Nuclei Acid Extraction

MAIN EXTRACTION TECHNIQUES
Two categories:
1) Remove everything that is not DNA.
2) Selectively capture DNA.
Both classes of methods are used. They share two initial phases:
a) Cell separation.
b) Lysis of the cells.
Then, one of the two methods is applied to the lysate.

CELL SEPARATION
Aside from lymphocytes, where there are DNA rearrangements, DNA is virtually the same in all the somatic cells of an individual. Mitochondrial DNA can have slightly different sequences, and these can be distributed differently in each tissue. In tumours, it is different: cancer cells are estimated to have 50-100 deleterious mutations and can be part of subclones with different sequences. Moreover, they can be mixed with normal cells (e.g. stromal fibroblasts). In general, the most accessible tissue is peripheral blood, which also, being liquid, provides cells that are already isolated. Human saliva is even easier to be obtained, and it contains cells exfoliated from the buccal mucosa (epithelial cells) and lymphocytes. In this type of sample, however, the DNA is certainly more degraded than in the blood.

LYSIS
It is the phase in which the cells are broken, since DNA is complexed with proteins, and it is enclosed in two membranes.
The first goal is to put everything in suspension. This aim can be achieved with three fundamental methods:
a) Use of a detergent: it lowers the surface tension, binds to the lipids of the plasma membrane and on the other hand binds to the water by "tearing" the membrane. Generally, SDS (sodium dodecyl sulphate) is used.
b) Use of enzymes such as proteases. The best known is the protease K which, in small quantity, degrades proteins down to single amino acids.
c) Chaotropic agents ("agents able to induce chaos"): they disturb ordered structures, having at least one very large charged atom (ion) which at high concentration interferes with the three-dimensional architecture of proteins. Examples: sodium iodide (NaI), guanidine thiocyanate (GuSCN).
The strong denaturant activity of guanidine thiocyanate is also useful to inactivate endogenous RNases released during RNA extraction, in addition to its ability to destroy cells and solubilize cell components. Guanidine thiocyanate homogenate is obtained in the first step of the most popular method RNA purification from cells, described by Chomczynski and Sacchi in 1987.
Mechanical lysis may also be required in advance, e.g. in the case of solid tumours of a very hard, wooden consistency (carcinomas).

DNA RECOVERY FROM THE LYSATE

1) EXTRACTION WITH ORGANIC SOLVENTS (classic method): phenol-chloroform. This method consists of several stages.
a) Extraction. It can be considered as a real "ex-traction", we separate the DNA from other undesired cell components; most of these are proteins. Organic solvents are generally apolar. DNA has negative surface charges, so it dissolves well in water. Proteins instead have a more complex structure, a carbon skeleton with unevenly distributed charges. DNA solubilizes only in the aqueous phase, and proteins globally are more suited for an apolar solvent. There is, therefore, a phase separation: DNA ends in the polar phase, while proteins go toward the apolar phase. This separation is done with a single centrifugation. Fats go into the apolar phase. RNA should end in the polar phase, but using basic phenol (pH>>7) causes hydrolyzation of the RNA (for the extra hydroxyl which has its sugar). DNA is comfortable in a basic environment, being very stable at pH 7-8. Therefore, for DNA extraction, phenol is mixed with a pH 8 buffer; thus, phenol for DNA is obtained. RNA should be extracted in water-saturated phenol, at low pH (~4, acid phenol). Phenol is heavier than water and therefore tends to go downwards, with a centrifugation it is therefore possible to separate the two phases easily. The solid phenol is liquefied at 60°C and is then stored under the adequate buffer (TrisHCl at pH 8 for DNA, water for RNA). Phenol is usually used together with another apolar solvent, chloroform, and with a small quantity of isoamylic alcohol as an antifoaming agent.
Following centrifugation, the upper polar, aqueous phase is taken out of the tube. To remove salt excess, and to reduce the sample volume, it is now possible to concentrate DNA by alcohol precipitation.

b) Precipitation. It is used to enrich and to concentrate DNA. Any nucleic acid dissolved in water may be re-precipitated by alcohol. A salt sodium (Sodium Acetate) is usually added before extraction or may be added now, to favour the precipitation of DNA molecules, that being negatively charged would repel each other. A cation may neutralize them by binding the phosphate groups.
Ethanol (CH3-CH2OH) has an apolar region (CH3-CH2) and a polar (OH) region, thus it creates an unstable environment for hydrophilic nucleic acids, favouring their precipitation.
If instead of ethanol isopropylic alcohol (isopropanol, 2-propanol) is used half of the dose it is sufficient, because it has three carbon atoms and not two like EtOH: 1 volume of the solution plus 1 volume of isopropyl alcohol. When using ethanol, two volumes of it must be added.
In any case, cold alcohol strongly favours precipitation, because low temperature decreases the solubility of the solute. Therefore, it is usual to maintain a bottle of alcohol (ethanol or isopropanol) in the freezer at -20°C, where it will remain liquid.
Thus, following the addition of cold alcohol, and centrifugation in a cold room or a refrigerated centrifuge, a pellet of sodium deoxyribonucleate is collected.
If there is little starting DNA (poor sample), little is found at the end of the extraction: to recover as much as possible nucleic acid, an exogenous source of nucleic acid may be used as a "carrier" to favour precipitation. Yeast tRNA (ala-tRNA) is used, which serves as an aggregation centre and does not affect subsequent analyses. It is estimated that even a few pg of human DNA can be recovered in this way.
c) Washing. It is necessary because precipitated DNA is still very rich in sodium which can cause interference with restriction enzymes or with polymerases. To wash DNA, 70-75% ethanol is used, this solution is very alcoholic but still a little aqueous, so it is still sufficiently apolar to pull down the DNA, but sufficiently polar to retain sodium, "extracting" it from the DNA.
d) Suspension. Following removal of the supernatant, the final DNA pellet is resuspended in one drop (50 μL) of sterile, bidistilled water. In the case of RNA, an RNAse inhibitor may be added, such as RNAsin (extracted from the placenta). Diethylpirocarbonate (DEPC)-treated water was also widely used in the past (however, this substance is toxic).

Note - Due to the presence of an additional hydroxyl group in the sugar (ribose) compared to DNA, RNA is, in general, much more reactive than DNA. Also, it can be cleaved by cellular RNases released from cells upon lysis. These enzymes are found on the skin too so that gloves are required during RNA extraction. RNases have strong intrachain disulfide bonds, therefore can be resistant to prolonged boiling and can refold quickly following denaturation. In order to prevent RNA degradation, RNases should be inactivated as rapidly as possible at the very first stage in the extraction process, which should be performed maintaining the tubes on ice and using RNase inhibitors. While RNases do not require divalent cations for their activity, DNases do, so the latter can be inhibited by adding ethylenediaminetetraacetic acid (EDTA), a chelating agent, i.e., able to sequester metal ions such as Ca2+.
Several types of RNA (e.g., almost all the mRNAs) have a poly(A) tail, that can be used for their selective isolation following binding with poly(T).

2) GLASS BEADS AFFINITY
In silicate gel (microscopic glass beads), DNA binds strongly to the beads in the presence of highly concentrated chaotropic agents (it has been known for decades). It is only needed to apply the lysate to these beads packed in a spin-column, and following a centrifugation the DNA remains attached to the beads. With a second washing at different molarity (low osmolarity), the DNA is detached and recovered.
It is a fast method, but less nucleic acid is usually recovered.

An agarose gel stained with ethidium bromide is prepared.
Ethidium bromide is an intercalating agent selectively staining nucleic acids. It can be visualized under ultraviolet (UV) irradiation, typically at a transilluminator with a 302 nm lamp.
Agarose gel can resolve two bands if they are related to polynucleotides different by at least 12 (in the best case)-50 bp. The agarose concentration should be adjusted for the best resolution (higher concentration, up to 2%, resolve smaller molecules, up to 100 bp; low concentrations, down to 0.3%, resolve larger molecules, of thousand bp.

Polyacrylamide gels may resolve nucleic acids molecules varying in size from 10 to 1500 bp and differing even by only 1 nucleotide (6% total acrylamide), this property is exploited for Sanger sequencing.
Acrylamide monomer is provided as a powder. Unpolymerized acrylamide is a neurotoxin. Upon addition of water, in the absence of oxygen, it polymerizes resulting in the formation of polyacrylamide. Gel size can be regulated by adjusting the concentration of acrylamide.
Bisacrylamide can form cross-links between two acrylamide molecules, thus creating gel pores of regular shape. Polymerization is triggered by a source of free radicals (ammonium persulfate) and a stabilizer (TEMED). The ratio of acrylamide to bisacrylamide is typically 19:1 for the analysis of nucleic acids.
Polyacrylamide gels are usually 1-2 mm thick and are cast vertically between two glass plates, to use a small quantity of this expensive reagent and to remove the air (oxygen), which hampers polymerization. However, systems have been marketed, offering the possibility to pour acrylamide gels horizontally.

DNA gel electrophoresis

2nd check - size: the pieces must be at least 20,000 bases. It can be estimated by comparison with a size marker.

3rd check - quality: DNA must not be degraded, e.g. showing different molecular weight bands scattered throughout the lane.

4th check - quantity: estimation of the DNA quantity based on the size and brightness of the bands.

The gel is more sensitive than the spectrophotometer in doing this estimation, because any bright signal is related to a nucleic acid, while spectrophotometer absorbance at 260 nm can still detect proteins (although most of them adsorb at 280 nm wavelength).
A ratio greater than 1.8 between the adsorbance value at 260 nm and the one at 280 nm is also considered as an estimation of a low level of contaminating proteins in the RNA preparation.

5th check - functionality, DNA can be effectively subjected to enzymatic treatment, e.g. digested by a restriction enzyme (e.g. EcoR1).

The method recovers any DNA polynucleotides, thus, non-human too. If there are viruses or bacteria in the sample, their nucleic acids will also be extracted.

This method can be done on samples of saliva, blood, body fluids; buccal, nasal, pharyngeal, ocular swabs; tissues, hair, sperm.

The final estimate of the quality of the extracted DNA is mainly made on the gel. For the quantitative estimation, it is important to load a "correct" quantity of sample, in fact, the shape of the bands is determined by physical factors, in particular from the quantity of DNA loaded, if this is excessive there is a dragging effect (trailing) with the formation of lateral "tails" on the upper edge of the band. If more than 200 ng of DNA are loaded in a 4-4.5 mm wide standard well, these effects begin to be obtained which compromise the definition of the bands, so that less molecular species are distinguished. On the other hand, loading less than 1 ng the bands will be invisible as this is the limit of detection for ethidium bromide. If there is a need to load a lot of DNA or RNA, wider wells should be used (10-tooth comb).

All mRNAs (or noncoding RNAs) are dispersed across the gel lane according to their size, each species being present at low concentration, because total RNA is made up of 85% rRNA, 10% tRNA, 1-5% mRNA.

References

The two most known books about protocols in Molecular Biology are often referred to after the name of their first Authors: Sambrook, and Ausubel, respectively. A shared feature is the discussion of the basic principles of the procedures presented.

Molecular Cloning: A Laboratory Manual
The first one-volume edition (1982) was authored by the molecular biologist Tom Maniatis, it has been known for many years as "The Maniatis" and has been present in virtually any laboratory of Molecular Biology and Genetics.
Subsequent three-volume editions were authored by Sambrook, Fritsch and Maniatis (1989) and by Sambrook and Russell (2001). Currently, the fourth edition is available (Green and Sambrook, 2012) at the Cold Spring Harbor Laboratory (CSHL) publisher.

Current Protocols in Molecular Biology (popularly referred to as "The Red Book") by Ausubel, Brent, Kingston, Moore, Seidman, Smith and Struhl consists of three looseleaf volumes and has been published by Wiley in 1988. Quarterly updates can be filed into the looseleaf.

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