Experiment

This page will show you all the experimental works that we have conducted

PRACTICAL 1: FRUITS DNA EXTRACTION
PRACTICAL 2: CHEEK CELL DNA EXTRACTION

Date of experiment : 23rd January 2009

PRACTICAL 1: FRUITS DNA EXTRACTION

Objective:

1. To carry out a DNA extraction technique.
2. To extract DNA from fruits.

Introduction:

Deoxyribonucleic acid (DNA) is the chemical inside the cells that contains the genetic code. Each DNA molecule is a long polymer of smaller chemical units called nucleotides. There are 4 different types of nucleotides nicknamed A,T, G and C. These occur in a particular sequence along the length of the DNA molecule in each individual’s cells. The order of the nucleotide sequence is slightly different in each organism in a particular species, making that individual unique. The order of nucleotides in DNA is what forms the genetic code. The procedure that will be use today will allow the DNA to escape the boundaries of the nucleus and the cell membrane and precipitate into a visible form that can be observed with the naked eye.
Scientists who study DNA and the gene sequences contained in the DNA molecules begin their study with an isolation technique. Then, it will be followed up by additional purification steps to remove all the protein and contaminating materials (including enzymes that can attack and destroy DNA in storage unless removed). After that, an additional procedure is carried out to isolate the particular region of DNA that contains the genes (DNA segments) they are intending to study.

Materials :

Equipments
Small plastic baggie.
Fresh fruits (Banana/Mango/Papaya/Kiwi/Plum).
Strainer.
100ml beaker.
Test tube.
Bamboo stick.

Reagents
DNA extraction buffer (one liter: mix 100ml of shampoo, 15g NaCl, 900ml water or 50ml liquid dishwashing detergent, 15g NaCl, 950ml water).
Ice-cold 95% ethanol or 95% isopropyl alcohol.

Methodology:

1. Each fruits (Banana, Mango, Papaya, Kiwi, Plum) is cut approximately 2cm x 2cm x 2cm

2. The fruits are placed in a plastic baggie.

3. The fruits are smashed with fist for 2 minutes

4. 10ml extraction buffer is added into the bag

5. The fruits are mashed again for one minute

6. The mixture of fruits and buffer is filtered through strainer into beaker

7. The filtrate is poured into test tube approximately 1/8 full

8. The ice-cold alcohol is poured slowly into the tube (half full)

9. The tube is slanted before the alcohol is added

10. The formation of DNA precipitate is observed

11. The DNA precipitate is spooled by using bamboo stick

Results :

Type of fruits Range of DNA appearance
Kiwi 4
Mango 2
Papaya 1
Banana 5
Plum 3

Table 1: The range of DNA precipitation appears according to the fruits.

(Range is based on 1- the most DNA precipitate formed to 5- the less DNA precipitate formed)

Discussion :

The procedure used in this experiment has the same essential elements as in the advanced laboratory DNA extraction procedure. It involves the mechanical and thermal disruption of cells, the liberation of DNA and the precipitation of DNA.

In this procedure, 5 types of fruits is used which are Kiwi, Mango, Papaya, Banana and Plum. The reason why fruits is used in observing the formation of DNA is because fruits are often polyploid which means they have more than two copies of every chromosome in each cell compared to human cell which are diploid. Thus, the formation of DNA precipitate would be much greater and easily observable. Other than that, the fruits that were used are already ripening. The ripening of the fruit will weakened the cell wall thus allowing for easier isolation process.

Before the DNA extraction for a fruits cell is carried out, the structure of the cell need to be understood. The fruits cell is considered as a plant cell. It consist of a cell wall composed of cellulose and hemicellulose, pectin and in many cases lignin, and secreted by the protoplast on the outside of the cell membrane. The cell membrane is a thin layer of protein and fat that surrounds the cell, but is inside the cell wall. The cell membrane is semi-permeable, allowing some substances to pass into the cell and blocking others. Within the cell is a vacuole, a golgi apparatus, a ribosomes, a mitochondrion, a rough and smooth endoplasmic reticulum and the important part is the nucleus that contain the DNA. The main building block of this structure is a protein and associated with other components. Thus, before the DNA could be extract, these components need to be removed.

The fruits cell walls are broken down by the mechanical mashing and the detergent dissolve the lipids in the cell membrane and nuclear envelope. No longer confine in the nuclear membrane, the DNA (highly soluble in water due to the phosphate group of each nucleotides carries a negative charge) goes into the solution. At this point, when the DNA is released from the cell membrane, it must be protected from DNAases that will cause shearing to the DNA obtained. DNAases or deoxyribonuclease is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Some DNases cleave only residues at the ends of DNA molecules (exodeoxyribonucleases, a type of exonuclease). Others cleave anywhere along the chain (endodeoxyribonucleases, a subset of endonucleases). Some method indicates about the use of heat to deactivate the enzymes. The temperature required is about 60oc but some research also mentioned that DNA also can be denatured at this temperature. Thus, to avoid from the denaturing of DNA by heat, this procedure is replaced by lowering the temperature by adding the ice-cold ethanol. In low temperature, the action of the enzymes will be slow. Other than that, the cold ethanol will help in increasing the amount of DNA precipitate.

The DNA extraction buffer also contains NaCl. The positively charge sodium ions from the salt NaCl (Na+) in the buffer are attracted to the negatively charged phosphate group of the DNA backbone, thus neutralizing the DNA’s electric charges. The neutralization allows the DNA molecules to aggregate with one another. The salt also caused the protein and carbohydrate present in the cell to precipitate (referring to the organelles). When the ice-cold 95% ethanol is added, the DNA clumps together and precipitates at the water-ethanol interface because the DNA is not soluble in ethanol. The addition of ethanol need to be done carefully. The test tube is slanted a little bit and the ethanol is placed on top of the detergent layer. The protein and fat in the cell debris will stay in the lower layer and the DNA will rise to the top and form an insoluble threads in the ethanol layer The appearances of DNA precipitates can be easily noticed by the formation of cloudy and whitish strand that sometimes form a cluster. By using a bamboo stick, the DNA threads can be wind up to be lift out from the test tube for study. The DNA can be dried and dissolved in water or kept as a precipitate in alcohol in a closed tube.

Based on the result obtained, by ranging the production of DNA precipitate in each fruits (1 is the highest DNA precipitate production and 5 the least production), the result showed that Papaya gave the most DNA precipitation followed by Mango, Plum, Kiwi and Banana. But, based on previous research, Kiwi should yield the most DNA precipitate compare to others fruits. The variation in the result would be most probably affected during the procedure been carried out. The way the fruits been mashed and the amount of extraction buffer applied to the fruits will affect the data. Thus, to obtain a more accurate result, a few precaution steps need to be done. First, the fruits need to be mashed by using blender. Second, the amount of filtered fruits needs to be the same for all fruits. Third, the amount of extraction buffer also need to be in the same amount, thus, the used of measuring cylinder is a vital.

Conclusion:

1. Based on the data, Papaya gave the most DNA precipitation and Banana gave the lowest DNA precipitate.

2. The DNA precipitation can be detected by observing the formation of whitish thread between the water-ethanol interface.

Reference:

1. Understanding DNA and Gene Cloning. A guide for the curious 4th edition. Karl Drlica. Public Health Research Institute. (ISBN 0000082841)
2. F:\TBS\DNA\DNA Extraction from Kiwi.htm Explorer’s Guide : Fruitful DNA Extraction.
Fruits DNA Extractions – Connecticut’s BioBus. www.ctbiobus.org

PRACTICAL 2: CHEEK CELL DNA EXTRACTION

Objective:

1. To carry out a DNA extraction technique.
2. To extract DNA from Cheek cell of human.

Materials :

Equipments Number required
Water bath 1
15ml tubes, each contain 3ml water 4
Pink micro test tube labeled “prot” 1
15ml tube labeled “lysis” 1
Disposable plastic transfer pipets 6
Permanent marker 1
Disposable paper cup 1

Reagents :

1.25ml of protease + salt.
10ml lysis buffer.
Ice-cold 95% ethanol or 91% isopropanol.

Methodology:

1. 15ml tube containing 3ml of water is obtained and is labeled with the experimenter initials

2. The inside of the experimenter mouth is gently chewed for 30 seconds

3. The 3ml water from the tube is inserted into the mouth and is rinsed vigorously for 30 seconds. The water is expelled from mouth into the 15ml tube

4. 1ml of lysis buffer is added into the tube by using a fresh disposable transfer pipet

5. The cap is placed back to the tube

6. The tube is gently inverted 5 times.

7. 5 drops of protease and salt solution is added to the cell extract. The cell extract tube is caped and is inverted 5 times to mix.

8. The cell extract tube is placed into a 50oc water bath for 10 minutes

9. 10ml of cold alcohol is added to the extract cell tube by using a disposable transfer pipet. The tube is slanted a little bit and the cap is screwed back

10. The 15ml tube is placed upright and is left at room temperature for 5 minutes

11. The test tube is observed.

Discussion :

Human mouth made up from thousands of cells and it can be obtained by scraping it gently and thoroughly with a brush. It is also known as “Stratified (stacked) Squamous (flat) Epithelium (external covering cells)”. A stratified squamous epithelium consists of squamous (flattened) epithelial cells arranged in layers upon a basement membrane. Only one layer is in contact with the basement membrane; the other layers adhere to one another to maintain structural integrity. Although this epithelium is referred to as squamous, many cells within the layers may not be flattened; this is due to the convention of naming epithelia according to the cell type at the end. This type of epithelium is well suited to areas in the body subject to constant abrasion, as the layers can be sequentially sloughed off and replaced before the basement membrane is exposed. Stratified squamous epithelium is further classified by the presence or absence of keratin at the apical surface. Non-keratinised surfaces must be kept moist by bodily secretions to prevent them drying out and dying, whereas keratinised surfaces are kept hydrated and protected by keratin.

The type of cells that line in the mouth divides very often, coming off easily as new cells replace them continuously. In fact, these cells are coming off and being replaced every time a person chew and eat food. The cheek cell collection is critical for success. Thus, ensure to spend the recommended time collecting and transferring the cells.

Before the DNA of the cheek cell is obtained, the cell need to be collected first. Following the procedures is breaking or lysing the cell. The cells need to be broken open to release the DNA. Detergent will dissolve the membranes of the cells, because cell and nuclear membranes are composed of fats and proteins. Dissolving the membranes results in the release of the DNA. The process of breaking open the cells are called lysis, and the solution containing the detergent is called lysis buffer.

After the cell have been lysed, it is crucial to remove the protein. DNA is packaged tightly around proteins. Like spools for thread, these proteins keep the DNA tightly wound and organized so that it does not get tangled inside the nucleus. In order to see the DNA, it is important to remove the proteins so that the DNA can first loosen and expand, then collect into a mass with the DNA from all the other cells. The lysed cheek cells need to be incubated with protease, which breaks down proteins so that they can no longer bind DNA.

Proteolytic enzymes or proteases catalyse the cleavage of peptide bonds in proteins. They are enzymes of class 3, the hydrolases, and subclass 3.4, the peptide hydrolases or peptidases. They constitute a large family (EC 3.4) divided as endopeptidases or proteinases (EC 3.4 21-99) and exopeptidases (EC 3.4.11-19) according to the point at which they break the peptide chain. These endopeptidases can be ordered further, according to the reactive groups at the active site involved in catalysis, into serine- (EC 3.4.21), cysteine- (EC 3.4.22), aspartic-proteinases or-endopeptidases (EC.3.4.23) and metalloproteinases or metalloendopeptidases ( EC 3.4 24 ). Enzymes whose reaction mechanism has not been completely elucidated are classed in the subgroup EC. 3.4. 99. Proteases differ in their ability to hydrolyze various peptide bonds and it works best at 50°C, which is the temperature of slightly hot water. The protease chews up the proteins associated with the DNA and also helps digest any remaining cell or nuclear membrane proteins.

In order to make the DNA visible, salt and cold alcohol is applied to bring the DNA out of solution, or precipitate it. Salt and cold alcohol create a condition in which DNA does not stay in solution, same as what is happening to the fruits cell procedure, so the DNA clumps together and becomes a solid mass that is observable. DNA is colorless when it is dissolved in liquid, but is white when it precipitates in enough quantity to see. As it precipitates, it appears as very fine white strands suspended in liquid (as shown in Figure 4). The strands are somewhat fragile can break if handled roughly, thus, a gently handling the solution is vital. Also, if a mass of precipitated DNA is pulled out of its surrounding liquid, it will clump together.

Conclusion :
1. Cheek cells enable the researchers to extract DNA more efficiently due to the lack of additional organelles within it.
2. The DNA precipitation can be observed by the formation of a very fine white strands suspended in liquid.

Reference:
1. Proteolytic enzymes: A pratical approach. Beynom,R.J., Bond, J.S. (eds).1989. Academic press. Oxford.
2. Understanding DNA and Gene Cloning. A guide for the curious 4th edition. Karl Drlica. Public Health Research Institute. (ISBN 0000082841)
3. Introduction to DNA Extraction. Lana Hays. F:\TBS\DNA\Introduction to DNA Extractions.htm