1.

0 INTRODUCTION

In clinical chemistry laboratory, a lot of tests are done to detect, screen and confirm the existence
of any diseases and abnormalities in human body. Besides that, the clinical chemistry laboratory
also responsible to monitor toxic cases and to monitor the effectiveness of certain drugs given to
certain patient.

Toxic cases occur because in way to treat any diseases, we dealing with medications
(drugs) which made up of chemical substances either the substances are organic or inorganic.
The substances used sometime are dangerous to normal peoples and also to the patients which
can cause hallucination, seizure and also can lead to fetal. So, when these medications are taken
overdose or wrongly used without prescription from authorized persons such as in suicide cases
and drug abuses, the person in-charge in laboratory responsible to do a series of tests in way to
help doctors to identify the type, amount and how to overcome of the drug problems. All the
process involved to determine drugs and their side effects toward human being is known as
toxicology in medical profession. This is because toxicology is a division of medical and
biological science which concerns with toxic substances, their detection, their avoidances, their
chemistry, their treatments and their adverse effects toward living organisms (Taber’s
Cyclopedic Medical Dictionary, 2005).

Since, medications or drugs can cause harmful effects toward humankinds, so it is

essential to monitor the usage of them and ensure the dosage introduced is the effective and
optimal dosage which non-toxic to the patients. The term used referred to the stage of dosage
monitoring of drugs is therapeutic drug monitoring (TDM). Actually, therapeutic drug
monitoring is the measurement of specific drugs at intervals in order to maintain a relatively
constant concentration of the medication in the bloodstream (Therapeutic Drug Monitoring,
2009). Therapeutic drug monitoring (TDM) is highly recommended in medical profession
because it ensures the human safety in drugs usage as medications. This is because the drugs
need by patients are vary from one person to other persons because the rate of absorption,
metabolism, utilization and elimination of drugs are different based on their age, health
conditions, genetic and the interference of other medications that taken by them. Besides that, the
amount of the drugs need by a patient also may be change at different time, day and weathers.
So, it is so difficult to maintain the steady state of the drugs intake by a patient to ensure their

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safety and it is become the duty for medical-linked professionals to do the best in their jobs to
maintain the patients’ safety and also for their own safety.

In therapeutic drug monitoring, a lot of tests are developed in way to ensure the right and
optimal concentrations of drugs are introduced into human body. High Performance Liquid
Chromatography (HPLC), Thin Layer Chromatography (TLC), Gas-Liquid Chromatography
(GLC) and Solid Phase Micro-extraction (SPME) are among the example of tests which always
done in therapeutic drug monitoring in laboratory. Besides that, polymerase chain reaction (PCR)
is also one of the important procedures in therapeutic drug monitoring because it can show the
effectiveness of certain drugs given to patient to treat certain diseases such as malignancy and
cytomegalovirus pulmonary infections in immuno-compromised patients.

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC) is used to separate two or more compounds in dried liquid
samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of
alumina or silica gel (stationary phase).

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High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is used to separate, identify and quantify
compounds such as drugs that are dissolved in solution. The HPLC become the first choice
because it needs small sample and the test easier to operate than GLC.

Gas-Liquid Chromatography (GLC)

The Gas-Liquid Chromatography (GLC) is used to separate vaporized samples with a carrier gas
(mobile phase) and a column containing a liquid or solid beads (stationery phase). But, the GLC
is not used frequently because it needs large volume of sample and the instruments involved are
too complex which need experiences to handle it properly.

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 2.0 ROLE OF POLYMERASE CHAIN REACTION (PCR) IN TOXICOLOGY AND
THERAPEUTIC DRUG MONITORING

Polymerase chain reaction (PCR) is a common technique used in clinical laboratory nowadays
although it is an old technology created more than 20 years ago but it is still relevant until today.
This is because the PCR is the simplest technique developed which can amplify the genetic
materials usually deoxyribonucleic acid (DNA) although it is in small fragments obtained from
body tissues such as skin and body liquids such as seminal fluid, saliva and bloods, then forming
large numbers of DNA fragments after several cycles.

The idea of construction of a technique nearly similar to PCR was firstly introduced in
year 1971. The technique constructed at that time however used two primers which function to
copy both stands of DNA, but it is not used widely throughout the world. However, the
development of a technique which later known as polymerase chain reaction (PCR) in year 1983
by American scientist, Kary B. Mullis and then he collaborated with Fred A. Faloona in his way
to improve and make the PCR more effective, and by year 1991, the PCR was used widely
throughout the world since it makes the work in laboratory easier.

2.1 Principle of PCR

The polymerase chain reaction (PCR) is a genetic technique which is done in vitro in laboratory
to allow the specific enzymatic amplification of specific DNA fragments. This technique is done
by using a DNA template obtained from body tissues or liquids, oligonucleotide primers (RNA
primers) and heat-stable DNA polymerase to duplicate many copies of the interest DNA
fragments. The DNA polymerase which usually used is Taq polymerase. The taq polymerase is
an enzyme originally obtained from a bacterium which can survive in high temperature condition
known as Thermus aquaticus. This bacterium can be found in a hot spring at the Yellowstone
National Park.

Actually, the PCR is done in laboratory in three major phases and the steps will repeated
for 30 to 40 cycles. The three phases involved are known as denaturation, annealing and

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extension, and every phase need different temperature to work properly and produce maximum
fragments which similar to the original DNA fragments.

At the denaturation phase, the original DNA template is heated at high temperature about
90°C to 95°C and usually about 94°C for 20 to 30 seconds. This phase actually function to break
down the hydrogen bonds which bind the two singe-stranded DNA forming double helix DNA
that can be found in living organisms such as human being. So, when the double stranded
(double helix) DNA are exposed to high temperature, it tends to dissociated resulting in the
formation of two single strands.

Then, it will followed by second phase which known as annealing phase. The annealing
phase is done at 50°C to 65°C and normally at 54°C for 20 to 40 seconds. At this phase, the
RNA primers (oligonucleotide) will bind by ionic bond to the single-stranded DNA of the
template strand. The attaches of primer to the single-stranded DNA will lead the DNA
polymerase (taq polymerase) to bind to the same strand. So, the addition of the polymerase will
cause the addition of free nucleotide to the DNA single-strands.

The next phase is extension phase which can be done at temperature about 72°C. The
temperature about 72°C is chosen because the polymerase react best at this temperature and at
the same time, the will cause the binding of primer and single stranded become weaker. At this
phase, the addition of free nucleotides complementary to the nucleotides on the DNA single
strands more progressively until to the end of the strand needed. After that, the same phases are
repeated for 30 to 40 cycles and every phase, the number of the fragments produced will double
from before.

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2.2 Type of PCR

Today, the PCR has undergoes some development to make more effective in its function. So,
varieties of new PCR have been introduced with a variety of names and make it more specific
and at the same time, the sensitivity of the PCR also increased. Some of the type of PCR which
can be found today are stated below:

i. Allele-specific PCR
ii. Asymmetric PCR
iii. Helicase-dependent amplification
iv. Hot-start PCR
v. Intersequence-specific PCR (ISSR)
vi. Inverse PCR
vii. Ligation-mediated PCR
viii. Methylation-specific PCR (MSP)
ix. Miniprimer PCR
x. Quantitative PCR (Q-PCR)
xi. Reverse Transcription PCR (RT-PCR)
xii. Touchdown PCR

2.3 Function of PCR

After the PCR was discover about two decades prior, the PCR become the famous technique
used in clinical laboratory. It has been used to solve a lot of cases in medical field and among the
main functions of the PCR are stated below :

i. Isolation of genomic DNA

In PCR, the DNA fragments can be isolated from the genomic DNA. The DNA can be
isolated because the primer used is specific to certain region on the single DNA strands
and it will bind to that region only. So, we can isolate the interest DNA sequences which
we want and then undergo the amplification process. The isolation of the interest gene
from a long DNA sequences is important in certain conditions such as recombinant DNA
technology, DNA fingerprint and in forensic cases.

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 ii. Amplification and quantitation of DNA
As we know, in PCR the DNA strands can be amplified forming large number of the
same DNA. So, it is important to undergo PCR process when the DNA sequences exist
only a minute sample which can limit the other tests which want to do to the same
sample. So, it is necessary to amplify it amount in way to make the other tests available
such as in forensic analysis and archaeologies’ DNA samples.

iii. Diagnosis of diseases

Via PCR, the dangerous diseases can be detected earlier than usual such as in cases of
malignancy and genetic diseases. The PCR can be used to diagnosis malignancy because
the malignancy is cause by the alteration in gene sequences. So, by using PCR, it can be
determine the interest gene associated with the malignancy.

iv. Identification of microorganism infections

The PCR can be used to identify the slow-growing bacteria such mycobacterium species.
This is because this type of bacterium needs a few weeks to grow on culture media. So,
via the PCR, the existence of this bacterium can be detected earlier since it needs body
tissues or body fluids from the suspected patient.
Besides that, the PCR also can be used to identify a variety of chronic viral infections
such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus
(HBV), human papilloma virus (HPV) and cytomegalovirus (CMV).

v. Drug monitoring
The PCR also used widely for drugs monitoring purposes. This is not mean, do the PCR
to the drugs itself because it is impossible to do that since it is not contain any genetic
materials. Actually, the drugs monitoring using PCR technique is done via using human
samples containing genetic materials such as body tissues and fluids which involve
directly or indirectly with the drugs taken by the patient. The drug can be monitor by
using PCR because in our DNA, the single nucleotide there different from person to
another person which known as single nucleotide polymorphism (SNP). Since, the SNPs

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 vary from one person to another, it can be used to monitor the drug response toward the
patients and from the information which can we get from the PCR, we can know either
the certain drugs is effective to the patient or not. At the same time, the drugs given also
can cause adverse effects to the patients when it alters the SNPs configuration results in
the DNA variation which then could alter the protein structures.

2.4 Advantages of PCR

Polymerase chain reaction become popular in a short time and used widely in medical fields
because it has own advantages which not have by other techniques. Several of the major
advantages are stated below to show the effectiveness of the PCR than other techniques.

i. Short time needed

The PCR procedure can be performed in a short of time usually within a few hours only.
Although the time needed by the PCR is short but it can reproduce a large number of
copies of the interest DNA fragment similar to the original templates. This is because in
every cycle of PCR, the single stranded DNA will double from the copies before and
after several cycles usually about 30 to 40 cycles, the copies of DNA fragments will
achieves million of copies.

ii. Ease of use

The PCR is easy to use than other techniques which more complicated which need high
technology machines, reagents, enzymes and many more complicated systems. In
addition, the components used in the PCR such as polymerase and primers can easily get
since it is prepared for commercial purposes. So, the person in-charge to run the PCR
doesn’t need to prepare the PCR’s components but it is already available in medical
market.
iii. Sensitivity
The PCR is a very sensitive method since a minute amount of DNA sequences can be
used as the sample. So, it is so useful in forensic analysis and archeological purposes
which usually only small DNA sequences obtained from the incident places.

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 iv. Robustness
The DNA can survive for long time up to 10 000 to 40 000 years. So, it becomes the
important part in molecular anthropology and paleontology studies. But, usually the DNA
sequences obtained is minute sample, so it needs to amplify forming a large amount of
similar sequences. But, the conventional techniques impossible to amplify it, however the
PCR allows the amplification of the interest sequences of the old and badly degraded of
DNA sequences.

2.5 Relationship Between PCR and Electrophoresis

Electrophoresis is a common technique used throughout the world for the separation of
deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein molecules. These DNA, RNA
and protein molecules are separated on a gel matrix based on their mass and charge carried by
them using an electric current. So, the molecules will run on the agar at different rate which the
smaller molecules will run faster and let the larger molecules behind result in the formation of
bands at different length on the agar. Meanwhile, since the electric current is applied, the
positively charged molecules will attract to the negatively terminal (anode) and at the same time,
the negatively charged molecules will attract to positively terminal (cathode).

The main component of the electrophoresis is the agar. There are two types of materials
which commonly used in electrophoresis for toxicity and therapeutic drug monitoring are
agarose gel and polyacrylamide gel. These two materials are specifically used in electrophoresis
for DNA and RNA (nucleic acid) but the agarose gel is the main material chosen rather than
polyacrylamide gel because it is safer since polyacrylamide is a neurotoxin and the cost
consumed much lower than polyacrylamide.

In toxicity and therapeutic drug monitoring, the DNA sequences which undergo
polymerase chain reaction (PCR) procedure in way to amplify its amount will undergo
electrophoresis to identify either the drugs given show any complications or effective against
certain diseases which can alter the DNA sequences.

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 The bands form on the gel matrix will be stained to make it visible and easier to make
comparison to the normal bands which supposed to be in normal persons. The stain used in
staining procedure is usually using ethidium bromide silver or coomassive blue dye.

The electrophoresis is done to the DNA sequences copied during PCR because it is used
to check the sequences either they are similar to the original templates. The electrophoresis will
shows the successful of the PCR since it can show either the product formed or not during PCR
because not every PCR successfully yields a product in its cycles such as in condition where the
primer doesn’t fit properly to the templates. Besides that, it will shows the size of product copied
to confirm the copies are 100% similar to original templates and for the correct copies, the bands
formed on agar should be only one band. This is because the copies should have the same
compositions since they are originally from the same template, so it is impossible it can form
several or a lot of bands on agar but it is possible to gain different bands if the primers bind to
different locations or more than two primers used.

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3.0 OTHER MOLECULAR TECHNOLOGIES

Actually, the electrophoresis is a technique performed for analytical purposes only in toxicology
and therapeutic drug monitoring and for further characterization and confirmation, there are a lot
of other molecular technologies developed such as Southern, Northern, Eastern and Western
blotting.

3.1 Southern Blotting

Southern blot is routinely done as a couple to the agarose gel electrophoresis and it function to
check the presence of DNA sequences in DNA sample. It is named as Southern blot because it
takes the name of its developer, Edwin Southern.

Procedure

i. Restriction enzyme is used to cut the DNA strands into smaller fragments and then place
in the well of the gel electrophoresis and it is separated throughout the agar based on their
size.
ii. Sometime, the DNA fragments will larger than 15 kb, so, the gel usually treated with an
acid such as diluted hydrogen chloride which functions to breaking the fragments into
smaller size.
iii. The DNA fragments also can be treated with alkaline solution such as sodium hydroxide
to breaking the fragments into smaller size.
iv. Then, the DNA is transferred to a membrane usually a sheet of nitrocellulose.
v. The membrane is then incubated in a vacuum or regular incubator at 80°C for 2 hours
with hybridization probe which actually a single DNA fragment with specific DNA
sequences. So, the probe will form base pair with complementary sequence of the DNA
on the membrane.
vi. After hybridization process, the excess probe is wash out from the membrane and the
pattern produced can be seen on X-ray film by using autoradiography or the production
of color on the membrane if use other methods such as chromogenic detection method.

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3.2 Northern Blotting

Northern blot was developed by Alwine and his colleagues at Stanford University in year 1977
and it is developed as RNA blotting technique.

Basically, the northern blotting is nearly same in its principle and procedure to the
southern blotting since it is undergoes gel electrophoresis separation, transferred to membrane,
hybridization with probe and detection phase. But, the northern blotting is different it is specify
developed for detection of RNA fragments. Since, the RNA fragments are usually exist as single
stranded, so its migration on the electrophoresis gel will affected. The northern blotting also can
be used in therapeutic drug monitoring because in certain cases the drugs given will cause the
alteration of the genetic materials which code for certain amino acids, hence the during DNA
translation, the mRNA forming is different mRNA which supposed to be translated. So, the
wrongly mRNA produced can be detected via using northern blotting.

Application of the northern blotting

i. Study of the mRNA level (messenger RNA transcripts).

ii. Detection of mRNA transcript size
iii. Study RNA degradation
iv. Study RNA splicing
v. Study RNA half-life
vi. Study IRES (internal ribosomal entry site) which essential to remove possibility of RNA
digestion against second cistron translation.
vii. Confirm and check transgenic in animal.

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3.3 Western Blotting

Western blotting is a technique used to detect the size of one protein in a mixture of proteins
either the protein are produced in-vivo or in-vitro by using high-quality antibody. Actually, the
function of the antibody used in this blot is same as the probe in the southern or northern blotting
when it is used specifically to bind to the molecules which want to be tested. The function of this
blotting is to show the amount of the protein accumulated in body’s cells.

Procedure

i. The proteins are separated according to their size on the sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE).
ii. The bands formed on the gel are transferred to a nitrocellulose membrane.
iii. Then, the membrane is incubated with a generic protein and then antibody carrying
enzyme or dye is added to bind to its specific protein.
iv. Then, to make it is available to see, the membrane is incubated again with a colorless
substrate which will attach to the enzyme and produced color product.

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3.4 Eastern Blotting

The eastern blotting is a technique developed as the extension to the western blot which firstly
developed to detect the proteins. But it is specifically designed to detect the post-translational
modification of protein. This mean, it is used to detect the proteins which have been undergo
certain other processes such as acetylation, acylation, alkylation, arginylation, formylation,
glutamylation, glycosylation, glycylation, hydroxylation, lipoylation, methylation and
phosphorylation. This technique introduced by S. Thomas at the Department of Pathology,
University of Texas Medical Branch, Galveston, Texas using the same procedure and principle
like in western blotting.

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4.0 ADVANCES IN MOLECULAR TECHNOLOGY

4.1 Amplified Fragment Length Polymorphism (AFLP)

Actually, the amplified fragment length polymorphism (AFLP) is also used the principle of
polymerase chain reaction (PCR) in its reaction. The AFLP can be used in genetic research as
well as in drug monitoring also since as we know, certain drugs can alter the DNA sequences by
alteration in the arrangement of single nucleotide.

4.2 Allele Specific Oligonucleotide (ASO)

Allele Specific Oligonucleotide (ASO) is a short fragment of DNA which produced synthetically
in vitro. Usually, the ASO is produced in length about 15 to 21 of single nucleotide and it is
produced specific for only one DNA sequence in way to increase its sensitivity toward the
specific sequence.

The ASO is important because it can be used to bind to other specific DNA sequence
which wants to test and if the specific DNA sequence has been change its nucleotide although
only one nucleotide, the ASO cannot bind to it.

So, it can be used in detection of genetic diseases, malignancy cases, toxicity and
therapeutic drug monitoring.

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5.0 CONCLUSION

Today, a lot of tests available to monitor toxicity cases occur in our society. So, with the variety
of high technology molecular tests developed, it hope can give benefits to human being in efforts
to increase the society health level. Besides that, the tests developed also can be used in
therapeutic drug monitoring which can screen and identify the effectiveness of drugs given to
patients and at the same time, to control the adverse effects of drugs.

As we can see here, polymerase chain reaction (PCR) plays a large role in determining
the diseases since it is can amplify the amount of DNA fragments. The copies of the fragments
produced by PCR are then used to run other tests for confirmation.

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6.0 REFERENCES

6.1 Books

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iii. Barbara, H.E & Anna, P.R . (2008) . Basic Clinical Laboratory Techniques . 5th Edition .
United State of America : Thomson Delmar Learning.

iv. W, Kamil . (2007) . Biochemistry : Lecture Notes . Malaysia : University Teknologi

MARA.

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 v. Andy, V . (08 November 1999) . Principle of the PCR . Retrieved on 11 April 2009 from
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vi. Richard A. G . (2009) . Polymerase Chain Reaction . Microsoft Encarta Online

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 xv. Protein Electrophoresis - Immunofixation Electrophoresis . (2009) . American
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