Isolation and estimation of DNA

BACKGROUND

The extraction of DNA from plant tissue involves breaking or digesting away the cell walls, followed by disruption of cell membranes to release DNA into the extraction buffer. This is normally achieved by cetyl trimethyl ammonium bromide (CTAB), a cationic detergent, which forms complexes with proteins. It acts on all but the most acidic polysaccharides does not precipitate nucleic acids.¹ 

Similarly extracting the intact genomic DNA from animal tissue is obtained by lysing the cells and inhibits DNase activity of cells by phenol-chloroform method. Since DNase activity has pH optima of 5.0 and depends upon Mg⁺⁺ ions. A buffer system having alkaline pH and with divalent cation chelator is used to inactivate DNase.

Hence, Tris-Cl buffer (pH 8.0), with EDTA is used to inhibit DNase in initial steps of homogenization. SDS is an anionic detergent used for lysis of the cell to releaseits contents.^1,2 RNase A is used to digest RNA. Proteinase K is used to digest protein in solution including RNase A.

To precipitate the proteins from the homogenate, phenol is used along with chloroform, by means of which the solution is separated into two distinct layers, upper aqueous phase contains nucleic acid (here only the DNA, as RNA is digested) and lower phase contains the organic compounds and the denatured proteins remain in the interface.³ 

The chloroform causes surface denaturation of proteins. Isoamyl alcohol reduces foaming and stabilizes the interface between the aqueous phase and organic phase, where protein collects. Then upper phase containing nucleic acid is separated and DNA is precipitated by the addition of absolute ethanol and sodium acetate. Sodium acetate reacts with phosphoric acid of DNA, which helps in precipitation of DNA and forms a thread-like appearance.

It is often difficult to measure the concentration of high molecular weight DNA by standard methods such as absorbance at 260 nm. This is because the DNA solution is non-homogenous and usually so viscous that it is impossible to withdraw a representative sample for analysis.

This problem can be minimized by withdrawing a large sample (10-20 µl) with an autopipette equipped with broad mouthed tip. The sample is then diluted with 0.5 ml of TE (pH 8.0) and vortexed vigorously for 1-2 minutes. The absorbance of the diluted sample can then read at 260, 270 and 280 nm in the standard way.^3,4

A solution with value of 1 (A₂₆₀ measurement) contains nearly 50 µg of DNA/ml.

Aim of the experiment is to isolate genomic DNA from animal tissue by Phenol-Chloroform method and to estimate the quantity and purity of the genomic DNA.

REQUIREMENTS

• Eppendorf tubes 1.5 ml – 50 numbers
• Reagent bottles
• Beakers 1 L – 2 sets; 0.5 L – 2 sets; 100 ml – 2 sets
• Micro tips 1 ml, 0.2 ml – 1 sets of each
• Pestle and mortar – 1 set of each
• Cooling centrifuge
• Autopipettes and Water bath
• Cyclomixture
• Spectrophotometer
• Autoclave
• Tissue paper
• Ice box
• DNA samples
• Cuvette

Reagents³

Autoclaved double distilled water (DDW)

Tris-Cl (1 M, pH 8.0): Dissolve 12.1 g of tris base in 70 ml of DDW. Adjust pH to 8.0 by slowly adding 2 ml of 6N HCL and make up the volume upto 100 ml with sterile water. Sterilize by autoclaving store at room temperature.

EDTA (ethylene diamine tetra-acetic acid), 0.5 M, pH 8.0: Add 18.6 g of disodium EDTA .2H₂O to 70 ml of DDW. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH ( ̴5 g of NaOH pellets). Adjust the volume of the solution to 100 ml and sterilize by autoclaving. The disodium salt of EDTA will not go into the solution until the pH of the solution is adjusted to ̴ 8.0 by adding NaOH.³

10% SDS (w/v): Also called sodium lauryl sulfate. Dissolve 10g of SDS in 90 ml of DDW. Heat to 68^°C and stir with magnetic stirrer. If necessary adjust the pH to 7.2 by adding a few drops of concentrated HCl. Adjust the volume to 100 ml with DDW. Store at room temperature.

TAE Buffer (50X) : Take the components i.e., 24.2 g Tris base, 5.71 ml glacial acetic acid and 10ml of 0.5 M EDTA (pH 8.0) and make up the volume 100 ml with DDW. Sterilize by autoclaving and store at room temperature.

Lysis buffer: Take the following components from the stock solution and make the desired final concentration by taking volumes given in the table below.⁴

Sl.no.	Components	Stock conc.	Volume (V)	Final conc.
1	Water	–	14.8	–
2	Tris-Cl, pH 8	1M	0.2	0.01
3	EDTA	0.5M	4.0	0.1
4	SDS	10%	1.0	0.5%

Sodium acetate, 3 M, pH 8.0: Dissolve 24.6 g of sodium acetate in 70 ml DDW. Adjust the pH to 5.0 with acetic acid and volume to100 ml.

Proteinase K – 3 mg/ml

RNase A – 10 mg/ml

Phenol

Chloroform

Isoamyl alcohol

Glacial acetic acid

Alcohol – absolute alcohol and 75% alcohol

T₁₀E₁: Prepare the solution with final concentration of 10mM Tris-Cl (pH 8.0) and 1 mM EDTA (pH 8.0) by taking stock solution 2 and 3 and store at 4^°C.

PROCEDURE

A 10% homogenate ( ̴0.05g tissue + 0.4 ml lysis buffer ) with the help of a mortar in a 1.5 ml micro centrifuge tube, mixed properly and left on bench for 5 minutes. To the homogenate 10 µl of 10 mg/ml RNase A is added and vortexed for 10 seconds and incubated at 37^°C for 1 h. Then the Proteinase K (final conc. 100 µg/ml) were added and vortexed for 10 secs, incubated at 50^°C for 1 h.

An equal volume of phenol (0.4 ml) was added and mixed properly by inverting up and down the tubes around 20 times. Then centrifuge the mixture at 12,000 rpm for 10minutes at 4^°C. the aqueous phase is collected and equal volume of PCI (phenol: chloroform : isoamyl alchol =25:24:1) were added to it and mixed gently by inverting up and down (20 times). Then the mixture was again centrifuged at 12,000 rpm for 10minutes at 4^°C.

Again the aqueous phase is collected and add equal volume of chloroform, again centrifuged at 12,000 rpm for 10miutes at 4^◦C. The aqueous phase is collected and two volumes of absolute alcohol and sodium acetate (final conc. 0.3 M) were added and mixed gently by inverting up and down (10 times) and kept at – 40^°C for 30 minutes.

Then, centrifuged at 12,000 rpm for 10 minutes at 4^◦C.The supernatant was discarded and wash (centrifuged at 12,000 rpm for 5 minutes at 4^°C) the pellets with 500 µl of 75% alcohol. The supernatant was decanted and the DNA pellet were left at room temperature for air dry until the whitish pellet turn to transparent (avoid over dry). The dried DNA pellet was dissolved in 20 µl of T₁₀E₁ and stored at 4^°C for further use.

For estimation

Observed OD × 50 µg/ml × Dilution factor = Amount of DNA (µg/ml)

A solution with value of 1(A₂₆₀ measurement) contains ̴ 50 µg of DNA/ml. Note that estimates of purity of nucleic acids based on OD₂₆₀ : OD₂₈₀ ratios are unreliable and that estimates of concentration are inaccurate if the sample contains significant amounts of phenol. Water saturated with phenol adsorbs with a characteristics peak at 270nm and an OD₂₆₀ : OD₂₈₀ ratio of 2. Nucleic acid preparations free of phenol (as in this protocol) should have OD₂₆₀ : OD₂₈₀ ratios of
̴ 1.2.^4,5 

CONCLUSION

From the above experiment it can be concluded that the DNA has been extracted and isolated properly with its estimated amount in the supplied genome.

REFERENCES

1. Practical biotechnology (Methods and Protocols ) ; S Janarthanan & S Vincent
2. Rice G. “DNA Extraction”; Educational resources, Microbial life. Montana state university .
3. https://www.ijsr.in
4. Sambrook j, Russel DW(2001). Molecular cloning: A laboratory Manual 3^rd Ed. Cold spring harbor lab press.
5. Yoshikawa H, Dogruman-Al F, “Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples”.

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