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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>