DNA EXTRACTION. Have you done DNA extractions with your class?

DNA EXTRACTION

• Have you done DNA extractions with your class?

1865 presented paper Experiments on Plant Hybridization,

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Johann Friedrich Miescher1844 -1895

1869: Characterizes a new substance in puspus (June 15, 1866-August 23, 1866).

“… the substance was derived from the nucleus of the cell. Hence, we call it nuclein.”

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Pál Plósz (1871) verified the presence of nuclein in the nucleated erythrocytes of birds and reptiles and its absence from the erythrocytes of mammals, which are devoid of a nucleus.

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1881: 1881: (Ludwig Karl Martin (Ludwig Karl Martin Leonhard)Leonhard) Albrecht Kossel Albrecht Kossel determines that nucleic acid determines that nucleic acid is composed of four bases.is composed of four bases.

Untersuchungen über die Nukleine und ihre Spaltungsprodukte (Investigations into the nucleins and their cleavage products)

19101910

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1893: Albrecht Kossel 1893: Albrecht Kossel determines that nucleic determines that nucleic acid is composed of four acid is composed of four bases.bases.

2 purines:2 purines: 2 pyrimidines:2 pyrimidines:

adenine (A)adenine (A) cytosine (C)cytosine (C)guanine (G)guanine (G) thymine (T)thymine (T)

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1889: Richard Altmann 1889: Richard Altmann

finds that nuclein is acidic finds that nuclein is acidic

and renames it and renames it nucleic nucleic

acid acid (nucleïnsäure).(nucleïnsäure).

Ueber Nucleinsäuren. Archiv für Anatomie und Physiologie. Physiologische Abteilung. Leipzig, 1889, 524-536.

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Phoebus Aaron Phoebus Aaron (Theodore)(Theodore) Levene Levene (1869-1940)(1869-1940)

1909: Phoebus Levene discovers 1909: Phoebus Levene discovers that DNA is made of 3 basic that DNA is made of 3 basic components: a sugar, an acid, components: a sugar, an acid, and an organic base.and an organic base.

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1944: Oswald Avery, Colin MacLeod, and Maclyn McCarty establish that Griffith's 1944: Oswald Avery, Colin MacLeod, and Maclyn McCarty establish that Griffith's transforming principle is DNA, and suggest that it may function as the genetic material.transforming principle is DNA, and suggest that it may function as the genetic material.Avery, O. T., MacLeod, C. M. & McCarty, M. 1944. Studies of the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus Type III. J. Exp. Med. 79:137-158.

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1949: Roger Vendrely, Colette Vendrely, and André Boivin find half as 1949: Roger Vendrely, Colette Vendrely, and André Boivin find half as much DNA in the nuclei of sex cells as they find in body cells, thus much DNA in the nuclei of sex cells as they find in body cells, thus paralleling the reduction in the number of chromosomes, making DNA paralleling the reduction in the number of chromosomes, making DNA look like the genetic material.look like the genetic material.

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Rosalind FranklinRosalind Franklin

X-ray diffraction of DNA

1951

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1952: Alfred D. Hershey & Martha Chase1952: Alfred D. Hershey & Martha Chase

DNA is DNA is the the

genetic genetic materialmaterial

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Martha ChaseMartha ChaseAwarded a blender Awarded a blender (?)(?)

Alfred D. HersheyAlfred D. HersheyAwarded the Nobel PrizeAwarded the Nobel Prize

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“We have formulated a structure for the nucleic acids… The structure involves three intertwined helical polynucleotide chains. Each chain… has approximately twenty-four nucleotide residues in seven turns of the helix. The helixes form a right-handed screw. The phosphate groups are closely packed about the axis… with the pentose residues… and the purine and pyrimidine groups projecting radially...”

Linus Pauling & Robert B. Corey (Nature, 1953, 171:346).

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Pauling, L. and Corey, R. B. 1953. A proposed structure for the nucleic acids Proc. Natl. Acad. Sci. USA 39:84-97.

What are the essential components of a DNA extraction

Procedure?

1. Maximize DNA recovery

2. Remove inhibitors

3. Remove or inhibit nucleases

4. Maximize the quality of DNA

How Much DNA Do We Need?

• The PCR reactions call for on average 1 ng of DNA (single or double stranded).

• Many of the commercially available kits are sensitive below 1 ng of DNA (100-250 pg).

Basic steps for DNA extraction1. Breaking the cells open, commonly referred to

as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding, sonicating or treating the sample with lysis buffer .

2. Removing membrane lipids by adding a detergent.

Lysis Buffer: 50 ml Lysis Buffer5 mM EDTA pH 8.0 0.5 M EDTA = 500 ul200 mM NaCL 5M NaCl = 4 mL100 mM Tris pH 8.0 1 M Tris-HCL ph 8= 5 mL0.2% SDS Sodium dodecyl Sulfate 10% SDS = 1 mLWater MQ water = 39.5 mL Add prior to 55C o/n icubation0.4 mg/ml Proteinase K 20 mg/mL Prot K = 6 ul per 300ul of Lysis

Buffer

Purposes of the Extraction Buffer

1. Dissolve cellular membranes

2. Inactivation of DNase and Rnase

3. Assist in the removal of contaminants

DetergentsChaotropic saltsCTAB

DetergentsMetal chelatorsReducing agents

Salts

CTAB PVP

Extraction/Precipitation Method

+

Plasma membrane(phospholipid bilayer) Detergent molecules

Use of Detergents to Lyse Cells:

Mixed micelle

SDS

3. Removing proteins by adding a protease (optional but almost always done).

4. Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.

Most Commonly used DNA Extraction Procedures

• Organic (Phenol-Chloroform) Extraction

• Non-Organic (Proteinase K and Salting out)

• Qiagen (anion exchange resin)

The method utilized may be sample dependant, technique dependant, or analyst preference

EXTRACTION

• Perhaps the most basic of all procedures in genetic engineering is the purification of DNA. The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform.

ORGANIC EXTRACTION REAGENTS

• Cell Lysis Buffer - Non-ionic detergent , Salt, Buffer, EDTA designed to lyse outer cell membrane, but will not break down nuclear membrane.

• EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.


• Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.


• Phenol/Chlorform - The standard way to remove proteins from nucleic acids solutions is to extract once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one.

• Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid preparation.

• Phenol is highly corrosive and can cause severe burns.

Step 1: Disruption of cell walls by grinding

Step 2: Lysis of cells in extraction buffer

Step 1+2: mechanical disruption and homogenization in extraction buffer


Grind sample into a fine powder to shear cell walls and membranes

Mix thoroughly with extraction buffer to dissolve cell membranes and inhibit nuclease activity

A homogenizer allows cells to be mechanically disrupted within the extraction buffer

Crude lysate

Crude lysate containing nucleic acids and other cell constituents

Mix thoroughly with an equal volume of organic solvent

e.g. phenol, chloroform, or phenol:chloroform

Centrifuge

The aqueous phase contains water-soluble molecules, including nucleic acids. Proteins and lipids become trapped in the organic phase, and are thus separated away. Insoluble plant debris become trapped in the interphase between the two layers

Perform additional extractions for increased purity

Collect aqueous phase

Extraction/Precipitation MethodStep 3: Organic extraction

Organic

Aqueous

Interphase

• Pellet down nucleic acids. • Pellet down nucleic acids.

• Wash pellet with 70% ethanol to remove residual salts and other contaminants.

• Pellet down nucleic acids.

• Wash pellet with 70% ethanol to remove residual salts and other contaminants.

• Discard ethanol and allow pellet to dry.

After

Add alcohol and salt to precipitate nucleic acids from the aqueous fraction

Supernatant

Pellet

70% EtOH

Dissolve pellet (H2O, TE, etc.)

Step 4: Nucleic Acid Precipitation


Before After

Centrifuge Wash Centrifuge

Concentrating DNA byAlcohol Precipitation

• The most widely used method for concentrating DNA is precipitation with ethanol. The precipitate of nucleic acid, forms in the presence of moderate concentrations of monovalent cations (Salt, such as Na+), is recovered by centrifugation and redissolved in an appropriate buffer such as TE.

• The technique is rapid and is quantitative even with nanogram amounts of DNA.

Concentrating DNAAlcohol Precipitation

• The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the speed at which it is pelleted.

• DNA a concentrations as low as 20 ng/ml will form a precipitate that can be quantitatively recovered.

• Typically 2 volumes of ice cold ethanol are added to precipitate the DNA.


• Very short DNA molecules (<200 bp) are precipitated inefficiently by ethanol.

• The optimum pelleting conditions depend on the DNA concentration. Relatively vigorous microcentrifuge steps such as 15 minutes at or below room temperature at 12,000 rpm are designed to minimized the loss of DNA from samples with yields in the range of a few micrograms or less.


• Solutes that may be trapped in the precipitate may be removed by washing the DNA pellet with a solution of 70% ethanol. To make certain that no DNA is lost during washing, add 70% ethanol until the tube is 2/3 full. Vortex briefly, and recentrifuge. After the 70% ethanol wash, the pellet does not adhere tightly to the wall of thetube, so great care must be taken when removing the supernatant.


• Isopropanol (1 volume) may be used in place of ethanol (2 volumes) to precipitate DNA. Precipitation with isopropanol has the advantage that the volume of liquid to be centrifuged is smaller.

• Isopropanol is less volatile than ethanol and it is more difficult to remove the last traces; moreover, solutes such sodium chloride are more easily coprecipitated with DNA when isopropanol is used.

Resuspension and Storage of DNA• TE Buffer - Tris-EDTA Buffer: 10 mM Tris-

HCl pH 8.0, 1 mM EDTA, or TE-4 which is 10 mM Tris, 0.1 mM EDTA. DNA is resuspended and stored in TE buffer. DNA must be stored in a slightly basis buffer to prevent depurination, and the EDTA chelates any Mg2+ helping to inactivate DNases.

• DNA can be stored at 4oC for extended periods, however for long term storage, - 20oC is preferable.

• Avoid repetitive freeze thawing of DNA, since this can cause degradation.

Assessing the Quality and Yield of Nucleic Acids

Running nucleic acid sample through an agarose gel is a common method for examining the extent of DNA degradation. Good quality DNA should migrate as a high molecular weight band, with little or no evidence of smearing.

genomicDNA

RNA(degraded)

Checking for DNA

Nucleic Acid Analysis via UV Spectrophotometry

By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak of 1 OD at ~260nm.

[dsDNA] ≈ A260 x (50 µg/mL)[ssDNA] ≈ A260 x (33 µg/mL)[ssRNA] ≈ A260 x (40 µg/mL)

DNA Absorption Spectra

How pure is nucleic acid sample?

Nucleic acids strongly absorb at 260 nm and less strongly at 280 nm while proteins do the opposite.

The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratios lower than 1.7 usually indicate significant protein contamination.

The A260/A230 ratio of DNA and RNA should be roughly equal to its A260/A280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates).

DNA EXTRACTION. Have you done DNA extractions with your class?

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