**2. Nucleic acid extraction methods**

Since nucleic acid extraction is a starting point in a vast array of downstream applications, the high quality of nucleic acids in the starting samples is a key factor for the success of the subsequent steps of analysis. Thus, nucleic acid extraction could be defined as a series of steps to obtain nucleic acid samples/materials of particular purity that are free of impurities and are suitable for different downstream application steps. The purpose of nucleic acid extraction methods is to disintegrate the cell envelope and achieve maximum elimination of lipids and proteins to obtain pure DNA and/or RNA. This is principally based on heat adsorption on silica membranes/beads, anion exchange chromatography, sedimentation/precipitation, and use of magnetic particles. These methods yield initial nucleic acid samples of different purity and concentration depending on the original sample (bacteria, viruses, tissues).

The choice of method – in view of optimal time/quality balance – depends on the aim of the study, the type of analysis, the type of nucleic acid, and the cost. It is important to provide appropriate conditions for nucleic acid extraction in order to avoid nucleic acid degradation due to oxidation by reactive oxygen species generated during respiration in vivo or, extrac‐ ellularly, by mechanisms involving metal ions [1–4]. Nucleic acid degradation can result from hydrolysis of the 3′-5′ phosphodiester bonds catalyzed by metal complexes as well as from the spontaneous breakage of these bonds due to transesterification via a nucleophilic attack at the phosphorus atom by an adjacent 2′-hydroxyl group [1].

What accounts for the differences between the methods for extraction of DNA and RNA is their different stability. RNA includes ribosomal RNA (rRNA) 80%, mitochondrial RNA (mtRNA), messenger RNA (polyadenylated – poly A+ in eukaryotic cells) (mRNA) 1–5%, transfer RNA (tRNA), and microRNA molecules (miRNA). There may be different amounts of mRNA in cells: from large quantities to just five copies per cell. In fact, mRNA is the RNA of choice in reverse transcription and cDNA synthesis. RNA molecules are susceptible to degradation by ozone in the air; ozone is highly reactive regardless of whether the RNA sample is liquid or solid [5]. Another factor that plays a role in RNA degradation is water, as it makes the transfer of protons possible and serves as a source of hydronium or hydroxyl ions. That is why dehydration has a protective effect against RNA degradation [6]. Nucleic acids, and especially RNA molecules, are also sensitive to nucleases. Therefore, in RNA extraction procedures, it is essential to ensure an RNase-free fraction and a means to quickly cool down the sample. This illustrates the importance of providing all necessary work facilities: use of BSL-1 or 2 laminar flow cabinets (depending on the type of biological material) is recom‐ mended for nucleic acid extraction; ultraviolet (UV) germicidal irradiation of both the premises and the laminar flow cabinets should be done, with irradiation of the premises done the night before (irradiation immediately before work may lead to degradation due to residual UV light). The same principle applies to UV germicidal irradiation of plastic labware, e.g., microtubes, pipette tips, etc. (if not commercially sterile, DNase- and RNase-free). Another important detail is that talc-free gloves should be used because talc may inhibit some downstream analyses such as PCR, reverse transcription, and real-time-PCR.

The quality of the extracted nucleic acids also depends on the quality of the starting sample. In fact, all manufacturers of nucleic acid isolation kits recommend that fresh starting material be used. If this is not possible – as is often the case with diagnostic samples – they can be stored for 24–48 h at 4°C, or for longer periods of time at –80°C or in liquid nitrogen, preferably using protective buffers, especially for samples intended for RNA analysis. There are paper matrices especially developed for storage and transport of blood samples at room temperature – dried blood spot sampling. In this train of thought, it has to be kept in mind that heparin, which is used as an anticoagulant, may inhibit some PCR reactions [7] and should, therefore, be avoided or removed. In the case of clotted blood samples, the coagulum can be treated as an organ sample. The spleen and the liver are transcriptionally active organs and they have a very high RNA content. Because of that, if the samples are intended for DNA analysis, they have to be treated with RNase prior to column purification. When the aim is RNA analysis, it is particu‐ larly important to protect the RNA against degradation, else the low-frequency transcripts could be lost and would not be detected in the downstream steps of analysis. Moreover, in microarray analysis, degraded RNA molecules may fail to successfully bind to the comple‐ mentary site due to loss of the complementary sequence. That is why, in RNA extraction, frozen samples should be mechanically processed (homogenized) prior to thawing, and fresh tissue samples should be ground in liquid nitrogen or by other means of cooling. This is not as essential in DNA extraction, since DNA molecules are relatively more robust, but is recom‐ mended.

**2. Nucleic acid extraction methods**

2 Nucleic Acids - From Basic Aspects to Laboratory Tools

Since nucleic acid extraction is a starting point in a vast array of downstream applications, the high quality of nucleic acids in the starting samples is a key factor for the success of the subsequent steps of analysis. Thus, nucleic acid extraction could be defined as a series of steps to obtain nucleic acid samples/materials of particular purity that are free of impurities and are suitable for different downstream application steps. The purpose of nucleic acid extraction methods is to disintegrate the cell envelope and achieve maximum elimination of lipids and proteins to obtain pure DNA and/or RNA. This is principally based on heat adsorption on silica membranes/beads, anion exchange chromatography, sedimentation/precipitation, and use of magnetic particles. These methods yield initial nucleic acid samples of different purity

The choice of method – in view of optimal time/quality balance – depends on the aim of the study, the type of analysis, the type of nucleic acid, and the cost. It is important to provide appropriate conditions for nucleic acid extraction in order to avoid nucleic acid degradation due to oxidation by reactive oxygen species generated during respiration in vivo or, extrac‐ ellularly, by mechanisms involving metal ions [1–4]. Nucleic acid degradation can result from hydrolysis of the 3′-5′ phosphodiester bonds catalyzed by metal complexes as well as from the spontaneous breakage of these bonds due to transesterification via a nucleophilic attack at

What accounts for the differences between the methods for extraction of DNA and RNA is their different stability. RNA includes ribosomal RNA (rRNA) 80%, mitochondrial RNA

transfer RNA (tRNA), and microRNA molecules (miRNA). There may be different amounts of mRNA in cells: from large quantities to just five copies per cell. In fact, mRNA is the RNA of choice in reverse transcription and cDNA synthesis. RNA molecules are susceptible to degradation by ozone in the air; ozone is highly reactive regardless of whether the RNA sample is liquid or solid [5]. Another factor that plays a role in RNA degradation is water, as it makes the transfer of protons possible and serves as a source of hydronium or hydroxyl ions. That is why dehydration has a protective effect against RNA degradation [6]. Nucleic acids, and especially RNA molecules, are also sensitive to nucleases. Therefore, in RNA extraction procedures, it is essential to ensure an RNase-free fraction and a means to quickly cool down the sample. This illustrates the importance of providing all necessary work facilities: use of BSL-1 or 2 laminar flow cabinets (depending on the type of biological material) is recom‐ mended for nucleic acid extraction; ultraviolet (UV) germicidal irradiation of both the premises and the laminar flow cabinets should be done, with irradiation of the premises done the night before (irradiation immediately before work may lead to degradation due to residual UV light). The same principle applies to UV germicidal irradiation of plastic labware, e.g., microtubes, pipette tips, etc. (if not commercially sterile, DNase- and RNase-free). Another important detail is that talc-free gloves should be used because talc may inhibit some downstream analyses

in eukaryotic cells) (mRNA) 1–5%,

and concentration depending on the original sample (bacteria, viruses, tissues).

the phosphorus atom by an adjacent 2′-hydroxyl group [1].

(mtRNA), messenger RNA (polyadenylated – poly A+

such as PCR, reverse transcription, and real-time-PCR.

Another key step is cell lysis, which – if incomplete – would result in reduced yield and column blocking and, in turn, in lower purity. There are different ways to aid the process of cell lysis: in the case of cell cultures or bacterial cultures, depending on the aim of analysis, they can be washed in PBS (phosphate buffered saline) or physiological saline and resuspended in ddH2O and/or subjected to several freeze/thaw cycles. In the case of mucous samples (nasal discharges, sputum, intestinal loops), it is good to first decrease the viscosity of the material (using a mucolytic –"mucus-dissolving" agent, e.g., acetylcysteine). The mechanical process‐ ing of samples from insects, plants, feces, organs requires 50–200 mg of sample in most kits. Gram-positive bacteria are treated with lysozyme; yeasts, with zymolyase or lyticase; and paraffin-embedded tissues are treated with xylene to remove the paraffin. Other approaches that can be applied to disrupt cell envelopes include: osmotic shock, which is suitable for Gramnegative bacteria, cell cultures and erythrocytes; chaotropic salts, for all types of samples with the exception of some Gram-negative bacteria, owing to the greater thickness of their pepti‐ doglycan layer; enzymatic degradation (lysozyme, proteinase K), which is often combined with osmotic shock or freeze/thaw cycles (for DNA extraction from hair, feathers etc.); and detergents, for tissue cultures. Should a sample remain not fully lysed, one way to overcome the problem is to centrifuge the mixture and use the supernatant prior to column loading or alcohol supplementation, in case the protocol includes such a step.

Sometimes the samples may be "old", i.e., stored for a long time at –20°C. In such samples, the chemical bonds in the DNA molecules may have become weaker. Then, in the vortexing steps in the column-based and solution-based methods, the DNA becomes, more often than not, ge the mix entation, in c

adequately m

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**F**

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degraded, resulting in smeared DNA bands (Figure 1). To avoid this, such samples should be kept frozen during the process of mechanical homogenization, and instead of vortexed, should be gently homogenized by slowly turning the microtubes upside down and back several times to adequately mix the reagents and, to a large extent, preserve the intactness of the DNA molecules. S samples, vortexing than not, should be should b ometimes th the chemic g steps in the ,degraded, r e kept frozen e gently hom he samples cal bonds in e column-ba resulting in n during the mogenized b may be "ol n the DNA ased and solu smeared DN process of m by slowly tu ld", i.e., sto molecules m ution-based NA bands ( mechanical h urning the m ored for a lo may have be methods, th (Figure 1). T homogenizat microtubes up ong time at ecome weak he DNA beco To avoid th tion, and ins pside down t 20°C. In ker. Then, in omes, more his, such sam stead of vort and back se n the mples exed, everal

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od samples.

T extraction and ones The methods n, solution-b that use ma s for prepar based metho agnetic partic ration of nu ods (homem cles. ucleic acid made and co samples ca ommercial k an be group kits), column ped into the n-based meth ermal hods, The methods for preparation of nucleic acid samples can be grouped into thermal extraction, solution-based methods (homemade and commercial kits), column-based methods, and ones that use magnetic particles.

T It can be method [ used; dilu Thermal extra e used to ext [8, 9], but th utions are pr action is a q tract DNA fr hey generall repared in U quick and low rom pure ba ly include th Ultra-pure 18 w-cost metho acterial cultu he following 8.2 MΩDNas od that does ures. There a g procedure: se/RNase-fre s not require are different cultures gro ee water (wh special reag variations o own 18–24 hen using cu gents. of the h are ulture Thermal extraction is a quick and low-cost method that does not require special reagents. It can be used to extract DNA from pure bacterial cultures. There are different variations of the method [8, 9], but they generally include the following procedure: cultures grown 18–24 h are used; dilutions are prepared in Ultra-pure 18.2 MΩ DNase/RNase-free water (when using culture broth, 1:40, or when using agar cultures, 0.5 McF in 1 mL); the samples are heated at 100°C for 5–15 min and then centrifuged; the supernatant is taken and stored at –20°С.

Despite its advantages, this method has some limitations: it cannot eliminate low molecular weight peptides and gives a low 260/280 nm ratio (purity), i.e., about 0.600. This limits the use of the extracted DNA only to conventional PCR.

Thermal extraction is part of the official VTEC *E. coli* diagnostic procedure, which is based on detection of *eae* gene fragments up to 384 bp in length (15). This method, however, appears inapplicable to amplification of larger fragments, especially the *E. coli* 16s RNA gene, which is 1,465 bp in size [10].

The next group of methods, the solution-based ones, in principle, includes the following basic steps: lysis, RNase treatment (if applicable), protein precipitation (two fractions are formed), separation of the fraction that contains the nucleic acids; their precipitation, washing, drying, and regeneration.

One of the earliest and most common solution-based methods is phenol–chloroform extrac‐ tion. It also has various modifications. Volkin and Carter [11] first developed a method for RNA extraction with 2 M guanidinе hydrochloride, chlorophorm, and alcohol, in which guanidine acts as a deproteination agent and protects the RNA molecules by denaturing the proteins and RNases [12]. After a series of different steps, there is an extraction step using guanidinium thiocyanate–phenol–chloroform [13]. The phenol–chloroform combination (in a 1:1 ratio) gives better protein denaturation (by forming two fractions following centrifugation: a bottom organic phase and an upper aqueous phase) and reduces the amount of poly(A)+ mRNA in the organic phase as well as of insoluble RNA–protein complexes in an intermediate phase [14]. What is more, chloroform prevents the retention of water in the aqueous phase (water can degrade RNA molecules; see above), which results in higher yield [15]. To avoid foam formation, isoamyl alcohol can be added (chlorophorm–isoamyl alcohol, 24:1). It is the acidic properties of phenol that actually determine the partitioning of DNA and RNA in a separate phase: at neutral and slightly alkaline pH (i.e., pH 7–8), DNA and RNA remain in the aqueous phase, since the phosphate diesters are negatively charged. At lower pH (optimal pH 4.8), DNA partitions in the bottom phase, whereas RNA remains in the aqueous phase. This is due to the fact that the phosphate groups in DNA are more prone to neutralization compared to those in RNA [16, 17]. This method is commonly used for DNA extraction from liquid samples, although it also gives good results with tissue cultures, cell cultures etc., provided that they are first homogenized and disintegrated using a lysis buffer (commercial kit or homemade, for example – 10 mM Tris, 1 mM EDTA (ethylenediaminetetraacetic acid) and 0.1 M NaCl) + 20–50 µL of 10–20 mg/mL proteinase K and 1–18 h of incubation at 50–60°С.

degraded, resulting in smeared DNA bands (Figure 1). To avoid this, such samples should be kept frozen during the process of mechanical homogenization, and instead of vortexed, should be gently homogenized by slowly turning the microtubes upside down and back several times to adequately mix the reagents and, to a large extent, preserve the intactness of the DNA

B

nd.

od samples. DNA bands kov, 2007–2 le Spongifor

**Figure 1.** DNA from blood samples. Smeared DNA bands (A), DNA with double-strand breaks (B) and normal intact DNA bands (C). First Report and Final Report of a research grant awarded to Dr. Ivo Sirakov, 2007–2008 – Medicine and Biotechnology – Aids and infectious diseases, Transmissible Spongiform Encephalopathies, funded by the World

The methods for preparation of nucleic acid samples can be grouped into thermal extraction, solution-based methods (homemade and commercial kits), column-based methods, and ones

Thermal extraction is a quick and low-cost method that does not require special reagents. It can be used to extract DNA from pure bacterial cultures. There are different variations of the method [8, 9], but they generally include the following procedure: cultures grown 18–24 h are used; dilutions are prepared in Ultra-pure 18.2 MΩ DNase/RNase-free water (when using culture broth, 1:40, or when using agar cultures, 0.5 McF in 1 mL); the samples are heated at 100°C for 5–15 min and then centrifuged; the supernatant is taken and stored at –20°С.

Despite its advantages, this method has some limitations: it cannot eliminate low molecular weight peptides and gives a low 260/280 nm ratio (purity), i.e., about 0.600. This limits the use

Thermal extraction is part of the official VTEC *E. coli* diagnostic procedure, which is based on detection of *eae* gene fragments up to 384 bp in length (15). This method, however, appears inapplicable to amplification of larger fragments, especially the *E. coli* 16s RNA gene, which is

The next group of methods, the solution-based ones, in principle, includes the following basic steps: lysis, RNase treatment (if applicable), protein precipitation (two fractions are formed), separation of the fraction that contains the nucleic acids; their precipitation, washing, drying,

One of the earliest and most common solution-based methods is phenol–chloroform extrac‐ tion. It also has various modifications. Volkin and Carter [11] first developed a method for

C Smeared DN (C). First R 2008 – Med rm Encephal

NA bands (A Report and dicine and opathies, fun

samples ca ommercial k

od that does ures. There a g procedure: se/RNase-fre

ep.

ored for a lo may have be methods, th (Figure 1). T homogenizat microtubes up tent, preserv

ucleic acid made and co

w-cost metho acterial cultu he following 8.2 MΩDNas

ration of nu ods (homem cles. quick and low rom pure ba ly include th Ultra-pure 18

NA from bloo mal intact D r. Ivo Sirak Transmissibl a, Switzerlan

s for prepar based metho agnetic partic action is a q tract DNA fr hey generall repared in U

molecules.

A **F** breaks (B grantawa infectiou of Scient

**Figure 1.**DN B)and norm arded to Dr us diseases, T tists, Geneva

The methods n, solution-b that use ma Thermal extra e used to ext [8, 9], but th utions are pr

of the extracted DNA only to conventional PCR.

es.

4 Nucleic Acids - From Basic Aspects to Laboratory Tools

T extraction and ones T It can be method [ used; dilu

that use magnetic particles.

1,465 bp in size [10].

and regeneration.

Federation of Scientists, Geneva, Switzerland.

A column b lysis: in t be washe and/orsub sputum, mucolyti samples positive embedde applied t bacteria, exception layer; en shock or tissue cu centrifug suppleme S samples, vortexing than not, should be should b times to molecule

Another key blocking and the case of c ed in PBS (p bjected to se intestinal lo ic –"mucusfrom insect bacteria are ed tissues are to disrupt ce cell culture n of some G nzymatic deg r freeze/thaw ultures. Shou ge the mix entation, in c ometimes th the chemic g steps in the ,degraded, r e kept frozen e gently hom adequately m

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– if incomp y. There are cultures, de e) or physio es. In the ca irst decrease g., acetylcys requires 50– e; yeasts, wi o remove the osmotic shoc haotropic sa owing to the oteinase K), raction from fully lysed, upernatant p es such a ste ld", i.e., sto molecules m ution-based NA bands ( mechanical h urning the m o a large ext

plete – woul e different w epending on ological salin se of mucou e the viscos steine). The –200 mg of ith zymolya e paraffin. O ck, which is alts, for all e greater thic , which is o m hair, feathe one way to prior to co

d result in r ways to aid th the aim of a ne and resusp us samples ( sityof the m e mechanica sample in m ase or lytica Other approa s suitable fo types of s ckness of the often combin ers etc.); an o overcome t olumn load

reduced yield he process o analysis, they pended in dd (nasal discha material (usi al processin most kits. G se; and para aches that ca or Gram-neg amples with eir peptidogl ned with osm nd detergents the problem ding or alc

d and of cell y can dH2O arges, ing a ng of Gramaffinan be gative h the lycan motic s, for m is to cohol

t 20°C. In ker. Then, in omes, more his, such sam stead of vort and back se ness of the D

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s not require are different cultures gro ee water (wh There are some specifics in RNA extraction procedures: to protect RNA molecules against RNase attack, chaotropic salts are added to extraction buffers. High-purity RNA can be obtained by guanidine treatment followed by gradient ultracentrifugation in CsCl [12], cesium trifluoroacetate, or LiCl. A disadvantage is the need to use an ultracentrifuge and the fact that the method is time-consuming (it takes about 16 h).

In the case of notoriously difficult samples, Birnboim [18] recommend the use of a combination of SDS (sodium dodecyl sulfate) and urea to more effectively inhibit leukocyte RNases.

During extraction, beta-mercaptoethanol can be added to denaturate RNases by reducing disulfide bonds and to aid the release of RNA from RNA–protein complexes.

For RNA extraction, phenol and guanidinium isothiocyanate mixtures (Trizol reagent) are available under different commercial brand names and chlorophorm is then added in the extraction process to separate the phases [19].

As a whole, solution-based methods, and especially their homemade versions, have the disadvantage that they use toxic reagents and, therefore, a fume hood is a must; moreover, precision in phase separation is difficult to achieve; the extracted nucleic acid samples are of lower purity (especially in homemade methods) compared to those obtained by column-based methods; the obtained samples have limited use – mainly for conventional PCR. Commercial kits and protocols, albeit validated for downstream applications, do not solve the problem of reagent toxicity and subjective factors related to the person who performs the procedure.

The main advantage of these methods is that they allow for a proportionally larger initial material to be used.

Column-based methods generally use the fact that DNA and RNA molecules are negatively charged to capture them by silica membrane and ion exchange chromatography. These methods include lysis in one or two steps (in silica membranes – chaotropes and proteolytic lysis, and in anion exchange – detergent and enzymatic digestion); then, loading the liquid sample onto a column and centrifugation for the purpose of nucleic acid binding in a high-salt environment; treatment of the column with lysis buffer and/or directly with wash buffer (containing alcohol and high salt); free centrifugation to eliminate the remaining buffer (ethanol\*); elution with ultra-pure RNase/DNase-free H2O (≈pH 8.0) or low-ionic-strength buffer in the case of silica membrane and higher-salt buffer (not suitable for most downstream applications) in the case of anion exchange; incubation of room temperature for 1–3 min and centrifugation. It is possible to repeat the washing and elution steps to achieve further purification (if the column is treated with DNase or RNase after the first elution) or to elute a higher amount of nucleic acids. The standard procedure takes about 20 min per sample or 35– 40 min in the case of DNase or RNase treatment. It may be possible to use more starting sample than recommended but it should be kept in mind that this could overload the purification column, resulting in lower yield and/or higher percentage of impurities.\* Ethanol remaining in the end-product may lead to the escape of the nucleic acid from the wells during gel electrophoresis and may block restriction analysis, PCR, and other enzyme reactions.

To directly extract mRNA, without an initial procedure of total RNA extraction, oligo(dT) affinity chromatography is used. In this method, oligo(dT) is bound to cellulose or paramag‐ netic particles. The essence of the method lies in binding of mRNA poly(A+ ) tails to the oligo(dT) fragments attached to the matrix in high-salt conditions. Then, high-salt wash buffer is applied, followed by low-salt wash buffer, and elution under low ionic strength. This method, however, is only limited to eukaryotic cells, since their mRNA is polyadenylated. Another drawback is that the samples may be selectively enriched in mRNAs with shorter poly(A) tracts. What should be avoided is using a maximum amount of sample and overload‐ ing, as indicated by higher viscosity and a mucus-like look of the medium.

Another approach to nucleic acid extraction is based on magnetic particles, i.e., the so-called charge-based method: at pH ≤ 6.5, the surface of magnetic particle is positively charged and binds nucleic acids. Depending on the purpose, the unwanted nucleic acid can be eliminated by DNase or RNase treatment. Then, the nucleic acid molecules remaining bound to the particles are released by changing the pH: at pH ≥ 8.5, the surface is neutral and the nucleic acid molecules are released.

In the case of DNA extraction, the comparative analysis of all these methods shows that the quantity/quality of extracted DNA is inversely proportional to the extraction time.

It is commonly accepted to assess the purity of extracted nucleic acid samples based on the ratio of nucleic acids to proteins (impurities). This ratio is determined by measuring the absorbance of the samples at 260 and 280 nm and calculating the 260/280 nm ratio (the absorbance at 260 nm reflects the mean absorbance of purines and pyrimidines [20, 21]. In the case of DNA samples, it is recommended to make a background correction at 320 nm, which accounts for turbidity. The sample purity reflects the degree to which different contaminants have been eliminated in the nucleic acid extraction procedure. There are different possible sources of contamination. These include the reagents – salts and residual buffer (especially the alcohol-containing wash buffer), as well as different compounds present in the starting material – polysaccharides, phenolic compounds, and DNA or RNA and proteins – nucleases. Elimination of these contaminants is an intrinsic part of the procedures in all the nucleic acid extraction methods. Moreover, there may be included additional purification steps: ultracen‐ trifugation to eliminate high molecular weight polysaccharides; use of beta-mercaptoethanol, dithiothreitol, sulfite, etc.; RNase or DNase can be added and care should be taken to protect the samples from exogenous RNases or DNases of different nature (the samples themselves, human skin, or the laboratory environment), by following the principles of Good Laboratory Practice. It is also recommended to use DEPC, which inactivates RNases in solutions, with the exception of solutions that contain primary amines, such as Hepes buffer and Tris, as they reduce its effect. It is noteworthy that autoclaving will not destroy RNase activity. In plant samples, contamination is often due to polysaccharides and polyphenols, which are eliminated by using polyvinylpyrrolidone. DNA and RNA are also considered contaminants: in DNA analysis, RNA acts as a contaminant and vice versa. Hence, DNase or RNase is used to eliminate contaminating DNA or RNA, respectively. This is so, first of all, because both DNA and RNA contribute to the total nucleic acid content, which – if too high – may block down‐ stream PCR. On the other hand, in gene expression analysis, the methods are sensitive to DNA contamination. For example, reverse transcription (RT) and microarray methods require high RNA purity, since small DNA fragments may anneal to the primers, giving a false positive result, whereas other contaminants (phenol, ethanol, and salts in RT) may react with enzymes, blocking the reaction or increasing the background signal. Conversely, methods such as Northern blotting are not as sensitive to contamination. Thus, sample purity may vary in a certain range – from 0.600 to over 3.0 (mostly for RNA) – depending on the aim of analysis, the extraction method used, the starting material, and the operator. Samples are considered to be of good quality if their 260/280 nm ratio is over 1.7, which is satisfactory for most down‐ stream applications.

The main advantage of these methods is that they allow for a proportionally larger initial

Column-based methods generally use the fact that DNA and RNA molecules are negatively charged to capture them by silica membrane and ion exchange chromatography. These methods include lysis in one or two steps (in silica membranes – chaotropes and proteolytic lysis, and in anion exchange – detergent and enzymatic digestion); then, loading the liquid sample onto a column and centrifugation for the purpose of nucleic acid binding in a high-salt environment; treatment of the column with lysis buffer and/or directly with wash buffer (containing alcohol and high salt); free centrifugation to eliminate the remaining buffer (ethanol\*); elution with ultra-pure RNase/DNase-free H2O (≈pH 8.0) or low-ionic-strength buffer in the case of silica membrane and higher-salt buffer (not suitable for most downstream applications) in the case of anion exchange; incubation of room temperature for 1–3 min and centrifugation. It is possible to repeat the washing and elution steps to achieve further purification (if the column is treated with DNase or RNase after the first elution) or to elute a higher amount of nucleic acids. The standard procedure takes about 20 min per sample or 35– 40 min in the case of DNase or RNase treatment. It may be possible to use more starting sample than recommended but it should be kept in mind that this could overload the purification column, resulting in lower yield and/or higher percentage of impurities.\* Ethanol remaining in the end-product may lead to the escape of the nucleic acid from the wells during gel

electrophoresis and may block restriction analysis, PCR, and other enzyme reactions.

netic particles. The essence of the method lies in binding of mRNA poly(A+

ing, as indicated by higher viscosity and a mucus-like look of the medium.

To directly extract mRNA, without an initial procedure of total RNA extraction, oligo(dT) affinity chromatography is used. In this method, oligo(dT) is bound to cellulose or paramag‐

oligo(dT) fragments attached to the matrix in high-salt conditions. Then, high-salt wash buffer is applied, followed by low-salt wash buffer, and elution under low ionic strength. This method, however, is only limited to eukaryotic cells, since their mRNA is polyadenylated. Another drawback is that the samples may be selectively enriched in mRNAs with shorter poly(A) tracts. What should be avoided is using a maximum amount of sample and overload‐

Another approach to nucleic acid extraction is based on magnetic particles, i.e., the so-called charge-based method: at pH ≤ 6.5, the surface of magnetic particle is positively charged and binds nucleic acids. Depending on the purpose, the unwanted nucleic acid can be eliminated by DNase or RNase treatment. Then, the nucleic acid molecules remaining bound to the particles are released by changing the pH: at pH ≥ 8.5, the surface is neutral and the nucleic

In the case of DNA extraction, the comparative analysis of all these methods shows that the

It is commonly accepted to assess the purity of extracted nucleic acid samples based on the ratio of nucleic acids to proteins (impurities). This ratio is determined by measuring the absorbance of the samples at 260 and 280 nm and calculating the 260/280 nm ratio (the

quantity/quality of extracted DNA is inversely proportional to the extraction time.

) tails to the

material to be used.

6 Nucleic Acids - From Basic Aspects to Laboratory Tools

acid molecules are released.

However, it is not only the quality of the extracted nucleic acids that is considered important but also their quantity. Depending on the downstream application, if the concentration of nucleic acid in a sample is low, this may, at least in part, be compensated for by using a greater volume of sample in the reaction mixture (the sample purity should be considered as well). For example, if there is insufficient concentration of nucleic acid, non-specific products may be amplified in PCR; or some expected fragments may appear missing in restriction enzyme analysis; or short read lengths may be generated in sequencing. Nucleic acid concentration that is too high may also have adverse effects: amplification of non-specific fragments in PCR or lack of product due to reaction blocking; retention of nucleic acid in gel wells during electrophoresis; incomplete digestion in restriction enzyme analysis (which may be compen‐ sated for by adding a proportionally higher amount of enzyme and/or extending the incuba‐ tion time); or high background in sequencing procedures.

To concentrate, purify DNA and reduce the salt content in DNA samples, precipitation is used (sample: 99% molecular grade ethanol (or isopropanol) 1:1 + 2 to 5% 3.5–7 М ammonium acetate) with 30–60 min incubation at –20°С, centrifugation at 4°C at maximum speed, washing in maximum volume of 70% ice-cold ethanol (–20°С), centrifugation, drying for 3–7 min and resuspending in a desired volume of ultra-pure water, ¼ ТЕ buffer or 1× ТЕ buffer, depending on the downstream application and the expected duration of storage.

Another way to determine the quality and quantity of extracted nucleic acids, apart from spectrophotometric analysis, is by gel electrophoresis (GE), which is informative of fragmen‐ tation and presence of impurities. Although, in some cases, GE may be sufficient when the researcher is experienced, it is still recommended to use both methods together.

The type of storage and its duration are crucial for the downstream applications. In the case of DNA samples, both the temperature storage and the buffer composition are important factors. Storage at –20°С in 1× PCR buffer for 100 days gives very good results [22]. There are reports that DNA stability can be enhanced by adding 50% glycerol, which limits the formation of ice crystals [23]. Overall, storage at –20°С in commercially available elution buffers (EB) gives stable DNA for use even after a year of storage (EDTA as a component of EB protects DNA against degradation), provided that repeated freezing and thawing are avoided. RNA samples are stored at –80°С in stabilizing buffers that contain EDTA; even storage microcap‐ sules have been developed [1].
