LTM 1: Detection of Nucleic Acid

Afrah (1406641792)
Group: 3

A. ABSTRACT
Nucleic acid is one of the biggest part of molecule in our life. The most common nucleic
acids are DNA and RNA, which contains a lot of genetic codes from a living thing. The
presence of DNA (or generally nucleic acids) can be tested by doing some types of
analysis. Besides, the amount of it can be also measured with some methods. In order to
test the presence of DNA, or to separate DNA fragments, we use qualitative analysis
method, such as electrophoresis, blotting, DNA hybridization and DNA sequencing. The
analysis also can be done by using quantitative analysis, for example using UV
spectroscopy, polymerase chain reaction and reverse transcription. Each analysis method
has different purpose and also different output data.
B. QUALITATIVE ANALYSIS OF NUCLEIC ACID
1. Electrophoresis
a. Introduction
Nucleic acid electrophoresis is an analytical method used to separate DNA
fragments based on their size in the presence of electric current. Nucleic acid
molecules are size separated by the aid of an electric field where negatively
charged molecules migrate toward anode (positive pole). The migration flow is
determined solely by the molecular weight where small weight molecules migrate
faster than the larger ones. In addition to size separation, nucleic acid fractionation
using agarose gel electrophoresis can be an initial step for further purification of a
band of interest.
b. Materials Needed
1) Nucleic acids, DNA samples or DNA size standards
2) Buffers, such as TAE, TPE and TBE
3) Coloring agent, example Ethidium bromide or SYBR green
c. Procedure
1) Making the agarose gel
Dissolving agarose in several amounts (ex: 0.5 g) and
adding 50 ml of TBE buffer, then mixing it. The result will
be agarose with 1% of concentration. To make different
concentration, it is needed to change the formulation of
agarose and TBE buffer.

Warming the mixture of agarose and TBE

in a microwave until it melts, then cooling
it down until the temperature reaches
50oC. Next, adding 1g/ml of ethidium
bromide and mixing it by swirling.

Pouring the agarose gel mixture into a

tray.

Making holes in the one end of the tray using

a gel comb. Choosing an appropriate gel is
important, in order to form a complete well.

2) Setting up the gel apparatus

Pouring the buffer into the electrophoresis
box, followed by the gel, then the gel will be
at the bottom of the box, while buffer will be
at the upper position.
3) Loading the sample of DNA into the gel
Adding dye to the sample with ratio 1:5
or 1:10.

Injecting the sample into the gel using a

micropipette or glass capillary tube.
Injecting the size standards in left and
right side of the gel.

4) Hooking up electrical current and running the gel

Connecting the electrophoresis box
with an electrical supply. The
negative electrode is in the buffer
solution, and the positive electrode is
in the other side. Tiny bubbles will
appear in both ends of the box
indicates that a current is running.
DNA is negatively charged, they are
repelled by the negative electrode and
migrate to the positive electrode.

5) Staining the gel

Staining the DNA fragments by
using ethidium bromide. The
purpose is to make the DNA easier
to be seen.

To make the DNA more visible, the

sample is placed in a UV light box,
with wavelength 300nm.

6) Analyzing the results

Determining the length of DNA
strands. The size of DNA molecule
affects the migration rate of DNA
fragments. The smaller fragments will
move faster than the larger ones,
since they can move more easily
through the gel.

d. Result analysis
Below is an example of DNA electrophoresis result:
1) By looking at the migration of the
DNA molecular weight standards, you
can tell that the migration of DNA
through an agarose gel is not linear with
respect to size. If you graphed the
distance traveled vs. the molecular
weight of the fragment, you would see
that there is a logarithmic relationship
(i.e. small fragments travel much faster
than large fragments).

2) You can see that there is a big difference between the way a plasmid as isolated
from the alkaline lysis prep will run vs. this same plasmid after it is cut with a
restriction enzyme and linearized. This is because the plasmid will be found in many
different supercoiled forms in the bacteria. When you isolate plasmid from a
bacterial culture, you isolate all the different supercoiled forms of the plasmid, and
each will migrate differently on the gel, giving you three major bands and many
minor bands. When this mixture of supercoiled plasmids is cut with a restriction

enzyme, the different forms linearize and unwind. As a result, they all become
identical and run at the same rate, and you see only one band on the gel.
3) The molecular size of an unknown piece of DNA can be estimated by comparison
of the distance that it travels with that of the molecular weight standards. This is only
true for linear DNA. None of the supercoiled forms will migrate at a rate relative to
linear DNA, which means that you can't use the DNA markers to estimate the
molecular weight of a circular DNA molecule. To estimate the molecular weight of a
plasmid, you must first linearize it. By looking at the gel above, the molecular size of
the plasmid can be estimated at approximately 3.0 kilobases (kb). A more accurate
estimate can be found by graphing the molecular weight of the standards (in base
pairs) vs. the distance traveled on semi-log paper and using this graph to determine
the molecular weight of the unknown. You will do this at the end of this experiment.
Molecular size is the most important information derived from the agarose gel and
the usual reason for running a gel.
e. Advantages and Disadvantages
f.

Advantages
Relatively simple to perform
Inexpensive

Disadvantages
The gel can be altered and provide
inaccurate results
The gel may melt when the electric
current is passed through it.
The buffer can become exhausted

Can test DNA from some type of

evidence (such as hair, blood, skin)
The gel does not denature the sample, Different form of genetic material may
it easily poured
run in unpredictable form
Application
DNA gel electrophoresis can be used in some purposes, such as:
1) To get a DNA fingerprint for forensic purpose
2) To get a DNA fingerprint for paternity testing
3) To get a DNA fingerprint to look for evolutionary relationships among organisms
4) To find genes associated with a disease
2. DNA Hybridization
a. Introduction
DNA Hybridization is the process of combining two complementary single-stranded
DNA or RNA molecules and allowing them to form a single double-stranded
molecule through base pairing. In a reversal of this process, a double-stranded DNA
(or RNA, or DNA/RNA) molecule can be heated to break the base pairing and
separate the two strands. DNA hybridization has a purpose to identify the presence
of a specific sequence in a living organism.
b. Procedures
1) Obtaining several DNA samples from different sources

(Left) is the example of DNA molecules which come from different

sources, and (right) is the closer look of a pair of double stranded
DNA containing paired nitrogen bases.

2) Heating up the double-stranded DNA up to 90oC

The pair of nitrogen bases is broken down (left) which will cause
the double stranded DNA separated from its pair (right).
3) Cooling down the temperature to about 65C
As the temperature
decreased, the single
stranded DNA will recombine
and paired with another
strand.

4) The labeled DNA probe will combine with a strand that has a similar nucleotide
sequence

3. Blotting
a. Purpose
Actually there are two types of blotting, southern and northern blotting. Southern
blotting has the similar purpose with electrophoresis, which is to separate DNA
fragments according to their size, in presence of electric current. Meanwhile,
northern blotting analysis reveals information about RNA identity, size, and
abundance, allowing a deeper understanding of gene expression levels.
b. Differences Between Southern and Northern Blotting
The table below shows the differences between southern blotting and northern
blotting:
Southern blotting
Separation of DNA
Need denaturation

Northern blotting
Separation of RNA
Do not need denaturation

Using nitrocellulose filter membrane

The DNA detected can be a single gene or can
be a part of larger piece of DNA

Using aminobenzyloxymethyl filter paper

membrane
Technique to detect specific RNAs separated
by electrophoresis and hybridization to a
labeled DNA probe.

c. Procedure
1) Southern Blotting

Explanation:
Step 1: DNA purification. Isolate the DNA from the rest of the cellular material in
the nucleus, next is incubate the specimen with detergent to promote cell lysis
(cell lysis frees cellular proteins and DNA). Proteins are enzymatically degraded
by incubation with proteinase, and DNA is purified from solution by alcohol
precipitation. The visible DNA fibers are removed and suspended in buffer.
Step 2: Restriction digestion. Cut the DNA into different sized fragments using
restriction endonucleases (RE).
Step 3: Gel electrophoresis. This step is exactly the same with electrophoresis
method, where nucleic acids have a negative charge, and move to the positive
charge. The smaller molecules will move faster, in contrast, the larger ones will
move slower. The rest of this step is also exactly the same with electrophoresis
method.
Step 4-5: Denaturation and blotting process. DNA is denaturated with an
alkaline solution (for instance, NaOH), which will cause the double stranded
become single stranded. The process of transferring the DNA from the gel to a
membrane is called blotting. Blotting is usually done on a sheet of nitrocellulose
paper or nylon. Then, DNA is neutralized with NaCl, in order to prevent rehybridization before adding the probe. The transfer process can be done by
either electro-blotting or capillary blotting. The blot is made permanent either by
drying at temperature ~80oC or exposing to UV radiation.

Step 6: Hybridization. The labelled probe is added to the membrane in buffer

and incubated for several hours to allow the probe molecules to find their
targets.
Step 7-8: Wash and autoradiography. Wash excess probe that are bound nonspecifically to the membrane. Blot is incubated with wash buffers containing
NaCl and detergent to wash away excess probe and reduce background. If the
probe is radioactive, the particles will expose x-ray film.
2) Northern Blotting

4. DNA Sequencing
a. Purpose
DNA sequencing is the process of determining the precise order of nucleotides
within a DNA molecule. It includes any method or technology that is used to
determine the order of the four basesadenine, guanine, cytosine, and thyminein
a strand of DNA. The advent of rapid DNA sequencing methods has greatly
accelerated biological and medical research and discovery.
b. Procedure
1) Denaturation of DNA to single strands with a heating process

2) Preparing of a mixture containing the single stranded of DNA, DNA polymerase,

four dNTPs and a single short primer

3) Placing the mixture in 4 different tubes, and each of the tube is added by
different ddNTP

The tubes before added

by ddNTP (top) and after
added by ddNTP (bottom)

4) If a single unit of DNA is inserted into the growing chain, the replication will
continue. In contrast, when a ddNTP is inserted, the replication will be
terminated.

The replication of DNA when a single unit is inserted (left) and when
the replication is stopped since a ddNTP is inserted (right).
5) Transferring the contents of the tubes to four lanes of an electrophoresis gel.
The fragments will be separated by size and nucleotide type. The shortest
fragment moves furthest down the gel

Each lane represents each tube (left), and the fragment test result
(right).
6) Reading the result from the bottom to the top base at a time, provides the
correct DNA sequence

C. QUANTITATIVE ANALYSIS OF NUCLEIC ACID

1. UV Spectroscopy
a. Purpose
Like in a common spectroscopy, the purpose of UV spectroscopy is to determine the
concentration of DN and RNA in a sample. Both RNA and DNA absorb UV light very
efficiently making it possible to detect and quantify either at concentrations as low
as 2.5 ng/l. The nitrogenous bases in nucleotides have an absorption maximum at
about 260 nm.
b. Procedure

Steps:
Step 1: Turn the machine on using the switch in the back of the machine (lower
right-hand side as you face the machine, turn on the monitor screen and printer.
Wait for the machine to go through its startup routine.
Step 2: Open the lid on the top of the machine. Choose the small cuvette holder
and place it in the holder in front of the light source. The word FRONT should be
toward the front of the machine.
Step 3: Click on the UV light key to turn the UV on. It takes about a minute for the
UV lamp to warm up. Quit the diagnostic screen by clicking on the word QUIT in the
upper right hand corner. The Main Menu will appear Choose Nucleic Acid from the
menu. When the nucleic acids analysis menu appears, make sure that the
measured absorbances are 260 and 280. Do not use the calculation functions.
Step 4: Use lens paper to clean the surfaces of the cuvette. Rinse the cuvette
chamber with 70% ETOH. Be sure to remove all of the ETOH after the wash. Place
100l sample of your blank (nH2O, TE, whatever your DNA or RNA sample is
dissolved in) in the cuvette chamber. Place the cuvette in the holder and place the
lid on the holder.
Step 5: Shut the lid of the machine.
Step 6: Click READ BLANK in the bottom left corner of the screen.
Step 7: Prepare a 1:100 dilution of the sample you want to read.
Step 8: After the machine has read the blank, remove the cuvette, remove the
blanking solution from the chamber, rinse and dry the chamber and place your
diluted sample in it.
Step 9: Click on READ SAMPLES in the upper left hand of the screen.

Step 10: After the machine has read your sample, the data will appear on the
screen. You do not need to print this each time you measure a sample. A machine
will collect data for you.
Step 11: Between samples clean the cuvette as described above. You do not need
to blank each time unless your samples are dissolved in different solutions. If you
use a different cuvette, you must run a blank.
Step 12: When you are finished reading samples, remove and clean the cuvette
and put it away. Print your results by clicking on PRINT at the top right of the screen.
QUIT the data screen. This will take you back to the main menu. Turn off the UV.
You can turn off the machine while the Main Menu screen is active. Turn off the
machine the monitor and the printer.
Step 13: Calculate the concentration of your RNA or DNA sample and the
OD260/OD280
c. DNA Purity Formula
You can calculate the concentration of the DNA or RNA in your sample as follows:
DNA concentration (g/ml) = (OD 260) x (dilution factor) x (50 g DNA/ml)/(1 OD260
unit)
RNA concentration (g/ml) = (OD 260) x (dilution factor) x (40 g RNA/ml)/(1 OD260
unit)
In contrast to nucleic acids, proteins have a UV absorption maximum of 280 nm,
due mostly to the tryptophan residues. The absorbance of a DNA sample at 280 nm
gives an estimate of the protein contamination of the sample. The ratio of the
absorbance at 260 nm/ absorbance at 280 nm is a measure of the purity of a DNA
sample; it should be between 1.65 and 1.85.
2. Polymerase Chain Reaction
a. Purpose
The polymerase chain reaction (PCR) is a technology in molecular biology used to
amplify a single copy or a few copies of a piece of DNA across several orders of
magnitude, generating thousands to millions of copies of a particular DNA
sequence. There are many variants of PCR method, such as
b. Procedure
Step 1: Separating the Target DNA Denaturation. During the first step of PCR,
called denaturation, the tube containing the sample DNA is heated to more than 90
degrees Celsius (194 degrees Fahrenheit), which separates the double-stranded
DNA into two separate strands. The high temperature breaks the relatively weak
bonds between the nucleotides that form the DNA code.
Step 2: Binding Primers to the DNA Sequence Annealing. PCR does not copy
the all of the DNA in the sample. It copies only a very specific sequence of genetic
code, targeted by the PCR primers. For example, Chlamydia has a unique pattern
of nucleotides specific to the bacteria. The PCR will copy only the specific DNA
sequences that are present in Chlamydia and absent from other bacterial species.
To do this, PCR uses primers, man-made oligonucleotides (short pieces of synthetic
DNA) that bind, or anneal, only to sequences on either side of the target DNA
region. Two primers are used in step two - one for each of the newly separated
single DNA strands. The primers bind to the beginning of the sequence that will be
copied, marking off the sequence for step three. During step two, the tube is cooled
and primer binding occurs between 40 and 60 degrees Celsius (104 140 degrees
Fahrenheit). Step two yields two separate strands of DNA, with sequences marked
off by primers. The two strands are ready to be copied.
Step 3: Making a Copy Extension. In the third phase of the reaction, called
extension, the temperature is increased to approximately 72 degrees Celsius (161.5
degrees Fahrenheit). Beginning at the regions marked by the primers, nucleotides in
the solution are added to the annealed primers by the DNA polymerase to create a
new strand of DNA complementary to each of the single template strands. After
completing the extension, two identical copies of the original DNA have been made.

3. Reverse Transcription
a. Purpose
Reverse transcription polymerase chain reaction (RT-PCR) is one of many variants
of polymerase chain reaction (PCR). This technique is commonly used in molecular
biology to detect RNA expression.
b. Procedure
In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA)
using a reverse transcriptase. The cDNA is then used as a template for exponential
amplification using PCR. There are 2 types of RT-PCT, the one step and two steps
RT-PCR.
1) One step RT-PCR
One-step RT-PCR combines the first-strand cDNA synthesis (reverse
transcription) reaction and PCR reaction in the same tube, simplifying reaction
setup and reducing the possibility of contamination. One-step RT-PCR allows
easier processing of large numbers of samples, and helps minimize carryover
contamination, since tubes are not opened between cDNA synthesis and
amplification. By amplifying the entire cDNA sample, one-step RT-PCR can
provide greater sensitivityfrom as little as 0.01 pg total RNA. One-step
reactions allow for the use of sequence-specific primers only.
2) Two steps RT-PCR
Two-step PCR begins with the reverse transcription of either total RNA or
poly(A)+ RNA into cDNA using a reverse transcriptase. Following first-strand
synthesis, the cDNA is transferred to a separate tube for the PCR step. Twostep RT-PCR is useful for detecting multiple messages from a single RNA
sample. Youll get greater flexibility when choosing primers and polymerase than
with one-step RT-PCR systems. When performing two-step RT-PCR, you have
the option of using either oligo(dT), random hexamer, or gene-specific primers,
and then PCR is performed with either Platinum Taq DNA Polymerase,
Platinum Taq DNA Polymerase High Fidelity, or your choice of PCR enzyme.

c. Comparison between one-step RT-PCR and two steps RT-PCR

Prime first-strand cDNA with

One step RT-PCR

Gene-specific primers

Two steps RT-PCR

Oligo(dT) primer

Random hexamers

Provides

Convenience

Amplifcation enzymes
premixed with reverse
transcriptase
Fewer pipetting steps
and reduced chances
of contamination

Ideal for analysis of large

numbers of samples

D. REFERENCES

Choice of primer

Choice of amplification
system

Ability to save some

RNA sample for later
use

Ability to optimize for

difficult RT-PCR
(combine with
Platinum enzymes
for higher specificity or
combine with
Platinum Pfx for
greater fidelity)

High sensitivity

Recommended uses

Gene-specific primers
Flexibility

Ideal for real-time

quantitative RT-PCR

Ideal for detection or

quantifying several messages
from, a single sample

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