Continuous Assessment

NAME- Debapriya Hazra; Roll No.-218

1.
Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the
English scientist Alec Jeffreys (whereas, Lalji Singh is called father of Indian DNA
fingerprinting or DNA profiling or DNA typing). It is used for the analysis of unique patterns
in DNA fragments in order to genetically differentiate between organisms – these patterns are
called Variable Number of Tandem Repeats (VNTRs). RFLP technique is used in DNA
fingerprinting study as well as in resolving a paternity conflict. The RFLP technique exploits
the differences in DNA sequences to recognize and study both intraspecies and interspecies
variation.
Principle
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:
1.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.
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

-so, in this way via using RFLP technique we can determine or confirm the source of a DNA
sample such as in paternity tests or criminal investigations.
2.
Gene expression is a highly regulated mechanism that controls the function and adaptability
of all living cells including prokaryotes and eukaryotes. Several techniques exist for studying
and quantifying gene expression and its regulation. The field of gene expression analysis has
undergone major advances in biomedical research. Traditional methods focused on
measuring the expression of one gene at a time. However, today, mRNA expression
techniques have led to improvements in gene identification and disease sub-classification.
Gene expression techniques: Analytical methods may be used to examine mRNA expression
levels or differential mRNA expression. Some examples of these techniques are listed below.
Techniques having less to mid impactness
Reporter gene
A gene contains two functional segments. One is a coding DNA sequence, which contains the
instructions for making a protein. The other is a DNA sequence called a promoter, which is
linked to this coding region and regulates the gene’s transcription, either by activating or
suppressing its expression. A reporter gene assay is used to determine the regulatory potential
of a DNA sequence that is unknown. This involves a promoter sequence being linked to a
detectable reporter gene such as luciferase, β-galactosidase or β-glucuronidase. Examples of
methods used to determine the expressed reporter gene protein are fluorescence, absorbance
and luminescence.
Northern blotting
This is a technique used to detect specific RNA molecules present within an RNA mixture.
Northern blotting is employed in the analysis of an RNA sample from a cell type or tissue so
as to determine the RNA expression of certain genes.
Western blotting
Western blotting is a technique for detecting specific protein molecules within a protein
mixture. This mixture might include all the proteins that are associated with a certain cell
type or tissue. The technique can help to determine a protein’s size, and how much of it is
expressed.
Fluorescent in situ hybridization (FISH)
This is a cytogenetic technique that can be used to identify and locate specific gene
sequences. FISH can be used to visualize copy number aberrations such as the deletion,
translocation or amplification of chromosomes. The technique is used in prenatal diagnosis
and also provides a useful tool in the diagnosis and predicted prognosis of various sarcomas.
The technique is also used in dermatology to help evaluate atypical moles.
Reverse transcription polymerase chain reaction (RT-PCR)
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

This is the most sensitive technique available for detecting and quantifying mRNA. Using
RT-PCR, extremely small sample sizes can be used in the quantification of mRNA and the
technique can in fact do this using just a single cell.

Techniques having higher impactness-

Serial Analysis of Gene Expression (SAGE)
SAGE is a technique used to create a library of short sequence tags which can each be used to
detect a transcript. The expression level of the transcript can be determined by assessing how
many times each tag is detected. This technology enables comprehensive expression analysis
across the genome.
DNA microarray
Also known of as biochip or DNA chip, a DNA microarray is a solid surface to which a
collection of microscopic DNA spots are attached. The microarrays are used to determine
expression levels across a large number of genes or to perform genotyping across different
regions of a genome.
RNA Seq
This refers to methods used to measure the sequence of RNA molecules. Examples include
shotgun sequencing of cDNA molecules acquired from RNA through reverse transcription
and technologies used to sequence RNA molecules from a biological sample so that the
primary sequence and abundance of each RNA molecule can be determined.
Tiling arrays
A tiling array is a type of microarray chip, with labelled DNA or RNA targets hybridized to
probes attached to a solid surface. However, the probes used differ to those used with
traditional microarrays. Rather than known sequences or predicted genes being probed, tiling
arrays probe for sequences known to be present in a contigious region. This can provide
information about regions that are sequenced but for which the local functions are largely
unknown.

3.
Molecular diagnostics provide a powerful method to detect and diagnose various neurological
diseases such as Alzheimer's and Parkinson's disease. The confirmation of such diagnosis
allows early detection and subsequent medical counseling that help specific patients to
undergo clinically important drug trials. This provides a medical pathway to have better
insight of neurogenesis and eventual cure of the neurodegenerative
diseases.Neurodegenerative disorders correspond to the disorders in the central nervous
system that are characterized by the progressive loss of neural tissues.
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

Behavioral symptoms can be utilized for the pre-mortem diagnosis of neurodegenerative

disorders. However, the major drawback of behavioral symptoms-based diagnosis is its
limitations to identify patients early in the course of their disease, when the pharmacological
intervention can significantly prevent further progression of the disease, if detected early. For
example, well-established behavioral tests like the ADAS-Cog that are regarded as the “gold-
standard” for AD diagnosis may give false-negative results for patients with mild symptoms.

To overcome these diagnostics challenges, current neuro-pathologic methods have been

combined with molecular biology techniques that have led to increased understanding of
neurodegenerative disorders along with biologically based classifications of these disorders.
Molecular diagnostics provide a powerful tool in the diagnosis of many neurological
diseases. For example, genetic testing of mutations in disease-causing-genes has been
leveraged to define and classify many of the heterogeneous inherited neurodegenerative
syndromes. Changes in pathologies, biochemistries and genetics of patients can give us
comprehensive information regarding the nature of a particular disease. However, molecular
testing may be performed only after careful consideration and a genetic counseling
Molecular Diagnostics Of AD by Using Genetic Biomarkers:
Less than 5% of all cases of AD can be accounted for by mutations in the following three
genes. Amongst them, mutations on the two homologous presenilin genes such as presenilin
1 and presenilin 2, are most common and are responsible for over half of the known familial
AD cases, whereas mutations in the gene for amyloid precursor protein are comparatively
less. The presenilin genes code for proteins known as presenilin, which control the APP
proteolysis into smaller peptides. An abnormal increase in the activity of APP can be due to
any missense mutation on one of these presenilin genes resulting into more Aβ peptides. The
first genetic mutation linked to AD was found on the βAPP gene. This βAPP gene, encodes a
glycosylated trans-membrane protein which contains 770 amino acids in its longest isoform.
This was confirmed by the fact that patients with Down's syndrome also developed similar
plaques and suffered Alzheimer encephalopathy in their later years. In addition to the
mutations mentioned above, which can cause AD, the E4 allele of the ApoE is associated
with the sporadic forms of AD. Although E4 allele was detected in about 40–50% of all AD
patients, but could not serve as a diagnostic marker based on the sensitivity criteria for
biomarkers. Therefore, ApoE is regarded as a risk factor indicator rather than an actual
genetic marker of AD. Along with positive family history, an early onset (in the 40s and 50s)
which is common to all these monogenic forms, should act as an indication for molecular
genetic diagnosis.

4.
Discovering new drugs has never been a simple matter. From ancient times to the beginning
of the nineteenth century, treatment for illness or disease was based mainly on folklore and
traditional curative methods derived from plants and other natural sources. but now a days
with the help of recombinant DNA technology the pharmacy industry nourished a lot and for
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

that the death toll is also reduced. The commercial exploitation of recombinant DNA
(rDNA) technology began in late 1970s by biotechnological companies to produce proteins
Recombinant DNA technology involves using microorganisms, macroscopic organisms, or
hybrids of tumor cells and leukocytes:
 to create new pharmaceuticals;
 to create safer and/or more effective versions of conventionally produced
pharmaceuticals; and also
 to produce substances identical to conventionally made pharmaceuticals more cost-
effectively than the latter pharmaceuticals are produced.
Recombinant DNA technology enables modifying microorganisms, animals, and plants so
that they yield medically useful substances, particularly scarce human proteins (by giving
animals human genes, for example).
The pharmaceutical products of rDNA technology are broadly divided into following three
types
1. Human protein replacements
2. Therapeutic agents for human diseases
3. Vaccines

rDNA Product Trade name Application / Uses

Insulin Humulin Diabetes

Growth hormone Protropin/Humatrope Pituitary dwarfism

Interferon Intron A Hairy cell leukemia

Recombinax HB/
Hepatitis B vaccine Hepatitis B
Engerix

TissuEplasminogen
Activase Myocardial infarction
activator

Factor vIII Kogenate/Recombinate Hemophilia

Dnase Pulmozyme Cystic fibrosis

Severe anemia with

Erythropoietin Epogen/rocrit
kidney damage

-Besides all Recombinant protein vaccines & DNA vaccines plays a great role in pharma
industry.
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

Recombinant DNA Technology is playing very important role in revolutionizing

medicinei.e., enabling mass production of safe, pure, more effective versions of biochemicals
that human body produces naturallyexamples includes a variety of products such as
hormones, Therapeutic proteins, Vaccines and various Enzymes and also to create new
pharmaceuticals.In this way biotech pharmaceuticals become unprecedented preventers and
relievers of human suffering.