OBJECTIVE

1. To extract and prepare Genomic DNA from blood sample;

2. To extract and prepare Genomic DNA from E.coli DH5alpha by using Alkaline

method and boiling method; and

3. To compare and contrast between the two different methods of DNA

extraction.

INTRODUCTION

There are a number of different procedures for the preparation of genomic

DNA. DNA extraction or isolation is a routine procedure to collect DNA for

subsequent molecular or forensic analysis. Nevertheless, the DNA is present in the

cell as an association with many proteins that we called as nucleoprotein complex. A

nucleoprotein is any protein which is structurally associated with nucleic acid either

DNA or RNA. The DNA must be separated away from these proteins prior to

characterization.

RNA and polysaccharides can interfere with DNA characterization methods,

so the DNA must always be purified away from these macromolecules. In addition, a

single cell may contain several different types of DNA molecule. The more simple

approach to the purification of DNA involves purifying the total DNA present in the

cell without regard the separation of different types of DNA molecules.

A) PREPARATION OF GENOMIC DNA FROM EUKARYOTE CELLS (BLOOD)

In preparation of genomic DNA from eukaryote cell (blood), there are three

basic and one optional step in a DNA extraction. First basic is breaking the cell open,

commonly referred to as cell disruption or cell lysis. It is important to expose the DNA

within. This is commonly achieved by grinding or sonicating the sample. The lysis

solution contains several components to help stabilize the DNA during the

purification process. A TE buffer which10mM Tris-C1 &1mM EDTA is included to

maintain pH at a constant value, usually around pH 7.5 to 8.0.

Next, the second basic is removing membrane lipids by adding a detergent to

release the contents of the cells into the solution. Then, the third basic is removing

proteins by adding a protease (optional but almost always done) and extractions with

organic solvents (generally phenol/or chloroform) that denature and unfold protein

and remove them from DNA. RNA that may contaminate DNA preparations is often

removed by treatment with enzymes that degrade RNA but not DNA.

Finally, for optional step which is to precipitate the DNA with an alcohol. It is

usually ice-cold ethanol. Since DNA is insoluble in these alcohols, it will aggregate

together, giving a pellet upon centrifugation. This step also removes alcohol-soluble

salt. The precipitated DNA can then be dried and resuspended in a small volume of

buffer.

C) Preparation of plasmid DNA from bacteria cells (E.coli) – Alkaline Method

Alkaline lysis was first described by Birnboim and Doly in 1979 (Nucleic Acids

Res. 7, 1513-1523) and has, with a few modifications, been the preferred method for

plasmid DNA extraction from bacteria ever since. Alkaline method or alkaline lysis is

a technique of DNA purification that based on differential denaturation of

chromosomal and plasmid DNA in order to separate both. Alkaline lysis depends on

a unique property of plasmid DNA. It is able to rapidly anneal after being denatured.

This is what allows the plasmid DNA to be separated from the bacterial

chromosome.

There are several general steps that must been conducted to prepare the

DNA from the E. coli. First is to extract the DNA from E. coli by lysing the cell wall.

Due to this process, DNA will be denatured. The next step is followed by

neutralization that allows only the covalently closed plasmid DNA to reanneal and to

stay solubilised whereas the chromosomal DNA remains denatured. Next, the

separated chromosomal DNA fragments will aggregate with the protein isolated from

the DNA by phenol-chloroform extraction and form precipitate. This precipitate will be

removed by centrifugation. The plasmid DNA will concentrate and further purified.

Lastly, the plasmid DNA will be further analysed by electrophoresis method.

Several reagents which are mixed in three different solutions are used to

perform this method. In Solution I, Glucose is used to help in stabilizing the DNA to

minimize random breaking or shearing due to the long-rod like molecule. It also

helps in giving osmotic shock that leads to the rupture of cell wall and bacterial

membrane. The Tris-C1 buffer is used to maintain pH at constant value which is pH8

via the ability to absorb counter ions (H+ and OH-). Next is Ethylene diaminetetra

acetic acid (EDTA). It eliminates divalent cations by chelating the Mg2+ that essential

for the integrity of the bacterial outer membrane and destabilizing the membrane.It

also inhibits DNAses to prevent the degradation of DNA. RNAse is also been added

to degrade the RNA.For the solution II, it is known as lysis buffer. The lysis buffer

contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl Sulfate

(SDS). SDS is there to degrade the cell membrane. In the alkaline lysis procedure,

bacterial cells are exposed to alkaline .This causes the cell walls and membranes to

burst and the contents of the bacteria are spilled out. But more importantly it disrupts

the hydrogen bonding between the DNA bases(due to high pH), causing the DNA

double strand to denature.Addition of potassium acetate in Solution III decreases the

pH of the mixture. Under these conditions the hydrogen bonding between the bases

of the single stranded DNA can be re-established. Next, phenol-chloroform

extraction is used. Both phenol and chloroform cause proteins to become denatured

and become soluble in the organic phase or interphase, while nucleic acids remain in

the aqueous phase. The ethanol is also used to precipitate the nucleic acid in order

to further purify the solution. (Dale &Schantz, 2002)

D) Preparation of plasmid DNA from bacteria cells (E.coli) – Boiling Method

Boiling method is another method used to extract DNA. This method is

adapted from Holmes and Quigley in 1981. The principle and the reagents that been

used in this method are the same with the alkaline lysis method. However, an

enzyme known as lysozyme is used to break the bacterial cells. Lysozyme is an

enzyme that naturally present in egg white and tears to break down the bacterial cell

wall (Dale &Schantz, 2002). Besides that, Triton X-100 is added which is a non-ionic

detergent. It reduces the levels of unwanted peroxides, carbonyl compounds and

salts. After that, the lysate is boiled to denature the nucleic acids and the proteins.

When the temperature is lowered, the bases of plasmid DNA will be reanneal. The

next procedure is to remove the protein and further purification of the nucleic acid by

applying the phenol-chloroform extraction and centrifugation. Sodium acetate is also

added to recover the plasmid. This followed by concentrating the nucleic acids by

ethanol precipitation.

MATERIALS/ APPARATUS

EXPERIMENT A:

A) Blood that were collected using Heparinized/EDTA VenoJet tube

DNA Isolation

B) Reagents:

1. 250 µL extraction buffer ( 10 Mm Tris-C1 pH 8.0, 0.1M EDTA, 20 μg/mL

RNAase A and 0.5% SDS),

2. 5 µL of Proteinase K (final conc. 200 μg/mL),

3. Saturated-Phenol

4. Chloroform

5. 3M Sodium Acetate

6. 100% of Ethanol

7. 75% of Ethanol

8. 50 µL sterile TE Buffer (10mM Tris-C1 &1mM EDTA),

9. 0.7% agarose gel electrophoresis

EXPERIMENT C& D:

A) E. Coli Culture

1. Single colony of transformed bacteria, Luria–Bertani (LB) medium: 5 g/L yeast

extract, 5 g/L NaCl, 10 g/L tryptone, Autoclave, and Appropriate antibiotics.

Plasmid Isolation

B) Reagents:

1. Solution I:

a. 50 mM Glucose,

b. 10 mm EDTA,

c. 10 µg/ml RNAse (Alkaline Lysis method)

d. 0.1% Triton-X 100 (Boiling method)

2. Solution II (Alkaline Lysis method)

a. 0.2 N NaOH (pH 12.0)

b. 1% Sodium dodecyl sulphate (SDS)

3. Solution III: 3 M Sodium Acetate (pH 5.5)

4. Water-saturated Phenol

5. Chloroform

6. 750 µL cold 70% ethanol

C) Enzyme

1. 25 μl Lysozyme (Alkaline Lysis method)

D) Buffers:

1. 25 mM Tris-Cl (pH 8.0)

2. 50 µL of sterile TE Buffer (pH 8.0)

MATERIALS

1. Boiling water 1000C (Boiling Method)

2. Ice

3. Water bath (Experiment A)

APPARATUS

1. 1.5 mL microfuge tubes

2. Incubation tool

3. Vortex

4. Micropipette

METHODS

EXPERIMENT A:

*Blood were collected using Heparinized/EDTA VenoJet tube.

Firstly, the blood is spun in 1.5 mL microfuge tube at 8,000 rpm for 10

minutes.Next, the supernatant is discarded. The pellet is then resuspended in 250 µL

extraction buffer (10mM Tris-C1 pH 8.0, 0.1M EDTA, 20 μg/mL RNAase A and 0.5%

SDS). After that, it is incubated at 37oC for 30 minutes.Then, 5 μL of Proteinase K

(final conc. 200 μg/mL) is added and the tube is swirled nicely and the suspension is

incubated in 50oC waterbath for 1 hour. The suspension is allowed to cool down at

room temperature for 5 minutes and then an equal volume of saturated-phenol is

added followed by soft vortex for 2 minutes.The suspension is again spun at 5,000

rpm for 5 minutes.Then, the upper phase is removed to a new sterile tube (fumehood

is used). Step 6 & 7 are repeated. After the upper phase is removed to a new sterile

tube, an equal volume of chloroform is added followed by soft vortex for 2 minutes.

Again, the suspension is spun at 5,000 rpm for 5 minutes.The upper phase is then

removed to a new sterile tube and step 9 & 10 are repeated.Finally, the upper phase

(DNA) is removed into a new sterile microfuge tube. After that, 0.1x volume 3M

Sodium Acetate & 2x volume 100% Ethanol (chilled) are added followed by gentle

swirling for 4-5 minutes at room temperature before the suspension is incubated at -

70oC for 10 minutes. After that, the suspension is spun at 12,000 rpm for 10 minutes.

The supernatant is then discarded and the pellet is washed with 75% Ethanol. Next,

the DNA is spun at 12,000 rpm for 10 minutes, supernatant is then removed and the

pellet is dried at room temperature for 5 minutes. At last, the DNA is dissolved in 50

μL sterile TE Buffer (10 mM Tris-C1 & 1mM EDTA) before the DNA is analyzed on

0.7% agarose gel electrophoresis.

EXPERIMENT C

First, a single colony of transformed bacteria is inoculated in 2mL of LB broth

containing appropriate antibiotic, and incubated at 37°C with vigorous shaking.1.5

mL of this overnight culture isthen transferred into a new sterile microfuge and

centrifuged at maximum speed for 30 seconds. After the supernatant is discarded,

the pellet is resuspended in 100µL of ice-cold solution I (50 mM Glucose, 25mM

Tris-Cl pH 8.0, 10 mM EDTA, 10 µg/mL RNase) followed by vigorous vortexing.

Then, 200µL of alkaline lysis solution II (0.2 N NaOH, 1% SDS) was added to the

suspension and mixed by inverting the tube 5-8 times until the suspension become

clear and sticky. Next, the solution is incubated on ice for 10 minutes.150µL of ice

cold solution III (3M Potassium acetate, pH 5.5) is then added to the viscous

suspension.The suspension is mixed thoroughly by inverting the tube 8-10 times,

and then, the suspension is incubated on ice for 5 minutes. After that, the

suspension is centrifuged at 10,000 rpm for 5 minutes and the supernatant is

transferred to a new sterile tube. Next, an equal amount of water – saturated phenol

plasmid is added and mixed thoroughly by vortexing to separate the organic and the

aqueous phase.The emulsion is then centrifuged at 13,000 rpm for 2 minutes at

+4°C, and then the aqueous layer containing plasmid DNA is transferred to a new

sterile microfuge tube. After that, the extraction processes are repeated with

chloroform twice and the aqueous phase is transferred to a new tube.The DNA is

then precipitated by adding 2 volumes of iced-cold 100% (v/v) ethanol and 0.1

volumes of 3 M Sodium Acetate (pH 5.5) followed by incubation at -70°C for 10

minutes. Next, the precipitate plasmid DNA is collected by centrifugation 13,000 rpm,

+4°C for 10 minutes. After that, the supernatant is removed carefully and 750µL of

cold 70% (v/v) ethanol is added before mixed by inverting the tube or by gentle

vortexing. The plasmid is then recovered by centrifugation at 13,000 rpm, +4°C for

10 minutes. Finally, the supernatant is discarded and the pellet is dried at room

temperature for 10 minutes before the plasmid is resuspended in 50µL of sterile TE

Buffer (pH 8.0) and store at -20°C.

EXPERIMENT D

1.5mL of this overnight culture is transferred into a sterile microfuge tube and

it’s centrifuged at maximum speed for 30 seconds.Then, the supernatant is

discarded and the pellet is resuspended in 100μL of the ice cold solution I (50 mM

Glucose, 25mM Tris-CI pH 8.0, 10mM EDTA, 0.1% Triton-X 100) and vigorously

vortex is followed. Next, 25μL of fresh prepared lysozyme solution is added into the

suspension and it is mixed by inverting the tube 5-8 times until the suspension

become clear and sticky.It is then incubated on boling water for 2 minutes before it is

chilled on ice for 5 minutes. Then, an equal amount of water-saturated Phenol is

added into the suspension and it is mixed thoroughly by vortexing to separate the

organic and the aqueous phase (the fume hood is used). After that, the emulsion is

centrifuged at 8,000 rpm for 2 minutes at +40C, and the aqueous layer containing

DNA is transferred to a new sterile microfuge tube.The extraction process is then

repeated with chloroform twice and the aqueous phase is transferred in to a new

tube. Next, the DNA is precipitated by 2 volumes of ice-cold 100% (v/v) ethanol and

0.1 volumes of 3 M Sodium Acetate (pH 3.5) are added. Then, it is incubated at -

700C for 10 min. After that, the precipitated DNA is collected by centrifugation 13,000

rpm at +40C for 10 minutes.The supernatant is then removed carefully and 750μL of

cold 70% (v/v) ethanol before it is mixed by inverting the tube or by gently vortexing.

Next, the DNA then is recovered by centrifugation at 13,000 rpm, +40C for 10 min.

Finally, the supernatant is discarded and the pellet is dried at room temperature for

10 min before the plasmid DNA is resuspended in 50μL of sterile TE buffer (pH 8.0)

and stored at -200C.

RESULT

A) PREPARATION OF GENOMIC DNA FROM EUKARYOTE CELLS (BLOOD)

6th line: a thin band and there is a smear below bands shown on 0.7% agarose gel.

The number of base pairs for the genomic DNA (as represent by the thin band) is

around or more than 23130 base pairs.

C) Preparation of plasmid DNA from bacteria cells (E.coli) – Alkaline Method

Alkaline lysis is a method to isolate plasmid DNA from the bacterial cells. For

group 6, based on the electrophoretic analysis, there are two different concentration

but continuous bands of DNA fragment are identified. The bands are referred as the

plasmid DNA of E.coli. This is ensured by comparing the bands to the marker as the

plasmid DNA have less than 23130 base pairs. Besides that, there is smear above

the bands.

D) Preparation of plasmid DNA from bacteria cells (E.coli) – Boiling Method

For group 6, the result shown that there are two distinct bands. The upper band

which is more than 23130 base pairs refers to chromosomal DNA, while the lower

band, less than 23130 base pairs refers to plasmid DNA. The intensity of the bands

is lower too. Besides that, there is smear below the bands.

DISCUSSION

EXPERIMENT A

In haploid human genome, there are more than 3 billion base pairs From the

result we obtained, the number of base pairs of the genomic DNA is around or more

than 23130 bps. Most groups got the similar result except the group1, 2, 4 and 5.

Group 2’s result has another band presence above the similar band share by most

groups. This may due to the presence of other cell residue. Group 1 has only the

upper band while for the group 4 and 5, there are none of band presence. Absence

of band may due to the errors done in the step which the proteins and lipids of the

cell is removed such as removing the upper phrase. These errors may cause the

genomic DNA removed unintentionally.Thus, during gel electrophoresis, there was

no band observed. Meanwhile, the smear observed may refer to the degraded RNA

as it was not completely removed.

EXPERIMENT C

In 1952, Joshua Lederberg introduced the term plasmid. A plasmid is an

extrachromosomal circular DNA molecules that replicates independently and distinct

from the normal chromosomal DNA. Plasmid is nonessential for cell survival under

non-selective conditions. Plasmids are nowknown to be present in most species of

Eubacteria that have been examined, as well as in Archaea and lower Eukarya.

Alkaline lysis and boiling method are widely used procedures for separating

plasmids from chromosomal DNA in bacterial cell extracts. Both methodsrequire

several steps in order to prepare the plasmid DNA from the E.coli. Both methods

involve the extraction of DNA from the cell by lysing the cell, degradation of RNA,

denaturation of DNA, renaturation of plasmid DNA, isolation of protein from the

nucleic acid, and purification and concentration of the plasmid DNA.

In Alkaline lysis, the key procedure in this method to is the process of alkaline

denaturation of DNA. Due to the lysis of the cell by the action of Sodium dodecyl

sulphate (SDS) and EDTA in E.coli, the chromosomal DNA exists as linear fragment

while plasmid remains undisrupted as covalently closed circular DNA. The plasmids

remain as intact supercoiled circular DNA. This process is continued by adding

sodium hydroxide in order to raise the pH. High pH will disrupt the hydrogen bond

and cause the linear strands of chromosomal DNA to dissociate. However, the

plasmids are tougher and not easily to be denatured. Although, the high pH disrupts

the hydrogen bonds, the two circular DNA strands remain interlinked as they cannot

separate physically. When the potassium acetate is added, the pH drops and the

interlinked plasmid strands will snap together and renature. The strands reform the

double-stranded plasmid. For the separated linear chromosomal fragments, they

cannot renature, instead they will aggregate into an insoluble network. The

chromosomal DNA will be removed by centrifugation, leaving the plasmids in

solution. It is important to get pure DNA plasmid. Thus, other cell components such

as cell wall and protein must be removed. The purity of the plasmid DNA is also must

be improved for further analysis by electrophoresis.

Same principle is applied in boiling method. However, lysozyme and Triton-X

100 are used to weaken the cell wall. By heating the solution, the hydrogen bonds

between the bases are disrupted, causing the chromosomal DNA to become

denature. Still, the plasmid DNA remain intact and become renature, which is the

same as what has been occurred to the plasmid DNA in alkaline lysis method.

Agarose gel electrophoresis is used as standard method for separating DNA

fragments. As DNA is negatively charged due to the phosphate, the DNA fragments

will migrate towards the positive electrode. The rate of migration depends on the size

of fragment. Thus, the smaller size or lesser the number of base pairs of the DNA

fragment, the more quickly it can progress through the agarose. The conformation of

the plasmid also affects the migration rate of the plasmid DNA.

Based on the result of experiment C, the migration rate is depending on the

conformation of the plasmid DNA. This is because the actual size and base pairs of

the plasmid can only be determined if the DNA is cut by restriction enzyme. Native

plasmids in nature are supercoiled circular molecules in covalently closed circle.

However, if a plasmid is nicked, the loose ends are free to rotate, and it has a

relaxed, open circular form. The other form of plasmid is in linear molecules which

are formed by double-strand breaks. Although all the three forms have same size or

number of base pairs, they move in different rate in a gel. The open circular plasmids

formed by single strand breaks migrate more slowly than the linear molecules and

supercoiled plasmids. However, there are two bands only, which the upper band is

from open circular plasmid while the lower band is from supercoiled plasmid.

Supercoiled DNA travel faster as this form has tight conformation compared to the

other two forms.

Besides that, the presence of smear is due to improper preparation when the

experiment is conducted.

EXPERIMENT D

Based on the result of experiment D, both chromosomal DNA and plasmid

DNA remain in the solution. The presence of upper band which has more than 23130

base pairs indicates the presence of genomic DNA as it contains more base pairs

compared to plasmid DNA. This maybe happen when some of renatured DNA

chromosomes are successfully pass through the detergent. This is also may refer to

the ability of boiling method which can work well with smaller plasmids with less than

15000 base pairs in size (Sambrook & Russel, 2001). The intensity of the bands is

lower compared to other bands from other groups. This may due to the DNA yield

from this method is low. The second band which indicates the plasmid DNA can be

used to determine the conformation of the plasmid DNA. Based on the shape of the

band, the plasmid DNA appears as nicked open circle plasmid. However, this cannot

be confirmed as it is hard to determine the conformation of the plasmid just by

looking to the shape of the band. The presence of smear below the band is referred

to the RNA. RNA still remains in the supernatant as there is no RNAse being used in

boiling method. As RNA has less base pair, it migrates faster. Other than that, the

boiling method will produce every single thing from the bacteria; the chromosomal

DNA, plasmid DNA, and RNA.

By comparing both methods (experiment C and D), we can see that the

alkaline method is the most appropriate technique to prepare the plasmid DNA. It is

most consistent plasmid purification protocol regardless of the strain. Besides that, it

is better suited for isolation of higher base pairs or low-copy number plasmid. Most

analysis and cloning procedures can be done on the plasmid without further

purification. For boiling method, it only works well with smaller plasmid, lower than 15

kb. Some strains of E.coli are not recommended for this method. Still, the principle of

denaturation and renaturationDNA is the same for both methods (Sambrook &

Russel, 2001)

Lastly, some precautions should be taken. Phenol is very toxic and should be

handled in a fume hood. After use, the waste phenol must be collected in a glass

toxic-waste container with a clear label and disposed of properly in compliance with

the local hazardous chemical disposal procedures. Besides that, talking is not

allowed as the saliva can contaminate the DNA sample. The container must been

kept in sterile, thus direct touch to the container is strictly prohibited.

CONCLUSION

In this experiment we have learn three difference method use in extraction of DNA

which depend on the types of the cell: eukaryote or prokaryote. The boiling method

more suitable to extract short chain genomic DNA such as plasmid DNA; the lysis

method more suit to extract the longer chain of genomic DNA.

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Wiley and Sons.

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