ASSIGNMENT #_1

TITLE: BIOLOGICAL MARKER

SEMESTER: 7

SUBJECT CODE : BNBM 2293

SUBJECT TITLE : MOLECULAR BIOLOGY II

PROGRAMME : BACHELOR OF BIOMEDICAL SCIENCES

(HONS)

STUDENTS NAME : REENA JOANNE A/P ROMADASS

ID NO. : 201606040009

LECTURER NAME : DR. SUHAILI MUSTAFA

LEARNING CENTRE : PETALING JAYA

INSTRUCTIONS TO STUDENTS

1) Answer the question based on the guideline given.

2) Plagiarism in all forms is forbidden. Students who submit plagiarised assignment will
be penalised.

3) This assignment carries a ____10____% weightage toward final grade.

Content Page

1.0 Introduction 3

2.0 Types of Biological Marker

2.1 CARDICA BIOMARKER 4

2.2 TUMOUR BIOMARKER 4

2.3 GENOMIC BIOMARKER 5

3.0 Function of Biological Marker 6

4.0 Principle of Biological Marker 7

5.0 Methodology and Techniques of Biological Marker 8

5.1 Creatine Kinase Assay Of Cardiac Biomarkers 8

5.2 Carcinoembryonic Antigen Test (Cea) 10
5.3 (RFLP) TECHNIQUE 11

6.0 Conclusion 12

7.0 Reference 13
 1.0 INTRODUCTION

Biomarkers are biological measures of a biological state. Its characteristic that is

objectively measured and evaluated as an indicator of normal biological processes,
pathogenic processes or pharmacological responses to a therapeutic intervention.
Biomarkers are the measures used to perform a clinical assessment such as blood
pressure or cholesterol level and are used to monitor and predict health states in
individuals or across populations so that appropriate therapeutic intervention can be
planned. Biomarkers may be used alone or in combination to assess the health or
disease state of an individual. A wide range of biomarkers are used today. Every
biological system for example the cardiovascular system, metabolic system or the
immune system has its own specific biomarkers. Many of these biomarkers are relatively
easy to measure and form part of routine medical examinations. An ideal biomarker has
certain characteristics that make it appropriate for checking a particular disease
condition. Ideally, an ideal marker should have the following features:

Safe and easy to measure

Cost efficient to follow up
Modifiable with treatment
Consistent across gender and ethnic groups

Biomarkers are used to predict serious illnesses such as diabetes and cardiovascular
disease. Each individual biomarker indicates whether there is a disease or health state and
can be combined to provide a detailed picture of how healthy a person is and whether or not
a diagnosis needs to be made. The principles of biomarkers in disease have been applied to
the detection, screening, diagnosis, treatment and monitoring of cancer. Traditionally, anti-
cancer drugs were agents that killed both cancer cells and healthy cells. However, more
targeted therapies have now been developed that can be directed to kill cancer cells only,
while sparing healthy cells. The assessment of a typical biomarker in cancer helps in the
development of therapies that can target the biomarker. This can minimize the risk of toxicity
and reduce the cost of treatment. In cancer research, genetic studies are valuable because
genetic abnormalities so often underlie the development of cancer. Certain DNA or RNA
markers may therefore help in the detection and treatment of specific cancers.
2.0 TYPES OF MARKERS

2.1 CARDIAC MARKER

Cardiac biomarkers are substances that are released into the blood when the heart is
damaged or stressed. Measurements of these biomarkers are used to help diagnose acute
coronary syndrome (ACS) and cardiac ischemia, conditions associated with insufficient
blood flow to the heart (Sans S, Kesteloot H, Kromhout D-1997). Tests for cardiac
biomarkers can also be used to help determine a person's risk of having these conditions or
to help monitor and manage someone with suspected ACS and cardiac ischemia.Cardiac
markers are used in the diagnosis and risk stratification of patients with chest pain and
suspected acute coronary syndrome (ACS). The cardiac troponins, in particular, have
become the cardiac markers of choice for patients with ACS. These is recommended that
cardiac biomarkers should be measured at presentation in patients with suspected MI, and
that the only biomarker that is recommended to be used for the diagnosis of acute MI at this
time is cardiac troponin due to its superior sensitivity and accuracy.

2.2 TUMOUR MARKER

Tumor markers are substances that are produced by cancer or by other cells of the body in
response to cancer or certain benign (noncancerous) conditions. Most tumor markers are
made by normal cells as well as by cancer cellshowever, they are produced at much higher
levels in cancerous conditions (Sturgeon C, Hammond E-2006). These substances can be
found in the blood, urine, stool, tumor tissue, or other tissues or bodily fluids of some
patients with cancer.An elevated level of a tumor marker can indicate cancer; however, there
can also be other causes of the elevation which is false positive values.Tumor markers can
be produced directly by the tumor or by non-tumor cells as a response to the presence of a
tumor.Most tumor markers are proteins. However, more recently, patterns of gene
expression and changes to DNA have also begun to be used as tumor markers.Many
different tumor markers have been characterized and are in clinical use. Some are
associated with only one type of cancer, whereas others are associated with two or more
cancer types( Schrohl AS, Anderson MH-2003). No universal tumor marker that can detect
any type of cancer has been found.There are some limitations to the use of tumor markers.
Sometimes, noncancerous conditions can cause the levels of certain tumor markers to
increase. In addition, not everyone with a particular type of cancer will have a higher level of
a tumor marker associated with that cancer. Moreover, tumor markers have not been
identified for every type of cancer.
 2.3 GENOMIC BIOMARKERS

Historically, successful genetic markers have been linked to single effects for both the
patient and the patients family for example cystic fibrosis testing, spinal muscular atrophy
testing and to monitor particular effects on large populations for example HIV mRNA, HCV
mRNA. Reproducible accessible genomic biomarkers are of diagnostic value, and may lead
to identification of causal factors. They therefore can be used clinically to screen for
diagnose, to monitor the activity of diseases, and also may be useful to guide molecularly
targeted therapy and personalised regimens or to assess therapeutic response. The
importance and the potential utility of GB has been recognized by substantial public and
private funding, and GB discovery efforts are now commonplace in both academic and
industrial settings (Brown MS, Goldstein JL-1976). For years GBs have been used to test for
single gene mutations predisposing to disease. Examples of these include familial
hypercholesterolemia and mutations in LDL receptors, high blood pressure, hyperkalemia
and mutations in WNK kinase genes and many other monogenic disorders. However, while
GBs give diagnosis, causality and pathophysiology information in monogenic disorders, they
seem to have a scarce or no detectable impact on polygenic and complex disorders.
Complex disorders are caused by multiple genetic and environmental factors, are
characterized by high population prevalence, lack of clear Mendelian patterns of
transmission, etiologic and phenotypic heterogeneity, and are involved in a continuum
between disease and non disease states (Wilkinson GR.-2005). Another complicating factor
in developing biomarkers for complex traits is the difficulty in understanding the role these
genetic factors play in the pathophysiology of disease. The identification of key genes
influencing complex traits such as heart disease, diabetes, and cancer will not only assist in
predicting those individuals who are predisposed to disease, but may potentially have
significant impact on the pharmaceutical industry as the identification of GBs for disease
susceptibility may lead to new targets, better classification of disease and more informed
clinical development (McCamish M, Izumi R-2006).
3.0 FUNCTION OF BIOLOGICAL BIOMARKERS

Biological markers is a molecular alterations that are measurable in biological media such as
human tissues, cells, or fluids. More recently, the functions has been broadened to include
biological characteristics that can be objectively measured and evaluated as an indicator of
normal biological processes, pathogenic processes, or pharmacological responses to a
therapeutic intervention. In practice, biomarkers include tools and technologies that can aid
in understanding the prediction, cause, diagnosis, progression, regression, or outcome of
treatment of disease. For the nervous system there is a wide range of techniques used to
gain information about the brain in both the healthy and diseased state. These may involve
measurements directly on biological media for example blood and cerebrospinal fluid or
measurements such as brain imaging which do not involve direct sampling of biological
media but measure changes in the composition or function of the nervous system.
4.0 PRINCIPLE OF BIOLOGICAL BIOMARKERS

There are two major types of biomarkers: biomarkers of exposure, which are used in risk
prediction, and biomarkers of disease, which are used in screening and diagnosis and
monitoring of disease progression. Biomarkers used in risk prediction, in screening, and as
diagnostic tests are well established, and they offer distinct and obvious advantages. The
classification of many neurological diseases is based on either standardized clinical criteria
or histological diagnoses. Biomarkers also have the potential to identify neurological disease
at an early stage, to provide a method for homogeneous classification of a disease, and to
extend our knowledge-base concerning the underlying disease pathogenesis. These
advantages have direct application to all types of clinical investigation, from clinical trials to
observational studies in epidemiology (Rohlff C-2003). In epidemiological, investigations,
biomarkers improve validity while reducing bias in the measurement of exposures for
neurological disease. Rather than relying on a history of exposure to a putative risk factor,
direct measurement of the level of exposure or the chromosomal alteration resulting from the
exposure lessens the possibility of misclassification of exposure. Such misclassifications not
only produce inaccurate and deceptive results but also reduce the power of studies to detect
health effects. Thus, the use of biomarkers improves the sensitivity and specificity of the
measurement of the exposures or risk factors. Molecular biomarkers have the additional
potential to identify individuals susceptible to disease. Molecular genetics have already had
an impact on neurological practice, leading to improved diagnosis. Classification of
populations in terms of the degree of susceptibility on the basis of such biomarkers produces
greater accuracy than relying on historical definitions of susceptibility. For example, a
biomarker will allow the stratification of a population on the basis of a specific genotype
associated with a disease rather than relying on a report of the family history of the
disease. The ability to quantify susceptibility in this way can be an extremely important
method for estimating disease risk among various populations (Muller U, Graeber MB-1996).
5.0 METHODOLOGY AND TECHNIQUES

5.1 CREATINE KINASE ASSAY OF CARDIAC BIOMARKERS

Method: Creatine kinase activity is determined in a coupled enzyme system utilizing

pyruvate kinase (PK) and lactate dehydrogenase (LDH). The following procedure is
essentially that described by Tanzer and Gilvarg (1959). One Unit is defined as the
conversion of one micromole of creatine to creatine phosphate per minute at 25C and pH
8.9 under the specified conditions.

Reagents

Reagent solution -- the required amount of solution should be prepared containing:

ATP 8.5 mM

NADH 1.22 mM

PEP 2.0 mM

LDH 15.0 u/ml

PK 7.0 u/ml

MgSO4 28.0 mM

Glutathione (reduced) 26.0 mM

pH adjusted to 7.4

Buffered Creatine: 0.40 M Glycine containing 53.2 mM creatine and 62 mM

potassium carbonate. Adjust the pH to 8.9 with NaOH.

Enzyme diluent: Freshly prepared 5 mM glycine pH 9.0

Enzyme

Prepare mg/ml in 5 mM glycine, pH 9.0.

Dilute to 0.1-10 g/ml in glycine buffer. (mg protein/ml = A280/ml x 1.142

Procedure

Adjust spectrophotometer to 25C and 340 nm. Pipette into each cuvette as follows:

Reagent solution 0.7 ml

Buffered creatine 2.2 ml

Incubate in spectrophotometer at 25C for 3-5 minutes to achieve temperature equilibrium

and establish blank rate, if any. Add 0.1 ml diluted enzyme and record decrease in A340 for 5-
8 minutes. An initial lag period may occur. Determine A340 from linear portion of the curve.

Calculation

Figure 1 : shows the example of the technique

 5.2 CARCINOEMBRYONIC ANTIGEN TEST (CEA)

The CEA test is a blood test performed by blood is usually drawn from a vein in patients
arm. The blood draw process, or venipuncture, usually involves the following steps:

A healthcare provider will clean the puncture site with an antiseptic. The site is usually
in the middle of patients arm, on the opposite side of the elbow.

A healthcare provider will wrap an elastic band around the upper arm to help make the
vein fill up with blood.

A needle is then inserted into the vein to collect blood into an attached vial or tube.

The band is unwrapped from patient arm.

A laboratory will analyze the blood sample.

A CEA test can help doctor find out if a cancer treatment is working. These treatments may
have included surgery, chemotherapy, radiation, or a combination of all three. Your doctor
might also use the CEA test to help determine if a cancer has come back, or recurred, after
finishing treatment. A CEA test is most useful after a diagnosis of a type of cancer thats
known to produce CEA. Not all cancers produce CEA. Increased levels of CEA may be
found in the following cancers:

breast cancer

cancer of the gastrointestinal tract

lung cancer

ovarian cancer

pancreatic cancer

The carcinoembryonic antigen (CEA) test measures the amount of this protein that may appear
in the blood of some people who have certain kinds of cancers.
 5.3 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) TECHNIQUE

Restriction endonucleases are enzymes that cut lengthy DNA into short pieces. Each
restriction endonuclease targets different nucleotide sequences in a DNA strand and
therefore cuts at different sites. The distance between the cleavage sites of a certain
restriction endonuclease differs between individuals. Hence, the length of the DNA
fragments produced by a restriction endonuclease will differ across both individual
organisms and species. RFLP is performed using a series of steps briefly outlined below:-

DNA Extraction - To begin with, DNA is extracted from blood, saliva or other
samples and purified.

DNA Fragmentation - The purified DNA is digested using restriction endonucleases.

The recognition sites of these enzymes are generally 4 to 6 base pairs in length. The
shorter the sequence recognized, the greater the number of fragments generated
from digestion. For example, if there is a short sequence of GAGC that occurs
repeatedly in a sample of DNA. The restriction endonuclease that recognizes the
GAGC sequence cuts the DNA at every repetition of the GAGC pattern. If one
sample repeats the GAGC sequence 4 times whilst another sample repeats it 2
times, the length of the fragments generated by the enzyme for the two samples will
be different.

Gel Electrophoresis - The restriction fragments produced during DNA fragmentation

are analyzed using gel electrophoresis. The fragments are negatively charged and
can be easily separated by electrophoresis, which separates molecules based on
their size and charge.

The fragmented DNA samples are placed in the chamber containing the
electrophoretic gel and two electrodes. When an electric field is applied, the
fragments migrate towards the positive electrode. Smaller fragments move faster
through the gel leaving the larger ones behind and thus the DNA samples are
separated into distinct bands on the gel.

Visualization of Bands - The gel is treated with luminescent dyes in order to make
the DNA bands visible.
6.0 CONCLUSION

Biomarkers of all types have been used by generations of epidemiologists, physicians, and
scientists to study human disease. The application of biomarkers in the diagnosis and
management of cardiovascular disease, infections, immunological and genetic disorders,
and cancer are well known. Their use in research has grown out of the need to have a more
direct measurement of exposures in the causal pathway of disease that is free from recall
bias, and that can also have the potential of providing information on the absorption and
metabolism of the exposures. Neuroscientists have also relied on biomarkers to assist in the
diagnosis and treatment of nervous system disorders and to investigate their cause. Blood,
brain, cerebrospinal fluid, muscle, nerve, skin, and urine have been employed to gain
information about the nervous system in both the healthy and diseased state. Many studies
using biomarkers never achieve their full potential because of the failure to adhere to the
same rules that would apply for the use of variables that are not biological. The development
of any biomarker should precede or go in parallel with the standard design of any
epidemiological project or clinical trial. In forming the laboratory component, pilot studies
must be completed to determine accuracy, reliability, interpretability, and feasibility. The
investigator must establish normal distributions by important variables such as age and
gender. The investigator will also want to establish the extent of intraindividual variation,
tissue localization, and persistence of the biomarker. Moreover, he or she will need to
determine the extent of interindividual variation attributable to acquired or genetic
susceptibility. Most, if not all of these issues can be resolved in pilot studies preceding the
formal investigation.
7.0 REFERENCE

Hulka BS. Overview of biological markers. In: Biological markers in epidemiology (Hulka BS,
Griffith JD, Wilcosky TC, eds), pp 315. New York: Oxford University Press,
1990

Blanke H, von Hardenberg D, Cohen M. et al. Patterns of creatine kinase during acute
myocardial infarction after nonsurgical reperfusion: comparison with conventional
treatment and correlation with infarct size. J Am Coll Cardiol. 1984;3:675 80.

Muller U, Graeber MB. Neurogenetic diseases: molecular diagnosis and therapeutic

approaches. J Mol Med 74: 7184, 1996.

Perera FP, Weinstein IB. Molecular epidemiology: recent advances and future
directions. Carcinogenesis 21: 517524, 2000.

Morelli RL, Carlson JC, Emilson B. et al. Serum creatine kinase MM isoenzyme sub-bands
after acute myocardial infarction in man. Circulation. 1983;67:128389.