Isolation and Characterization of

DNA

Table of contents:

• Introduction• Sample disruption (Isolation)• Quantification of DNA• Staining• Visualization• Methodology• Results and discussion• Conclusion

Introduction

DNA

• Deoxyribonucleic acid

• Hereditary material in humans and almost all other organisms.

• Carries information needed to direct replication and protein synthesis.

• Watson and Crick

Published the first description of the structure of DNA.

DNA Structure

DNA ComparisonProkaryotic Eukaryotic

Single circular chromosome Multiple linear chromosomes

Lacks histones With histones

Stored in the cytoplasm or area called nucleoid

Stored in the nucleus

Genomic DNA constitutes the total genetic information of an organism. (except virus)

Genomic DNA molecules are generally large, and in most organisms are organized into DNA–protein complexes called chromosomes.

The size, number of chromosomes, and nature of genomic DNA varies between different organisms

Bacterial plasmids are closed circular molecules of double-stranded DNA that range in size from 1 to >200 kb.

They rely upon enzymes and proteins provided by the host for their successful transcription and replication.

Once purified, plasmid DNA can be used in a wide variety of downstream applications such as sequencing, PCR, expression of proteins, transfection, and gene therapy.

Disruption/Isolation

Complete disruption and lysis of cell walls and plasma membranes of cells and organelles is an absolute requirement for all genomic DNA isolation procedures.

Incomplete disruption results in significantly reduced yields.

Eg. (Lysis buffer, Disruption using rotor–stator homogenizers, Disruption using bead mills, Disruption using a mortar and pestle)

Sample disruption for extraction of genomic DNA

Lysis buffer

Contains a detergent (for breaking down cellular membranes) and a protease (for digestion of protein cellular components).

The choice of protease depends on the lysis buffer used. Some sample types require additional treatment for efficient lysis

•Many bacterial cell cultures can be efficiently lysed using lysis buffer and protease or proteinase K.

•Bacterial DNA can also be isolated from a wide variety of clinical samples. Bacterial cells should be pelleted from biological fluids, and the DNA isolated as for bacterial cell cultures

Quantification

Reliable measurement of DNA concentration is important for many applications in molecular biology.

Spectrophotometry and fluorometry are commonly used to measure both genomic and plasmid DNA concentration

Quantification of DNA

• Spectrophotometry can be used to measure microgram quantities of pure DNA samples (i.e., DNA that is not contaminated by proteins, phenol, agarose, or RNA).

• Fluorometry is more sensitive, allowing measurement of nanogram quantities of DNA, and furthermore, the use of Hoechst 33258 dye allows specific analysis of DNA.

• The concentration of DNA and RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer.

• For accuracy, absorbance readings at 260 nm should fall between 0.15 and 1.0.

• Pure DNA has an A260/A280 ratio of 1.8–2.0 in 10 mM Tris·Cl, pH 8.5.

Spectrophotometric measurement of DNA concentration 

• Strong absorbance at A280 resulting in a low A260/A280 ratio indicates the presence of contaminants, such as proteins.

• Strong absorbance at 270 nm and 275 nm may indicate the presence of contaminating phenol.

Note: Phenol has an absorbance maximum of 270–275 nm, which is close to that of DNA. Phenol contamination mimics both higher yields and higher purity, because of an upward shift in the A260 value.

Agarose gel analysis enables quick and easy quantification of DNA, especially for small DNA fragments (such as PCR products).

Agarose gel (Gel Electrophoresis)

As little as 20ng DNA can be detected by agarose gel electrophoresis with ethidium bromide staining.

Standard of roughly the same size of fragment is to be use to ensure reliable estimation of the DNA quantity, since large fragments interchelate more dye than small fragments and give a greater band intensity.

Gels allow separation and identification of nucleic acids based on charge migration.

Migration of nucleic acid molecules in an electric field is determined by size and conformation, allowing nucleic fragments of different sizes to be separated.

However, the relationship between the fragment size and rate of migration is non-linear, since larger fragments have greater frictional drag and are less efficient at migrating through the polymer.

Agarose gel analysis is the most commonly used method for analyzing DNA fragments between 0.1 and 25 kb.

Staining

To allow visualization of the DNA samples, agarose gels are stained with an appropriate dye. The most commonly used dye is the intercalating fluorescent dye ethidium bromide, which can be added either before or after the electrophoresis

Note: Ethidium bromide is a powerful mutagen and is very toxic.

Staining

Visualization

Ethidium bromide–DNA complexes display increased fluorescence compared to the dye in solution. This means that illumination of a stained gel under UV light (254–366 nm) allows bands of DNA to be visualized against a background of unbound dye.

Visualization

Materials, Reagents, and Procedures

Materials

• Centrifuge• Centrifuge tubes• Bacteria culture• Vortex mixer

• Electronic balance• P1000 pipettor• P10 pipettor

Reagents

• Lysis Solution

• RNAse Solution

• Protein Precipitation Solution

• Isopropanol

• 70% Ethanol

• DNA Rehydration Solution

• Saline Citrate Buffer

• TAE Buffer

• Ethidium Bromide Solution

Procedure

Isolation and purification of DNA from bacteria sample

Characterization of DNA using Spectrophotometer

Quantitation of DNA using Gel Electrophoresis

Isolation and Purification of DNA from bacteria

1ml of bacteria culture to

microcentrifuge. Centrifuge

5,000rpm for 5 mins.

Discard supernatant and resuspend cell

pellet in 600μl of Lysis Solution

Gently pipet cells. Cool tube at room temperature for 5

mins.

Add 3μl of RNAse solution and invert 2-5 times to mix.

Incubate at 37°C for 15-60 mins.

Cool the sample to room temperature

for 5 mins.

Incubate at 80°C for 5 mins.

Pipet until cells are thoroughly

resuspended

Cool tube contents to room

temperature for 5 mins.

Add 200μl PPS and vortex at high speed for 20

seconds.

Incubate sample in ice for 5 mins and

centrifuge at 15,000rpm for 3

mins.

Transfer supernatant to microcentrifuge

containing 600μl of isopropanol

Mix by inversion. Centrifuge at

15,000rpm for 3 mins.

Isolation and Purification of DNA from bacteria

Pour off supernatant and drain tube on clean absorbent

paper. Do not allow DNA pellet to dry

out

Add 600μl of 70% Ethanol and invert tubes to wash the

DNA pellet. Centrifuge at

15,000rpm for 3 mins.

Pour off ethanol and drain tubes on clean

absorbent paper.

Mix the solution by gently tapping the tube. Store DNA at

2-8°C

Add 100μl DNA RH and incubate at 65°C for 1 hour

Allow the pellet to air dry for 10-15

minutes

Characterization of DNA using Spectrophotometer

Dissolve small quantity of DNA in 3ml of 0.1 X SSC

Turn on and blank UV spectrophotometer at

220nm. Determine the absorbance.

Change wavelength to 240nm and determine

absorbance

Increase wavelength by 20nm and repeat

blanking and measuring

absorbance until reading are up to

300nm.

Compute absorbance ratio from 260-

280nm.

Quantitation of DNA using Gel

Electrophoresis

Methodology

1• Wash the gel tank, casting gel tray and the comb thoroughly. Rinse with

ethanol to remove grease. Set the comb into appropriate slot of the tray

2

• Prepare 0.75% agarose in 75 mL TAE buffer. Boil the solution until agarose to casting gel.

3• Cool to about 50°C and pour agarose to casting gel

Methodology

After solidification, remove the

combs gently and set the gel in the gel

tank. Cover the gel with TAE buffer.

Place 3 microlit

loading dye in Paraffin

wax

Place 3 µL of DNA sample and mix it

with loading dye, through

pipettor back and

forth.

Load sample into second lane and the remaining samples.

Place 4 microlit of

DNA marker to the first

lane.

Methodology

Cover the apparatus and run 100 V for 30 mins until the tracking dye has run about two thirds of gel length.

After 30 mins remove gel and transfer to a staining pan containing erhidium bromide solution and stain for 5 mins.

After 5 mins, rinse gel with tap water and visualize DNA bamds using GEL DOCUMENTATION SYSTEM

RESULTS AND DISCUSSION

Isolation and purification of DNA from Escherichia coli

• What is the rationale of homogenizing the samples using Saline Citrate buffer?

It stabilizes red blood cells and prevent them from lyzing or clotting.

• What are the substances found in the supernatant liquid that must be discarded?

Soluble proteins and other membrane bound organelles of the cell

• What is the importance of salt in the set-up? Why do you have to suspend the cells in cold salt solution?

• To make proteins and carbohydrates precipitate while making the DNA remained in solution. 

• What is the importance of salt in the set-up? Why do you have to suspend the cells in cold salt solution?

• Suspending cells in cold salt solution will provide the DNA with favorable environment since salt contributes atoms that neutralize the normal negative charge of DNA. • The NaCl Solution provides Na ions that will block charge from

phosphates on DNA. The Na ions will form an ionic bond with negatively charges and allow DNA molecules to come together.

In what ways have the discovery of DNA useful to science?

• Modern Medicine and Genetic Research

Improved ability to diagnosis disease, detect genetic predisposition to disease, create new drugs to treat disease, use gene therapy as treatment, and design "custom drugs" based on individual genetic profiles.

• Forensics

DNA is used in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual called DNA profiling and DNA Fingerprinting.

• Bioinformatics

manipulation, searching, and data mining of DNA sequence data.

In what ways have the discovery of DNA useful to science?

• Genetic Engineering

Recombinant DNA , a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms (GMO) of desired and appropriate format.

• DNA Nanotechnology

uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties.

What is the rationale of using the ff. reagents?

•Pancreatic ribonuclease A• It specifically degrades single stranded RNA at C and U residues. It cleaves the phoshodiester bond between the S ribose of a nucleotide and the phosphate group attached to the 8’ ribose of an adjacent pyrimidine nucleotide

What is the rationale of using the ff. reagents?

•Pronase• It is used in order to break down proteins

removing them from the DNA. It is also used to purify the isolated DNA.

What is the rationale of using the ff. reagents? (cont.)

•Sodium Lauryl Sulfate•Used to aid in lysing cells during DNA extraction and for unraveling proteins. • It is commonly used in preparing proteins for electrophoresis. This compound works by disrupting non-covalent bonds in the proteins, denaturing them, and causing the molecules to lose their native shape (conformation).

What is the rationale of using the ff. reagents? (cont.)

•Chloroform : isoamyl alcohol• Proteins and restriction enzymes are removed by chloroform in disrupting protein secondary structure. • Chloroform is an organic solvent that very efficiently denature and cause the precipitation of proteins. • Isoamyl alcohol reduces the foaming of proteins that would normally be generated by the mechanics of the extraction procedure.

What is the rationale of using the following reagents?

• Cell Lysis Solution

It disrupts the cell membrane and the nuclear envelope, causing the cells to burst open and release their DNA.

What is the rationale of using the following reagents?

•Protein Precipitation SolutionThe protein precipitation step causes all the proteins (cellular debris) to aggregate together so they can be separated by centrifugation.

What is the rationale of using the following reagents?

•Addition of hydration solutionHydration solution of DNA plays important role in its structure,

conformation, and function. Of significance to the function

is the selective recognition by DNA of small molecules

Characterization of DNA using spectrophotometer

Fill in the Table 10.1 and supply a title describing the contents of the tableTable 10.1. The Absorbance of Isolated DNA from Escherichia coli

Wavelength Absorbance

300 0.046

280 0.032

260 0.056

240 0.006

220 0.065

TRIAL 1

Plot the absorbance spectrum of your sample.

Table 10.1. The Absorbance of Isolated DNA from Escherichia coli

Wavelength Absorbance

300 0.032

280 0.032

260 0.056

240 0.006

220 0.065

TRIAL 2

The absorption spectra of the isolated DNA from Escherichia coli

Compute the absorbance ratio of your sample

20μl DNA

980μl SSC

Absorbance:

260=0.056 0.056

280=0.032 0.032

Conc. DNA= 1.75 μl/ml

260:280

Abbreviation: SSC: NaCl-trisodium citrate buffer

Quantitation of DNA using Gel Electrophoresis

Is you DNA sample pure? Justify your answer.

•No, the DNA sample is not pure. • Pure sample of DNA the ratio of absorbencies at

260 nm and 280 nm (A260/A280) is 1.80 to 1.90. This is because proteins absorb maximum UV light at A280. Ratio of less than 1.80 which shows that there are lots of proteins in the sample - indicative of protein contamination

Draw the band patterns of the isolated DNA

3, 5,6,7,8, and 9 bands are positive with S. typhi

Gel Electrophoresis Concept:

• The DNA fragments moved from their origin, the sample wells (or: slots), through the gel towards the positive electrode that’s from top to bottom in the picture. The gel matrix acts as a sieve: smaller DNA molecules migrate faster than larger ones, so DNA molecules of different sizes separate into distinct bands during electrophoresis. 

What is the role of ethidium bromide in the visualization of DNA bands?

• Ethidium bromide is a large, flat basic molecule that resembles a DNA base pair. • Because of its chemical structure, it can intercalate (or insert)

into a DNA strand. • Ethidium bromide is commonly used in molecular biology

laboratories to stain electrophoresis gels. The compound forms fluorescent complexes with nucleic acids and these can be viewed under UV light.

Plasmid DNA and gel electrophoresis

Plasmid DNA can exist in three conformations: supercoiled, open-circular (oc), and linear (supercoiled plasmid DNA is often referred to as covalently closed circular DNA, ccc).In vivo, plasmid DNA is a tightly supercoiled circle to enable it to fit inside the cell. 

Conclusion

It is concluded that base on the result of the experiment all the learning objectives were met, the DNA of prokaryotic cell and eukaryotic cell were differentiated base on the presence of histone, difference in the chromosome and area where DNA is stored.

Conclusion

The DNA were isolated base on the different extraction procedures. It was observed that each step is critical in isolating a pure sample. One way of determining the average concentrations of DNA and its purity is through spectrophotometric analysis. It is based on the principle that nucleic acid absorbs UV light in a specific pattern. In using this method, the Beer-Lambert law is used to determine unknown concentrations.

To quantitate the DNA, gel electrophoresis is used by observing the fragment. The principle behind gel electrophoreses is also observed. The smallest fragment of 564 basepairs (1) is hardly visible, while the biggest fragment of more than 23.000 basepairs (2) shows a very bright band.

Band 3 contains smaller DNA fragments than band 2, but is still much brighter. This is because there is more (nanograms of) DNA in 3 than in 2 (the number of molecules in 3 must be much higher than in 2).

Reference:

 Sample & Assay Technologies, DNA Protocols & Applications, QIAGEN, Data retrieved from: <http://www.qiagen.com/knowledge-and-support/spotlight/protocols-and-applications-guide/dna/#Spectrophotometry>