Dna Isolation, Restriction, Visualitation, And Quantification

Report of DNA isolation, restriction, visualitation, and quantification Percobaan Isolasi, Pemotongan, Visusalisasi, dan penghitungan DNA Written by: Sonianto kuddi (40420060017) Barto minggu using (4112006001) Dewa Komang Tri Mahayana (40420060028) Dewa made dwi kamayuda (40420060027)

EMBI1

Pelita Harapan University Faculty of education – Teachers College Lippo village 2009

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DNA Asolation, Restriction, Visualization, and Quantification I. Aim 1. To know how to isolate DNA using simple materials 2. To study the way of writing notation of restricted enzyme and the various restricted anzyme 3. To study the cutting process of DNA by restricted enzyme 4. To study the basic technique of gel electrophoresys. 5. To study how to visualize and quantify DNA II. Introduction a. DNA DNA is nucleate acid that contains genetic material and functions to manage the development of all forms of biological life on mobile. DNA found in the nucleus, mitochondria and chloroplast. Differences between the three are: DNA and linear-shaped nucleus associate very closely with the histon protein, whereas DNA mitochondria and chloroplast shape circular and not associate with protein histon. In addition, DNA mitochondria and chloroplast typically, the only hand that nature come from the mother. This is very different from the DNA nucleus that has a pattern of inheritance of both the nature of parents. View from its organism, prokaryote structure of DNA with different DNA structures eukaryote. b. Overview of DNA isolation technique DNA does not have prokaryote protein histon and shaped circular, whereas linear DNA eukaryote shaped and has a protein histon. DNA structure has torsion rope double anti pararel with the components, namely sugar pentose (deoksiribosa), phosphate group, pair and base. Base pair on the DNA consists of two kinds, namely purin and pirimidin. 'Purin consists of adenin (A) and guanine (G) which has the structure of the double-ring, while pirimidin of cytosine (C) and timin (T) which has a single-ring structure. When Guanine binds with Cytosine, so will the three hydrogen bonds, while when Adenin binds with Timin will then only two hydrogen bonds. One 2

component builders (building block) DNA consists of a sugar pentosa, a phosphate group and one pair is called nucleotide base. c. Restricted enzyme (endonucleus, exonucleus) This age is usually called the age of molecular biology because studying an organism genome is the one of the most popular research in scientist ream. But the problem they are faced is how to reproducibly cut a genome’s DNA into fragments that were small enough to handle? It was a significant problem. So many methods have been tried to isolate DNA and then randomly cut it up using chemical or mechanical means. Unfortunately, this random cutting was not a satisfactory way to obtain smaller pieces of DNA, since it was impossible to tell what the original order of the DNA fragments were, an important point since the specific order of DNA is essential for its function. The biologists were stuck. It was discovered that a type of bacterial enzyme was found to have the ability to cut DNA. It was named as restriction endonuclease because they cut double stranded DNA at restricted sites, were discovered as a natural part of the bacterial machinery. In a bacterial cell, restriction endonucleases (often referred to as restriction enzymes) act as a kind of immune system, protecting the cell from the invasion of foreign DNA, as would occur when a virus attempted to infect a bacterial cell. d. Gel electrophoresis Garose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the positive pole:

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To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. After cooling the solution, it is poured into a casting tray containing a sample comb and allowed to solidify at room. After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. DNA will migrate towards the positive electrode, which is usually colored red. The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. When adequate migration has occured, DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and RNA. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. e. The quantification of DNA concentration This is the formula used to quantify the DNA concentration.

In this quantification, we will have λ DNA hind III which serves as standar quantification to find the concentration by comparing them in ratio the intensisty/ brightness of light (fragment molecule of DNA) produced as result of electrophoresis by UV ray 4

III. Tools, Materials and Procedures Samples, detergent, regular buffer, meat tenderizer, ethanol 96%, enzyme EcoRI, HIndIII, λ DNA, 1 Kb ladder, DNA sample, water, buffer R, buffer EcoRI, micropipette, microtube, loading dye, gel 1 %, UV Transilluminator, Enlenmeyer flask, agar, running buffer, loading buffer (loading dye), parafilm paper, spatula, balance, electrophoresis tray, electrophosis tank, comb, and microwave. Procedures DNA Isolation Sample is crushed into pieces by using scissors, and then the sample is put into a tube and added with buffer 50 μl. The mixture is crushed again by using the edge of tips. Regular buffer as much as 350 μl and 150 μl of detergent are added into the mixture and vortexed for a minute. Sample and buffer are incubated at the temperature of 65 0 C for 15 minutes. Add 1 ml of ethanol 96%, the tube is turned upside down, and then incubated at the temperature 0f -200 for 30 minutes. By using a tootpick, lift the DNA precipitation whick looks like a soft thread. DNA restriction The composition of the cutting Enzyme (10μ/μl)

1 μl

Buffer 10 x

2μl

Sample (2μg/μl)

5μl

Water

12 μl

Total Volume

20 μl

All the solution is put into 1.5 ml microtube, and then tapped so that the solution is mixtured evenly. The mixture is briefly dicentifugated by using spindown. The mixture is incubated for an hour at the temperature of 370 C. C. DNA Visualization 5

DNA as a result of restriction is analyzed in gel elektrophoresis 1%. To rpoduce 1 % the gel, 0.4 mg agar is needed and then it is dissolved in 40 ml running buffer. Agar is dissolved by heating it in microwave. As soon as agar dissolved, gel is shaped in electrophoresis tray and gel comb. Let the gel harden for about an hour. The hardened gel is then put on electrophoresis tank and filled with running buffer; afterwards gel comb is lifted up from gel, to form well. Running buffer is filled until the gel is sunk. Parafilm paper is prepared as media to mix the DNA sample and loading dye. As much as 10 μl of DNA sample is mixed with 2 μl loadin dye. The mixing is done by using micropipette. Having been homogen, the mixture is loaded into well. Gel is given electic current as much as 100 volt for 20 minutes. Afterwards, gel is soaked for 15 minutes in Ethidium bromide. DNA agar will glow is seen through UV ray. D. DNA Qualification The result of DNA electroforesis is then photographed under the UV ray and the concentration of DNA sample is analyzed. DNA concentration is gained by comparing the tighness and intensity of the brightness of the thread of DNA marker (λ Hind III) with the DNA sample. The comparison result is shown through ratio of the comparison. Based on the comparison ration then the DNA concentration can be quantified by using the formula as follows:

IV. Result and discussion 6

A. DNA isolation DNA isolations technique generally: There are two principles in conducting DNA isolation, namely centrifuges and precipitation. Centrifuges main principle is to separate substances based on molecular weight species in a way to provide centrifugal force so that the substance of the more serious will be in basic, while the substance of a more light will be located at the top. Centrifuges techniques are performed in a machine called centrifuges with the engine speed is varied, for example, 2500 rpm. To isolate the DNA there are 5 techniques that are able to use for it, such us Tissue isolation, Membrane and cell wall are removed, Extraction of solution, Purification, and Precipitation. First step which can be done by isolating tissue that we want to use, it is Blood. The next step is removing membrane and cell wall by using blood cell solution. After the incubate blood which has been mixed and centrifugation in 10 minutes long at speeds of 2500 rpm. Next supernatant that is created will be removed and then be done in the extraction solvent. It aims to be obtained extracts nucleus white blood cell. The next stage is purification. This stage aims to clean the white blood cells from other substances-substances, and the last stage, namely precipitation, has aims to precipitate protein histon, so that the strand-strand DNA is no longer round up (coiling) and bind with histon protein, which causes DNA to become visible. Tissue isolation step is to isolate the white blood cell tissue, so the blood which contains complete component need to be separated one another so that blood cell still exists. Because of that, the tube that is containing blood was given blood cell pelisses solution that is hypertonic solution. Because the solution is hypotonic, it will happen hemolytic. Pelisses solvent red blood cells consist of EDTA (ethylenediamine tetra acetic acid), which will form a complex (chelate) the metal ion, such as Mg2 + which is a cofactor DNAse. Next tube be inverted with the movement to play the 8-digit form that integrates with the solution can be perfect for 10 minutes. Blood was mixed with pelisses red blood cells and centrifugation for 10 minutes at speeds of 2500 rpm, and then supernatant will be removed. To remove cell membrane and the nucleus membrane of white blood cells isolated before, given the solution pelisses white blood cells consisting of EDTA and SDS (Sodium Dodecyl sulfate) that 7

serves to damage the lipid in the cell membrane so that it can be destroy the leukocyte. The next stage is namely purification. Purification aims to clean the white blood cells from other substances-substances; to earlier in the solution and then given RNAse and incubated for 15 minutes at a temperature of 37 ° C. This work aims to optimize the enzyme that is strongly influenced by temperature. The next step is precipitation. Precipitation step is done by dropping the protein precipitation solution, the aims by doing this is to homogeneous the solution. Protein precipitation solution consists of ammonium acetate. Where if its bind with protein will be produce the new compound that has lower solubility, so that it can cause protein precipitation. After that, using centrifugation again by using its solution in 15 minutes at speeds of 3000 rpm. Supernatant containing the DNA and then poured into a tube containing isopropanol and cold tube be inverted back to the figure 8. Giving isopropanol has aim for DNA visualization. Then again tubes for 5 minutes with a speed of 3000 are sentrifugated. The result of sentrifugaiton is the occurring of stratified DNA at the bottom of the tube. After that the stratified will be mixed with 70% of ethanol solution and be inverted. Giving ethanol aims to clean up DNA from the polluters, after the mixed of solution, then the tube is sentrifugated again during 5 minutes long with a speed 3000 rpm. The end result is the DNA that is located on the edge basic tube. Last step is by giving the Tris-EDTA which aims to soluble again DNA for preservasi step. Function of the solutions Buffer solution (NACL and baking soda) NaCl has a important role in separate or removes protein and carbohydrate in DNA and also has a function as lysing buffer. NaCl is also used to solve the DNA, because of DNA consist of ion Na+ which is conceived by the NaCl that is able to be a blocker (form binding) with negative DNA phosphate pole that is usually cause the attraction or throw of the molecule one another so that at the same time when the Na + ion form the binding of negative DNA phosphate pole, DNA will get together. So obviously seen that beside to remove the protein and carbohydrate and stabilize PH, NaCl also helps the process of density effect of DNA, while the 8

baking soda function is to reduce the acidity of solution, and also be able to remove the carbon dioxide (Co2). Giving ethanol aims to clean up DNA from the polluters. Detergent Has a function to remove barrier cells as a substitute for the chemical compound that is capable of damaging the cell wall and membrane, among other lisozim can digest compound that causes stiffness polymeric cells endiamin tetra and ethyl acetate (EDTA) which serves to remove the Mg2 + ion is important to maintain overall structure of the jacket cells, and prevent enzymes which can damage cellular DNA (Mg2 + ion is important for cofactor that DNAse usual "eat" DNA). Detergent can cause damage to the cell membrane lipid and protein emulsified cells and interrupts the interaction of polar cell membrane to unite as detergent containing disodium EDTA and lauryl sulfate which have the same functionality with dodesil sulfate. Treatment given Samples were small with the scissors, and then inserted into the tube. After that tubes are mixed with 50μl of buffer solution. After that the mixture is destroyed again by using the tip of a thin object. Regular buffer as much as 350μl and 150 of detergent are added into the mixture and vortexed for a minute. After that, sample and buffer are incubated at the temperature of 65 0 C during 15 minutes. Add 1ml of ethanol, and turned upside down, and then incubated at temperature of 200 for thirty minutes. Using a toothpick, lift precipitation DNA and DNA visible as fine threads of colored tape on the cornea stem toothpick.

Result of practicum

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From the experiment conducted, we got a result that is threadlike DNA-fine white thread and stick close enough to the mixer shaft.

B. DNA restriction How Restriction Enzymes Work and are Used The way the enzymes cutting DNA is varied. Some of them cut the foreign DNA randomly. While others recognize a particular DNA recognition sequence and then either cut within this DNA sequence or several nucleotides away from it. Where a restriction endonuclease cuts within a DNA molecule is one of the primary characteristics by which it is classified. These enzymes bind to DNA at any position and then travel along the strand of DNA until they reach a recognition sequence. Tight binding of the enzyme at the recognition site causes its structure to change, bringing the parts of the enzyme necessary for DNA cleavage into close proximity of the DNA strand. Once accomplished the “backbone” of the DNA molecule can be cut to produce two DNA fragments from one (see Figure 2). It is the recognition sites that provide the key to how one DNA fragment can be cut into two in a specific manner. These sites can vary in size; with some restriction endonucleases recognizing sequence motifs in DNA that are four nucleotides long, while others recognizing sequences that are twenty nucleotides long. The nucleotide pattern that is recognized by different restriction enzymes is quite variable, although it is frequently palindromic. A palindromic sequence is the same when read in

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5′ to 3′ direction on either complementary strand of DNA, an example being the palindromic sequence recognized by the restriction enzyme known as EcoRI (see Figure 2).

Figure 2. An EcoRI restriction enzyme. EcoRI recognizes a six-nucleotide pattern that reads GAATTC from the 5′ to the 3′ end of the DNA molecule. The complement of this sequence (on opposite DNA strand) also reads GAATTC when read from 5′ to 3′. In figure 2 you can see an illustration of an EcoRI molecule binding to and cleaving a strand of DNA, here you can see the palindromic recognition for the molecule. In this scenario the cleavage of the DNA molecule by EcoRI is symmetrical-with the enzyme cutting at the same point inside the sequence (between the G and the A when reading 5′ to 3′) on both strands. An important feature of this cleavage is the “overhanging” ends of the DNA molecule that are produced. These overhangs are often referred to as “sticky ends,” since they can bind, or stick, to a complementary sequence of DNA (in this case 5′-AATT-3′). This stickiness is often utilized in the process of DNA cloning to help the adhesion of two DNA fragments.

Results. 1. EcoRI = 10μ/μl > 0.2 μl + 0.8 μl > 0.5 μl 11

2. Hind III = 10μ/μl > 0.2 μl + 0.8 μl > 0.5 μl 1 unit = 1μg DNA, I hour, 370 c 50 μl of recommended buffer λ DNA = o,3 μg /μl } > is cut by using two enzymes above •

λDNA

•

Enzyme = 0.5 μl

●

Buffer

•

Water

= 3μl

= 2 μl =14.5 μl

● λDNA

= 3 μl

●Enzyme 1 = 0.5 μl ●Enzyme 2 = 0.5 μl +

●Buffer

= 2 μl

●Water

=14 μl

=20 μl

+

= 20 μl

Enzyme

EcoRI

HincII

HindIII

HpaII

AluI

Recognition

Source

Escherichia

Sequencea

and Cleavage Sitesb coli

of

Recognition

Sequences per Chromosome of Phage λ

SV40 Virus

5

1

34

7

6

6

750

1

143

34

GAA. TTC

strain RY13

CTT

Haemophilus

GTPy

influenza strain Rc

CAPu

Haemophilus

Number

AAG PuAC PyTG

AAG CTT

influenza strain Rd

TTC GAA

Haemophilus

C C GG

parainfluenza

GG C C

Arthrobacter

AG CT

luteus

TC GA 12

C. DNA visualization 1. Function of materials: Substances: •

Samples of λ DNA Hind III, Escherichia coli, frog, corn, onion, and plastic.

•

Gel 1%: 0.4 mg agar and dissolve in 40 ml running buffer.

•

Agarose: A long chain polysaccaride isolated from seaweed. It is similar to collagen in that when it is heated (to dissolve it) and then cooled it forms a matrix (gel) with buffer solution trapped inside and as a solid substrate to contain culture medium for microbiological work.

•

Running buffer: Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications and provide ions to support conductivity.

•

Loading dye: to enable tracking the progress of the electrophoresis.

•

Ethidium bromide: When exposed to ultraviolet light, it will fluoresce with an orange color.

•

UV lamp or UV lightbox or other method to visualize DNA in the gel, which is used to visualize ethidium bromide-stained DNA in gels.

•

Power Supply: electric.

Tools: •

Microwave: to heat and keep the certain temperature.

•

Electrophoresis tray: used to shape the gel poured.

•

Gel comb: around which molten agarose is poured to form sample wells in the gel.

•

Parafilm paper: is used to mix DNA sample and loading dye. 13

•

Micropipette: to mixture substances.

•

Nitrile rubber gloves: protect us from danger chemicals.

2. Result of electrophoresis:

D. DNA Quantification The result of DNA electroforesis is then photographed under the UV ray and the concentration of DNA sample is analyzed. DNA concentration is gained by comparing the tighness and intensity of the brightness of the thread of DNA marker (λ Hind III) with the DNA sample. The comparison result is shown through ratio of the comparison. Based on the comparison ration then the DNA concentration can be quantified by using the formula as follows:

•

Marker size = 23.130

•

Marker concentration = 0.045 μg/μl (0.000045 ηg/μl)

•

μl marker = 10 μl

•

Total size of marker = 48.5

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•

μl sample = 5 μl This is the DNA quantification result gained based on the formula used above: Row

Sample

Concentration ng/μl

1

Λ DNA Hind III

0.000042922

2

Eschericia coli

0.000007154

3

Corn

0.00001073

4

Onion

0.00001073

5

Plasmid

0.000014307

Note. In this experiment, λ DNA hind III serves as a standard measurement.

1. Eschericia coli

2. Corn

3. Onion

4. Plastic

V. Conclusion To isolate the DNA there are 5 techniques that are able to use for it, such us Tissue isolation, Membrane and cell wall are removed, Extraction of solution, Purification, and Precipitation. 15

The function of the solution in DNA isolation: NaCl has a important role in separate or removes protein and carbohydrate in DNA and also has a function as lysing buffer, remove the protein and carbohydrate and stabilize PH, NaCl also helps the process of density effect of DNA, while the baking soda function is to reduce the acidity of solution, and also be able to remove the carbon dioxide (Co2). Giving ethanol aims to clean up DNA from the polluters. Enzyme restriction is the enzyme that cut double stranded DNA at restricted sites so that we could obtain smaller pieces of DNA and it help us in observation. There are many kinds of enzyme restriction and each of them has varied ways in cutting DNA such as EcoRI produced by Escherichia coli strain RY13, HincII produced by Haemophilus influenza strain Rc, HindIII from Haemophilus influenza strain Rd, HpaII from Haemophilus parainfluenza and AluI from Arthrobacter luteus. Gel electrophoresis is a powerful tool for the separation of macromolecules with different sizes and chares. DNA molecules have an essentially constant charge per unit mass; thus, they separate in agarose and acrylamide gels almost entirely on the basis of size or conformation. Techniques of gel electrophoresis until visualization are (1) prepare a semisolid agarose gel with wells for DNA samples. (2) Remove comb and sealing tape after agarose solidifies, and place gel in the electrophoresis chamber. (3) Load DNA solutions in wells of gel. (4). Attach power supply and begin electrophoresis. (5) Remove gel from chamber, stain with ethidium bromide, and photograph under UV illumination. DNA quantification can be done by comparing (in ratio) the fragment of DNA molecule produced by UV ray. Light intensity produced by those DNA molecules, differenciate the length of the DNA strain. The DNA which does not have any band like frog in this case tells us that it does not have any DNA quantification.

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VI. Appendix

A glass plate is used eventually to receive the gel you make. This must be sealed at all borders with tape to prevent the liquid gel from leaking out. 17

A buffer solution is used this acts as a conductor for the electrical current that will eventually be used. Prepare an adequate amount of this buffer solution to fill the electrophoresis tank and to prepare the gel.

Heat the agarose solution in a microwave oven and allow all of the grains to dissolve. This will only happen with heat.

Place the plate and gel in the electrophoresis tank till it is covered by approximately 2mm of solution. The tops of the wells made by the comb should be submerged.

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Mix the prepared samples and controls with the correct amount of loading buffer and pipette it carefully into the wells made previously by the comb. (All those pictures are taken from Karen Braun, New Mexico state University, http://students.ncwc.edu/bio401/2004/team_b/what_is_gel_electrophoresis.htm).

The finished gel, after having been fully processed can be viewed for results under a UV light. These can then be identified, distances calculated and the results used in a myriad of ways from mapping a gene to helping decide which species is the more viable to keep and try to protect.

VII. References Braun, K. (2006). What is gel electrophoresis. New Mexico State University. Retrieved on April 18, 2009 from: http://students.ncwc.edu/bio401/2004/team_b/what_is_gel_electrophoresis.htm Detection of exonuclease activities in restriction endonuclease preparations using an enforcement plasmid for kanamycin-resistance selectionhttp. (1995). Retrieved April, 20, 2009 from www.sciencedirect.com/science DNA Extraction: A Laboratory Using Common Ingredients (n. d). Retrieved on April 18, 2009 from: http://www.spiritcrow.com/helix/StudyOfLife/a2.htm Isolasi dna dan kromosom. (n. d). Retrieved on April 18, 2009. From: http://rismakafiles.wordpress.com/?s=dna+isolasi. Word Press. Rbowen. (2000). Agarose Gel Electrophoresis of DNA. Retrieved on April 18, 2009 from: http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/index.html Snustad, P., Simons, M. (2006). Principles of genetics: forth edition. John Wiley & Sons, Inc: USA.

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