the yield and purity of desired product,

reproducibility, and scalability of the molecules as well as the

speed, accuracy, and reliability of the assay, while minimizing

the risk of cross-contamination. Overall, to generate an

efficient DNA extraction method, even though proposed methods are

seems to be simple, there are critics and rationales with the

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methods in respect to the purpose. To pin-point and understand

more about the crucial steps involved in different methods will

make the way to generate a finely tuned method, will be a common

strategy suitable to the laboratories and industries. In this

overview, the important steps involved in the DNA extraction and

purification methods formulated in the past, present and a

direction towards future developments are narrated. This study

is necessary due to the active participation of DNA molecules in

several instances in wide range of studies.

DNA is a long polymer of repeating sub-unit called

nucleotides, and each nucleotide consists of three sub-units of

pentose or five-carbon sugar, a phosphate group and an organic

nitrogenous (nitrogen-containing) group (Cseke et al, 2004). DNA is

a complex molecule that contains all of the information necessary

to build and maintain an organism. In fact, nearly every cell in

a multicellular organism possesses the full set of DNA required

for that organism. However, DNA does more than specify the

structure and function of living things (Mitnik et al., 2012).

Whenever organisms reproduce, a portion of their DNA is passed

along to their offspring, this transmission of all or part of an

organism's DNA helps to ensure a certain level of continuity from

one generation to the next, while still allowing for slight

changes that contribute to the diversity of life (Buckingham &

Flaw, 2007). DNA encodes the genetic information used to assemble

proteins in similar to the way the letters on books encode

information (Blin & Stafford, 1976). Unique among macromolecules,

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nucleic acid are able to serve as templates to produce precisely

copies of them.

2. Discovery of DNA as main building block

Even though, initially the first nucleic acid discovery was

realized by a few people, nucleic acid was first identified in

the year 1869 by the Swiss physiological chemist, Friedrich

Miescher that was called as "nuclein" resides in the nuclei of

human white blood cells. The term "nuclein" was later changed to

"nucleic acid" and eventually to "deoxyribonucleic acid," or "DNA

(Whitford, 2005). At first, Miescher's plan was to isolate and

characterize the protein components of leukocytes or white blood

cells. For that, he made arrangements for local surgical clinics

to send him the used, pus-coated patient bandages for his

research. Once he received the bandages, he planned to wash them,

filter out the leukocytes, extract and identify the various

proteins within the white blood cells until he came across a

substance from the cell nuclei that had chemical properties

unlike any proteins, including a much higher phosphorous content

and resistance to proteolysis, Miescher realized that he had

discovered a new substance (Dahm, 2008).

Other scientists, Phoebus Levene from Russian biochemist

have continued to investigate the chemical nature of the molecule

formerly known as ‘nuclein’ and publishing more than 700 papers

on the chemistry of biological molecules over the course of their

career (Haines et al, 2005). Levene was the first person to

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discover the order of three major components (phosphate-sugar-

base) of a single nucleotide and the first discovered the

carbohydrate component of DNA (Brent, 1998). Levene also proposed

that nucleic acids were composed of a series of nucleotides, and

that each nucleotide was in turn composed of just one of four

nitrogen-containing bases, a sugar molecule, and a phosphate

group. Levene made his initial proposal in 1919, discrediting

other suggestions that had been put forth about the structure of

nucleic acids (Kojima & Ozawa, 2002).

Erwin Chargaff, an Australian biochemist was one of a

handful of scientists who expanded Levene's work by uncovering

additional details of the structure of DNA, thus further paving

the way for Watson and Crick model, which demonstrated that

hereditary units, or genes, are composed of DNA. As his first

step in this search, Chargaff set out to see whether there were

any differences in DNA among different species (Dahm, 2004).

After developing a new paper chromatography method for separating

and identifying small amounts of organic material, Chargaff

reached two major conclusions. First, he noted that the

nucleotide composition of DNA varies among species. In other

words, the same nucleotides do not repeat in the same order, as

proposed by Levene. Second, Chargaff concluded that almost all

DNA, no matter what organism or tissue type it comes from,

maintains certain properties, even its composition varies (Kojima

& Ozawa, 2002). In particular, the amount of adenine (A) is

usually similar to the amount of thymine (T), and the amount of

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guanine (G) usually approximates the amount of cytosine (C). This

second major conclusion is now known as "Chargaff's rule."

Chargaff's research was vital to the later work of Watson and

Crick model, but Chargaff himself could not imagine the

explanation of these relationships and specifically, that A bound

to T and C bound to G within the molecular structure of DNA

(Brooks, 1998).

Chargaff's realization that A = T and C = G, combined with

some crucially important X-ray crystallography work by English

researchers Rosalind Franklin and Maurice Wilkins, contributed to

Watson and Crick's derivation of three-dimensional, double-

helical model for the structure of DNA. Watson and Crick's

discovery was also made possible by recent advances in model

building, or the assembly of possible three-dimensional

structures based on known molecular distances and bond angles.

(Sambrook & Russel, 2001).

3. DNA extraction discovery

As stated above, Friedrich Miescher, the first scientist

attempted to isolate DNA while studying the chemical composition

of cells. Firstly, he was aimed in solving the fundamental issue

or principles in life by determine the composition of the cell.

In 1869, he tried to isolate cells in lymph nodes but the purity

of lymphocyte was impossible to obtain, thus he switch to use

leukocytes that he has collected from the samples on fresh

surgical bandages and conducted experiments to purify and

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classify proteins in these cells. He was mainly concentrating in

various proteins that make leukocyte, but during his experiments

he accidently identified a novel substance in the nuclei, and he

called “nuclein” (Dahm, 2004).

He has developed two protocols to separate cell’s nuclei

from cytoplasm and to isolate this novel compound, a so-called

DNA, which is differed from proteins and other cellular

substances. He noticed that a substance precipitated from the

solution, when acid was added and dissolved, when alkali was

added. Thus, for the first time he had obtained a crude

precipitate of DNA (Brooks, 2002). However, his first protocol

was believed to be failed to yield enough material to continue

with further analysis. Then, he had developed a second protocol

to obtain larger quantities of purified nuclein, which had been

named as ‘nucleic acid’ later by his student, Richard Altman.

This scientific finding, together with the isolation protocols

being standardize and was published in 1871 in collaboration with

his mentor, Felix Hoppe-Seyler. However, in the year 1958,

Meselson and Stahl have developed a routine laboratory procedure

for DNA extraction. They performed DNA extraction from bacterial

samples of Escherichia coli using a salt density gradient

centrifugation, resulting in DNA extraction techniques that can

perform on various types of biological sources (Melkonyan et al.,

2008).

These developments formed the basis for DNA extraction

methods developed in the later stages, which follow the important

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facts that mentioned earlier, as they need effective disruption

of cells, denaturation of nucleoprotein complexes, inactivation

of nucleases and other enzymes, removal of biological and

chemical contaminants, and finally obtained the pure DNA as

precipitants. Most of the conventional methods or other modified

methods follow similar basic steps and included the use of

organic and non-organic reagents and centrifugation. These basic

steps finally entered into varieties of automated procedures and

commercially available kits.

4. Conventional extraction methods

After the Miescher achievement to obtain DNA from cell, many

scientists interested and followed the lead, eventually to

further advancements in the DNA isolation and purification

protocol were attained. The first laboratory procedures developed

for DNA extraction were from density gradient centrifugation

strategies. This was proposed by Meselson and Stahl in the year

of 1958 to demonstrate semi-conservative replication of DNA

(Buckingham & Flaws, 2007). Later protocol was mainly based on

the use of differences in solubility of large chromosomal DNA,

plasmids, and proteins in alkaline buffer, now-a-days there are

many specialized methods of extracting pure DNA. General

extraction protocols are divided into solution-based and column-

based and most of these protocols have been implemented in

commercial kits that ease the DNA extraction processes.

4.1. Phenol-chloroform extraction

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Phenol-chloroform is a  liquid-liquid extraction  technique

in biochemistry and molecular biology studies to purify nucleic

acids and eliminate the proteins. In brief, aqueous samples are

mixed with equal volumes of phenol:chloroform mixture. After

mixing, the mixture is centrifuged and two distinct phases are

formed, because phenol:chloroform mixture is immiscible with

water. The aqueous phase is on the top, due to its less density

than the organic phase (phenol:chloroform). Proteins will

partition into the lower organic phase while the nucleic acids

(as well as other contaminants such as salts, sugars, etc.)

remain in the upper aqueous phase. The upper aqueous phase is

pipetted off and this procedure is often performed multiple times

to increase the purity of the DNA (Chomczynski & Sacchi, 2006).

By mixing two phases, and allowing the phases to be

separated by centrifugation, chloroform and phenol mixture is

more efficient, as it denature the proteins than either reagent

is alone. The phenol-chloroform combination reduces the

partitioning of poly (A)+ mRNA into the organic phase and reduces

the formation of insoluble nucleic acid and protein complexes at

the interphase (Dederich et al., 2002). Moreover, phenol retains

about 10-15% of the aqueous phase, which results in a similar

loss of nucleic acid. Chloroform also prevents this retention of

water and thus improves the yield (Massart, 1981). Typical

mixtures of phenol to chloroform are 1:1 and 5:1 (v/v). At acidic

pH, 5:1 ratio results in the absence of DNA from the upper

aqueous phase; whereas 1:1 ratio, providing maximal recovery,

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will maintain some DNA present in the upper aqueous phase (Gjerse

et al, 2009). For example, a very polar solute such as urea is

soluble in highly polar water, less soluble in fairly polar

methanol, and almost insoluble in non-polar solvents, such as

chloroform and ether (Esser et al., 2006). Nucleic acids are polar,

because of their negatively charged phosphate backbone, and

therefore nucleic acids are soluble in the upper aqueous phase

instead of the lower organic phase (water is more polar than

phenol) (Woodard et al., 1994). As contrast to protein, contain

varying proportions of charged and uncharged domains, producing

hydrophobic and hydrophilic regions (Buckingham & Flaws, 2007).

In the presence of phenol, the hydrophobic cores interact with

phenol, causing precipitation of proteins and polymers (including

carbohydrates) to collect at the interface between two phases

(often as a white flocculent) or for lipids to dissolve in the

lower organic phase (Massart, 1981).

The pH of phenol determines the partitioning of DNA and RNA

between the organic phase and the aqueous phase (Arnold et al,

2005). At neutral or slightly alkaline pH (pH 7-8), the phosphate

diesters in nucleic acids are negatively charged, and thus DNA

and RNA both partition into the aqueous phase. DNA is removed

from the aqueous layer by lowering the pH to 4.8. At this acidic

pH, most proteins and small DNA fragments (<10 kb) fractionate

into the organic phase and large DNA fragments and some proteins

remain at the interphase (Arnold et al, 2005; Whitford, 2005).

Acidic phenol retains RNA in the aqueous phase, but moves DNA

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into the phenol phase, because the phosphate groups on the DNA

are more easily neutralized than those in RNA and an acid pH also

minimizes RNase activity (Watson et al, 2004). Isoamyl alcohol is

sometimes added to prevent foaming (typically in a ratio of 24

parts chloroform to 1 part isoamyl alcohol). Guanidinium salts

are also used to reduce the effect of nucleases (Puissant &

Houdebine, 1990).

4.2. Alkaline lysis

Alkaline lysis is the method of choice for isolating

circular plasmid DNA, from bacterial cells. It was first

described by Birnboim and Doly in 1979 and with a few

modifications, been the preferred method for plasmid DNA

extraction from bacteria. It has been proven to work well with

all strains of E. coli and with bacterial cultures ranging in size

from small scale (1 mL) to large scale (500 mL) in the presence

of Sodium Dodecyl Sulfate (SDS) (Cui et al, 2003).

The principle in the alkaline lysis procedure, bacterial

cells is exposed to Sodium hydroxide (NaOH) and SDS which are

strong detergents. Eventually, this will cause the cell walls and

membranes to burst and the contents of the bacteria are spilled

out (Feng et al., 2004). An acidic solution of sodium acetate is

then added to neutralize the solution. At this point, most of the

cell membrane material and the genomic DNA precipitate to form a

phlegm-like mass. The cell contents (including plasmids) can be

separated from this material by centrifugation. The resultant

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supernatant is then extracted to purify the plasmid DNA (Tinay et

al., 1998; Dai et al., 2001; Cui et al., 2006). It is one of the most

general useful techniques as it is a fast, reliable and

relatively clean way to obtain DNA from cells. In this method,

rapid anneal the following denaturation, allows the plasmid DNA

to be separated from the bacterial chromosome.

4.3. CTAB extraction method

This method has been shown to give intact genomic DNA from

plant tissue (Simon, 1996). The initial step that needs to

extract the plant genomic DNA is to grind the sample by freezing

it with liquid nitrogen to break down the cell wall material and

allow access to DNA while harmful cellular enzymes and chemical

remain inactivated. After grinding the sample, it can be re-

suspended in a Cetyltrimethylammonium bromide (CTAB) buffer. CTAB

is a non-ionic detergent that can precipitate nucleic acids and

acidic polysaccharides from low ionic strength solutions

(Sambrook & Russel, 2001). In order to purify DNA, insoluble

particulates are removed through centrifugation while soluble

proteins and other material are separated through mixing with

chloroform and centrifugation. DNA must then be precipitated from

the aqueous phase and washed thoroughly to remove contaminating

salts. The purified DNA is then re-suspended and stored in TE

buffer or sterile distilled water (Schott & Arnold, 1994). This

method has been shown to give intact genomic DNA from plant and

other tissues. To check the quality of the extracted DNA, a

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sample is run on an agarose gel, stained with ethidium bromide

and visualized under UV light.

4.4. Ethidium bromide (EtBr)-Cesium Chloride (CsCl) gradient

centrifugation

The CsCl gradient centrifugation is a complicated,

expensive, and time-consuming method compared to other

purification protocols and requires large scale bacterial culture

to perform. Therefore, this protocol is not suitable for the

mini-preparation of plasmid DNA (Cseke et al., 2004). The desired

nucleic acids can be concentrated by centrifugation in an EtBr-

CsCl gradient after alcohol precipitation and re-suspension. The

main principle is the intercalation of EtBr that will alters the

density of the molecule in high molar CsCl. Thus, the closed

circular molecules will accumulate at lower densities in the CsCl

gradient, because they incorporate less EtBr per base pair

compared to linear molecules. The hydrophobic EtBr is then

removed with appropriate hydrophobic solvents after extraction.

The purified nucleic acid then will be re-precipitated with

alcohol (Wink, 2006).

5. Solid-phase nucleic acid extraction

Solid-phase nucleic acid purification is an alternative

toward a conventional method and can be found in most of the

commercial extraction kits in the market. This system also

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promises for quick and efficient purification compared to

conventional methods (Esser et al, 2005). There are many drawbacks

with liquid-liquid extraction such as incomplete phase

separation, repeated centrifugation step and others. Via solid

phase system such as silica, glass bead, it can absorb nucleic

acid in the extraction process depending on the pH and salt

content of the buffer (Guarerro et al., 2010). The absorption

process is based on the hydrogen-binding interaction with a

hydrophilic matrix under chaotropic conditions.

In this system, there are four key steps involved such as

cell lysis, nucleic acids adsorption, washing, and elution

(Kojima & Ozawa, 2002). The initial step in a solid phase

extraction process is to condition the column for sample

adsorption. This can be done by using certain type of buffer at a

particular pH to convert the surface or functional groups on the

solid into a particular chemical form (Gjerse et al., 2009). Next,

the sample then being degraded by using lysis buffer and the

desired nucleic acid will absorb to the column with the aid of

high pH and salt concentration of the binding solution (Smith et

al., 2002). Other compounds, such as protein being removed from

the desired product by using the washing buffer containing a

competitive agent (Padhye et al., 1997). Finally, for the elution

step, TE buffer or distilled water is introduced to release the

desired nucleic acid from the column by breaking the bond of DNA

backbone from the surface of the solid compound, so that it can

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be collected in a purified state (Smith et al., 2002). Normally,

rapid centrifugation, vacuum filtration, or column separation is

required during the washing and elution steps of purification

process.

7.1. Silica matrices-based nucleic acid purification

The basis for most of the products related to nucleic acid

purification is the unique properties of silica matrices for

selective DNA binding. There are many types of silica materials

can be used including glass particles, such as glass powder,

silica particles, and glass microfibers prepared by grinding

glass fiber filter papers, and including diatomaceous earth

(Padhye et al., 1997). Hydrated silica matrix, which was prepared

by refluxing silicon dioxide in sodium hydroxide or potassium

hydroxide at a molar ratio of about 2:1 to 10:1 for at least

about 48 hours, had been introduced in DNA purification (Woodard

et al, 1994).

The principle of silica matrices purification is based on

high affinity of negatively charged DNA backbone towards

positively charged silica particles (Esser et al., 2006). Sodium

plays a role as a cation bridge that attracts the negatively

charged oxygen in the phosphate backbone of nucleic acid (Feng et

al., 2004). Sodium cations break the hydrogen bonds between the

hydrogen in water and the negatively charged oxygen ions in

silica under high salt conditions (pH ≤ 7). In the presence of

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high salt buffer, DNA will binds to silica particles (Arnold et

al., 2005). Then the silica with adsorbed DNA will be washed using

washing buffer to remove salt and impurities from the original

sample, and the clean DNA will eluted under low ionic strength

(pH ≥ 7) either in water or TE buffer (Chen et al., 2007). This

isolation method of DNA using silica is said to be faster

(approximately 20 min) and easier to perform than the other

organic-based extraction method. This method also replaces the

Potassium Iodide (KI) based procedure, where free Iodine may

modify the purified DNA. When using silica adsorption method for

isolating DNA from agarose gels, it is important to note that use

of TBE buffer (Tris-borate-EDTA) can inhibit the ability of DNA

to bind silica, thus lowering recovery efficiency. 

Silica extraction works well with a wide size range of DNA

and allows efficient recovery (90%) of product. By using silica,

it can purify DNA as small as 100 bp (Salgueiri˜no-Maceira et al,

2006). There is no upper size limit on recovery efficiency of

DNA. However, precautions should be taken during purification of

longer DNA fragments (20 kb) to avoid shearing (Woodard et al,

1994). Proteins and RNA do not bind to silica and are eliminated

during washes and for this reason it is also an ideal tool to

purify and concentrate DNA directly from various reaction

mixtures. Other advantage is, because silica does not bind to

oligonucleotides with high efficiency. This method can also be

used to remove low molecular weight oligonucleotides or

nucleotides from DNA. Silica can also be used to remove RNA from

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DNA, because RNA does not bind to silica and will yield only the

pure DNA as a final result (Esser et al., 2006). The purified DNA

is suitable for any molecular biology procedures such as

restriction digestion, cloning, sequencing and etc. Small amounts

of silica do not inhibit enzymatic reactions, therefore silica

bound DNA can be used directly for PCR or enzymatic cleavages

without prior elution of DNA (Woodard et al., 1994).

7.2. Magnetic bead-based nucleic acid purification

Magnetic bead technology is recognized as combining high

quality genomic DNA (gDNA) production with compatibility for

high-throughput processing. Furthermore, automation of magnetic

bead methodology reduces user to be exposed to bio-hazardous

human materials. Magnetic separation is known to be a very

simple and efficient way which used in purification of any

desired nucleic acid. Currently, many magnetic carriers are now

commercially available. Particles having a magnetic charge may be

removed by using a permanent magnet in the application of a

magnetic field. Usually, magnetic carriers often immobilized with

affinity ligands or can be prepared from biopolymer tend to show

affinity toward the target nucleic acid, are commonly used for

the isolation process (Dederich et al., 2002).

For example, magnetic particles that are produced from

different synthetic polymers, biopolymers, porous glass or

magnetic particles based on inorganic magnetic materials, such as

surface modified iron oxide are preferred to be used in the

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binding of nucleic acids, but magnetic particulate materials such

as beads are more preferable to be a support in isolation

process, because of their larger binding capacity. The nucleic

acid binding process may be assisted by the nucleic acid

surrounding support and a permanent magnet can be applied to the

side of the vessel to aggregate the particles near the wall of

the vessel and the sample or desired DNA can be eluted using TE

buffer or distilled water (Berensmeier, 2006).

Particles having magnetic or paramagnetic properties are

employed in an invention where they are encapsulated in a polymer

such as magnetizable cellulose (Nargessi, 2005). In the presence

of certain concentrations of salt and polyalkylene glycol,

magnetizable cellulose can bind to nucleic acids. Small nucleic

acid required higher salt concentrations for strong binding to

the magnetizable cellulose particles. Therefore, salt

concentration can be selectively manipulated to release nucleic

acid bound to magnetizable cellulose on the basis of size. The

magnetizable cellulose which bound with nucleic acid will be

washed with suitable wash buffer before they contact with a

suitable elution buffer to separate out the desired nucleic acid

with cellulose. Separation of magnetizable cellulose from

supernatant during all the purification steps can be done by

applying a magnetic field to draw down or draw them to the side

of the vessel (Nargessi, 2005). The magnetizable cellulose used

in this invention has an iron oxide content of up to around 90%

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by weight of the total mass of the cellulose. The magnetic

component of cellulose can also be substituted by other magnetic

compounds such as ferrous oxide or nickel oxide (Berensmeier,

2006).

There are many extraction kits that are currently implies

this technology, available commercially in the market (Sasso et

al., 2012). The special part of this kit is that the reagents

provided are intended for use with magnetic tools, such as in

GeneCatcher™ extraction kits and other kits that currently

available in the market. Magnetic beads are a simple and reliable

method of purifying genomic, plasmid and mitochondrial DNA. Under

optimized conditions, DNA selectively binds to the surface of

magnetic beads, while other contaminants stay in solution (Keeley

et al., 2013). Purified DNA can then be used directly (recovered by

using elution buffer) for many applications, such as sequencing

or restriction digestion. The major advantage of this method is

that there is no need for centrifugation or vacuum manifolds,

which can be a bottle-neck in many automated processes.

Separation can be done manually, semi-automated or fully

automated in 24, 96 and 384 well plates. Equipment necessary for

this technology includes the bead purification kits, magnetic

particle processing systems, magnetic separators, tubes, columns

and flasks. Features to be looked are narrow size distribution of

the beads and an adequate magnetic mass susceptibility (Sasso et

al, 2012).

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There is also another extraction kit that has the same

principle as the extraction described above, which used the

magnetic-particle technology for nucleic acid purification that

combines the speed and efficiency of silica-based DNA

purification with the convenient handling of magnetic particles

(QIAGEN Inc., QAsymphony_ DNA Handbook, QIAGEN,Alameda, Calif,

USA, 2008). A magnetic rod protected by a rod cover is used for

the capture of magnetic particles. It enters a vessel containing

the samples and attracts the magnetic particles. Then, the

magnetic rod cover is positioned above another vessel and the

magnetic particles are released (Applied Biosystems, MagMAXTM

Total Nucleic Acid Isolation Kit, Applied Biosystems, Foster City, Calif,

USA, 2008).

Other than magnetic bead, zirconia bead also can be used in

nucleic acid purification. These micro-spherical paramagnetic

beads have a large available binding surface and can be dispersed

in solution. This characteristic allowed thorough nucleic acid

binding, washing, and elution. The extraction kits that use

zirconia beads mainly adapt the guanidinium thiocyanate-based

solution protocol that not only releases nucleic acid but also

inactivate nuclease in the sample matrix (Ma et al., 2013). After

the lysis step, dilution of samples is done by isopropanol.

Paramagenetic beads are added to the samples for the nucleic acid

binding purpose. The mixture of beads and nucleic acid are

immobilized on magnets and washed to remove protein and other

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contaminants. Removal of residual binding solution is done with a

second wash solution and finally the nucleic acid is eluted in

low-salt buffer (Ma et al., 2013).

7.3. Glass Particle-based nucleic acid purification

Glass particles, powder and beads are useful for nucleic

acid purification. The adsorption of nucleic acid on the glass

substrate occurs most likely based on the mechanism and principle

that similar to adsorption chromatography (Dederich et al, 2002).

Nucleic acid purification can also be done on silica gel and

glass mixture. This invention has discovered that a mixture of

silica gel and glass particles can be used to separate nucleic

acid from other substances in the presence of chaotropic salts

solution (Padye et al., 1997).

8. Diatomaceous earth

Diatomaceous earth, which is also known as kieselguhr or

diatomite, has silica content as high as 94% (Little, 1991). It

has been useful for the purification of plasmid and other DNA by

immobilizing DNA onto its particles in the presence of a

chaotropic agent. There are several methods regarding

purification of plasmid DNA using this technique has been

described in detail. The principle behind this purification

technique is almost similar as other purification techniques. The

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desired DNA will binds to the silica dioxide, which is the major

component of the diatomaceous earth. In the presence of

chaotrophic salts such as guanidine hydrochloride, NAL or

guanidine isothiocyanate, the chaotropic salts will denature the

protein and will be washed out using washing buffer. The resin-

bound DNA will be collected in plastic column using vaccum

manifold and eluted using low salt buffer or in distilled water

(Vrancken et al, 1995).

9. Anion-exchange materials

Ion exchange chromatography has emerged as a reliable

alternative to classic CsCl-ethidium bromide gradients for

isolating nucleic acids with the highest purity. The principle is

based on the interaction between positively charged

diethylaminoethyl cellulose (DEAE) groups on the resin’s surface

and negatively charged phosphates of the DNA backbone. Most of

the anion-exchange resin consists of defined silica beads with a

large pore size and some of the anion-exchange might contain a

porous silica that modified with diethylaminoethanol that give a

hydrophilic surface coating with a high charge density (Endres et

al., 2003). The resin works over a wide range of pH conditions (pH

6–9) and/or salt concentration (0.1–1.6 M) which can optimize the

separation of DNA from RNA and other impurities (Knudsen et al,

2001).

24

In this technique, salt concentration and pH conditions of

the buffers play the main role that determine whether nucleic

acid is bound or eluted out from the column. DNA can bind to the

DEAE group over a wide range of salt concentrations. Other

unwanted impurities such as protein and RNA usually washed from

the resin by using medium-salt buffers, while DNA remains bound

until eluted with a high salt buffer (Endres et al., 2003). There

are lots of commercially available strong or weak positively

charged anion exchanger materials that can be used with selected

solutions of known ionic strength for adsorption and elution of

desired DNA. The ionic strength for elution is generated by using

known salt concentration, which mixed with a buffer to control pH

strength, ideally corresponding to the lowest ionic strength at

which the nucleic acids will completely elute (Selingson and

Shrawder, 1990).

Based on the research, plasmid DNA usually isolated well

using this technique. Plasmid purification method based on a

unique anion exchange membrane (IEXM) was developed for the

production of superior quality plasmids (Marion & Warren, 1989).

This method was simpler and more efficient as compared to the

conventional bead-based methods. Plasmids were extracted from

bacterial cells through alkaline lysis and the crude lysate was

clarified by a sequential filtration device that not only removed

cell debris but micellar aggregates as well (Prazeres et al, 1998).

The clarified lysate was mixed with an extraction solution and

25

loaded into a spin column containing IEXM. The binding, washing,

and elution conditions were optimized to achieve efficient

isolation of plasmids from the impurities. IEXM had an

exceedingly highly dynamic binding capacity, excellent

selectivity, and a near 100% recovery for plasmids (Prazeres et

al., 1998). These products are thought to be cost-effective,

disposable, minimize cross contamination and sophisticated

chromatographic instrument is not required.

10. All-in-one biomolecules extraction

Generally, the extraction or purification techniques or

kits available in the market can only allow the extraction of one

type of nucleic acid, either DNA or RNA, or protein from a

targeted organism. When the sample material is limiting, it is

desirable to extract DNA, RNA and protein from the same source.

For example, the clinical sample, such as bone tissue of fluids

that are painful to collect from the patient should be used

wisely. A variation on the single-step isolation method of

Chomczynski and Sacchi in 1987 shows that the guanidinium

thyicyanate homogenate is extracted with phenol:chloroform at

reduced pH, allows the preparation of DNA, RNA and protein from

tissue or cells. This method involves the lysis of cells with

guanidine isothiocyanate and phenol in a single phase solution

and the second phase forms after the addition of chloroform that

result in DNA and proteins extracted and leaving RNA in the

26

aqueous supernatant. The DNA and proteins can be isolated from

the organic phase by precipitation with ethanol or isopropanol

and the RNA precipitated from aqueous phase with isopropanol

(Sambrook & Russel, 2001).

Currently, as the technologies develop, extraction process

also becoming easier, formulated as all-in-one extraction kits

have been introduced in the market. For example, a column-based

extraction kit that designed to purify genomic DNA, total RNA and

total protein from a single biological sample simultaneously,

without the usage of toxic substances such as phenol: chloroform

and alcohol precipitation and usage of small sample size. The

targeted sample does not need to be separated before the

purification step (Prazeres et al., 1998). Other than that,

solution-based 3-in-1 extraction kit is another example of non-

organic solutions kit that can extract and purify DNA, RNA and

protein, from different organisms in any types and sizes. The

principle is the same as three simple steps protocol, less time

consuming (15-30 min) and thus, provides a fast and easier way to

do the extraction of different biomolecules and gives higher

yield of desired results (DeRiPRO, DNA, RNA and Proteins

Extraction Technology).

11. Automated Extraction System

27

When come across automated extraction system, people will

think about large, expensive and complex instrumentation designed

for high-throughput sample processing. To handle such instrument

does need for professional expertise to handle. To overcome the

drawbacks from complicated to user friendly instruments,

researchers tried to developed many system to simplify the

isolation of nucleic acids (Loeffler et al., 2004). Thus, now-a-

days with high technology, this system was designed for medium to

large laboratories which has grown in presence over recent years

(Boyd, 2002).

There are many extraction system that is available in the

market has met the requirements stated above. Some of the

applications, such as forensic department required lot of sample

to be process at once. Thus, forensic laboratories need to be

fast and reliable sample processing along with high-quality

automated DNA purification (Mijatovic et al, 2005). Paramagnetic-

particle handling system usually used to process the sample in

large amount and provide consistent yield and purity as there is

no detectable cross-contamination between samples. The whole

extraction process reported takes about 20 min. There are three

steps are needed in extraction using paramagnetic-particles. It

start with the addition of liquid samples to reagent cartridge,

next, place the reagent cartridges into the machine and lastly to

press the start button on the machine. At the last step, the

28

desired DNA is then being eluted into elution buffer (Okamoto et

al., 2007).

Another example of automated system that is flexible and

efficient for extraction of nucleic acids and proteins has been

introduced by using various starting materials or sample and

those sample can be processed by using this system at once using

the same machine and extraction protocol. This system was

designed not only for small scale sample and also for medium

scale sample. In this system, the purification step using the

benefit of the surface functionalized paramagnetic particles to

adsorb the isolated nucleic acid (Okamoto et al., 2007). It is

reported that the flexibility of this system allows the

extraction of nucleic acid up to twelve samples simultaneously

and only takes about 20 to 40 min to complete, depending on the

application. These kits useful in extracting not only genomic DNA

but also cellular RNA, viral and bacterial nucleic acids. As

conclusion, automating nucleic acid extraction processes are

potentially beneficial for a number of reasons, including reduced

working time, decreased labor costs, increasing worker safety and

in the midst provides opportunity in increasing reproducibility

and quality of results (Boyd, 2002). Besides, it is a key

solution to increase the laboratory efficiency.

12. Future directions

29

Now-a-days, the extraction protocol has been standardized

and can be easily performed by everyone that gives great benefit

and save time. Even though, there were lots of extraction kits

are available that does not require tedious lab-based extraction,

still there are lots of drawbacks to be improved. Biomolecules

extraction for example is the first step that needs to be

performed for the downstream analysis or manipulation processes.

The liquid handling requirement is the most challenging aspect in

extraction process. Therefore, an automatic system must include

not only automatic equipment for each extraction step, but also

equipment for automating the transfer of liquid between machines.

This might reduce the consequent of doing mistakes that will lead

to low purity result (Wallace, 1987). Automation is currently

highly aided in increasing the throughput and improving the

reliability of the process, but these systems are still designed

for the use in laboratory environments, which mean in a small

scale. Some of the nucleic acid extraction systems that are

available in the market, suitable for large scale and to process

the higher number of samples, currently require manual pre-

processing stages by laboratory staff with technical expertise

(Goedecke et al., 2004). Therefore, the urge for robotic

workstations in large scale nucleic acid extraction should be

fulfill with a fully automated process. A combination of all-in-

one biomolecules extraction solution and method with fully

automated extraction system can be a prospective invention in the

future.

30

There is also need that the purification of DNA, RNA or

protein from various organisms can be performed simultaneously

using the same type of extraction system with just a single

extraction method. It is often inconvenient that targeted

biomolecules sample from an animal, plant or even a clinical

sample must be sent to the laboratory for it to be extracted and

analyzed separately. Current technique requires different

protocols that lead time consuming and will eventually destroyed

the sample. For some precious sample, such as clinical specimen

need to be refrigerated and transfer to the expertise laboratory

to be processed, will sometimes reduce the yield of final result.

Hence, a portable biomolecules extraction system that could be

perform anywhere without the need of expertise really in demand

as its brings several advantages such as reduced labor, reduced

waste and increased speed of extracting process (Thomsin, 2007).

The combination of portable extraction system with DNA, RNA, or

protein analyzer can be build up in the future to help

researchers in reducing working time and increasing the work

efficiency. Thus, continued improvement in miniaturization will

be the future trend of robotic automation in the laboratory

(Thomsin, 2007). Besides, this automation system can be

implemented at relatively low cost, improving the turnaround

times and also reduce the labor costs.

Introduction to microfluidic

31

Other than that, the manipulation of fluids in channels with

dimensions of tens of micrometers, microfluidics has emerged as a

distinct new field. Microfluidics has the potential to influence

subject areas from chemical synthesis and biological analysis to

optics and information technology. Microfluidic is the science

and technology of systems that process or manipulate small (10–9

to 10–18 liters) amounts of fluids, using channels with dimensions

of tens to hundreds of micrometers. The main applications of

microfluidic technologies have been in analysis as they offer a

number of useful capabilities such as the ability to use very

small quantities of samples and reagents, carry out separations

and detections with high resolution and sensitivity, low cost,

short times for analysis and small footprints for the analytical

devices (Cho et al., 2007).

Now-a-days, microfluidics technology not only based on

photolithography, but also association of photolithography in

silicon microelectronics and in micro electromechanical systems

(MEMS) that would be directly applicable to microfluidics.

Beginning work in fluidic microsystems uses silicon and glass,

but these materials have largely been displaced by plastics. This

is due to unnecessary or inappropriate fabricated device in glass

and silicon used for analysis of biological samples in water.

Silicon, on the other hand, is expensive and opaque to visible

and ultraviolet light, thus cannot be used with conventional

optical methods of detection. It is easier to fabricate the

components required for micro analytical systems, especially

32

pumps and valves in elastomers than in rigid materials. Thus,

microfluidic devices in exploratory research have been carried

out in a polymer which is called as poly (dimethylsiloxane), or

PDMS. The properties of PDMS are entirely distinct from those of

silicon (Sia & Kricka, 2008). PDMS is an optically transparent

and soft elastomer. Microelectronic technologies however, been

indispensable for the development of microfluidics, and as the

field has developed, glass, steel and silicon have again emerged

as materials which to build specialized systems that require

chemical and thermal stability (Daar et al., 2002).

Figure 1 shows Scheme describing rapid prototyping of

microfluidic systems. A system of channels is designed in a CAD

program. A commercial printer uses the CAD file to produce a

high-resolution transparency (~5000 dpi). (a) This transparency33

is used as a photomask in contact photolithography to produce a

master. A master consists of a positive relief of photoresist on

a silicon wafer and serves as a mold for PDMS. (b) Liquid PDMS

pre-polymer is poured over the master and cured for 1 h at 70°C.

(c) The PDMS replica is peeled from the master. (d) The replica

is sealed to a flat surface to enclose the channels. The overall

process takes ~24 h. (McDonald & Whitesides, 2002).

Microfluidic for DNA extraction

A microfluidic system must have a series of generic

components from a method of introducing reagents and samples,

methods for moving the fluids around on the chip, combining and

mixing the fluids until various other devices such as detectors

for most micro analytical work, and components for purification

of products for systems used in synthesis. The field has been

centered on demonstrating concepts for these components. Two

particularly important contributions have been the development of

soft lithography in PDMS as a method for fabricating prototype

devices, and the development of a simple method of fabricating

34

pneumatically activated valves, mixers and pumps on the basis of

soft-lithographic procedures (Gravesen et al., 1993; Dolnik &

Jovanovich, 2000). These methods have made it possible to

fabricate prototype devices that test new ideas in a time period

much shorter than that which could be achieved using silicon

technology which is non specialized specialists that would

consume longer period of time.

Figure 2 shows an example of the chip that was used for the

extraction experiments. The microchannels have been milled

directly into a polycarbonate substrate (blank DVD). To

illustrate the extraction process DI water dyed with ink has been

injected into the chip (outlet port 4a is blocked and therefore

filled with air). The different colours denote the different

buffer solutions used for the extraction process. Red: lysis and

binding buffer including the cell sample and the magnetic beads,

blue: washing solution and green: elution buffer.

35

Since in the last century, the miniaturization of electronic

devices or microelectronics has been regarded as the most

significant enabling technology in human history. With the

integrated circuits and progress in information processing,

microelectronics has revolutionized the way we work, live and

play. Miniaturization concepts have recently been brought into

the fluidics since the introduction by Manz et al., at the 5th

International conference on Solid-State Sensors and Actuators

(Transducers ‘89): microfluidics, which appeared as the name for

the new research discipline dealing with transport phenomena and

fluid-based devices at microscopic length scales (Manz et al,

1990).

One of the most impressive developments of microfluidics in

life sciences is ‘Bed-side analyses’ or ‘Point-of-Care testing

(POCT)’ such as glucose test meter, pregnancy strip test and

others, which is defined as diagnostic testing at or near the

site of patient care to make the test convenient and immediate

(Sia & Kricka, 2008). Patients can now receive the testing result

within minutes. Such devices can be used in hospitals, at a

doctor office or simply by patients themselves at home without

any professional knowledge or particular skill. Thus, currently,

miniaturized devices are likely to impact economy and improve

public health significantly (Daar et al, 2002).

Other than contributed in biomedicine application,

microfluidic also being applied in biotechnology application such

as in continuous DNA extraction. The detection of DNA and its

36

variation is critical for many fields, including clinical and

veterinary diagnostics, industrial and environmental testing,

agricultural researches and forensic science. Disease diagnosis

and prognosis are based on effective detection of chronic disease

conditions, such as in cancer or contagious disease such as HIV

and genetic markers. However, DNA analysis from original

specimens is a complex process involving multiple chemical

compositions as well as multistep reactions. Conventionally this

procedure is time consuming, labor intensive and contamination

prone, it is not compatible with high throughput or field testing

requirement. To integrate the automatic sample pretreatment

functions will reduce reagent consuming, assay time and

contamination risk. More importantly, it will not only affect the

efficiency and reliability but also determine the feasibility of

a final product. In the past decade, about 3,000 papers published

about on chip DNA tests. Almost all of them need off-chip sample

preparation and reagent handling. A full function system with

sample-in–answer-out capability is still rare (Easley et al.,

2006). By using microfluidic DNA extraction can be performed

anywhere and by anyone without the need of professional

assistance. The concentration of DNA analyte in the test samples

is usually not yet high enough for direct detection. Therefore,

DNA amplification is a required step to raise the concentration

of the target sequence. There is many articles and review paper

regarding combining the continuous DNA extraction using

microfluidic together with DNA amplification. The integration of

37

extraction and polymerase chain reaction (PCR) in the same device

becoming the future revolution in POTC (Dineva et al, 2007).

A B

Figure 3 shows the scheme of mixer construction. Adapted from

[219]. (A) Schematic picture showing the PDMS active mixer design

and construction. The overall chip size is 3 cm by 1.5 cm. (B)

Schematic of integrated device with two liquid samples and

electrophoresis gel present.

As conclusion, the development of microfluidics still need

several steps to go. A number of factors suggest that there are

many early-stage applications of microsystems containing fluids,

38

including the exploration of fluidic optics and cells, the

development of new types of organic synthesis in small-channel

systems, the continuing development of technologies based on

large arrays of detectors and on high-throughput screening, the

fabrication of microrobotic systems using hydraulic systems based

on microfluidics, other fluidic versions of MEMS, and work on

biomimetic systems with microfluidic components. Different types

of micro/nano-fluidic technologies have facilitated DNA

purification, amplification and detection to be integrated into

one chip which combine the advantages of small sizes, much

shorter reaction times, less manual operation and reduced cost.

Successful DNA amplification and detection on chip depends on the

optimization of several parameters, which is a cumbersome task

due to many variables (conditions and components) typically

involved and requires comprehensive knowledge of multi-subject

intersecting molecular biology, chemistry, physics, mechanics and

micro/nano-fabrication technologies.

13. Conclusion

DNA plays many roles now-a-days, not only for the interest

of the researcher but it is also important for the phylogenic

tree to trace the species of new things. For forensic

applications, to trace criminals and also for medical purposes

with hereditary and unknown diseases. Thus, DNA extraction

process has attract the biggest interest as re-cap back when the

39

first DNA isolation was successfully done by Friedrich Miescher

in 1869 and the initial DNA extraction developed from density

gradient centrifugation strategies by Meselson and Stahl in 1958,

many techniques for biomolecules purification has been developed

afterwards. From the phenol-chloroform extraction to the column-

technology has widely used in nucleic acids extraction.

Biomolecules extraction has helped researchers and scientists in

manipulating subsequent molecular biology analysis in order to

have a better understanding about the biological materials. The

automated nucleic acid extraction system has been developed due

to the influence of rapid growth of automation technology.

Automating nucleic acid extraction process is potentially

beneficial for a number of reasons including to reduce working

time, decrease labor costs, increase worker safety and at the

same time provides opportunity in increasing reproducibility and

quality of results. However, improvement with the currently

available instruments needs to be conducted all the time. In the

meantime, an all-in-one biomolecules extraction system, or the

invention of a miniature and portable extraction system can

become a prospective development in the future.

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