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Molecular diagnostics: nucleic acid and protein based diagnostic methods
Genomics- the branch of molecular biology concerned with the structure, function, evolution,
and mapping of genomics using methods such as next generation sequencing and
fluorescent in situ hybridization
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Personalized medicine
Personalized medicine is based on using an individual's genetics profile to make the best
therapeutic choice by facilitating predictions about whether that person will benefit from a
particular medicine or suffer serious side effects
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Medical decision making based
on empirical data relies on the law of averages, whereas personalized medicine recognizes
the differences between individual patients
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In the first
step, ourines will react with dimethyl sulfate and pyrimidines will react with hydrazine in such
a way as to break the glycosidic bond between the ribose sugar and the base, displacing the
base
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The use of these elective reactions to DNA sequencing then
involved creating a single-stranded DNA substrate carrying a radioactive label on the 5’ end
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The
reactions are then loaded on high percentage polyacrylamide gels and the fragments
resolved by gel electrophoresis
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Wherever a labelled fragment stopped on the gel the radioactive tag would expose the film
due to particle decay-autoradiography
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Limitations of the
maxam-gilbert's method include the use of large amounts of radioactive material and the
neurotoxin hydrazine
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A
DNA primer is designed to be a starting point for DNA synthesis on the strand of DNA to be
sequenced, four individual DNA synthesis reactions are performed
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The four reactions can be named A, G, C and T according to which of the four
ddNTPs was included
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This is because the ddNTP molecule lacks a 3’ hydroxyl group, which is required
to form a link with the next nucleotide in the chain
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Following synthesis, the
products of the A, G, C and T reactions are individually loaded into four separate lanes of a
single gel and separated using gel electrophoresis, a method that separates DNA fragments
by their sizes
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, including bands in all four lanes
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Automated fluorescence sequencing
Leory Hood introduced an automated sequencing method using fluorescence-labelled
dideoxy-terminators
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Once the reactions were complete, the four reactions were
pooled and run together in one lane of a polyacrylamide sequencing gel
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The
fluorescence signature of each fragment was then sent to a computer where the software
was trained to perform base calling
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Each of the four
dideoxynucleotide chain termination is labelled with fluorescent dyes, each of which with
different wavelengths of fluorescence and emission
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SNPs are the smallest
unit of genetic variation
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SNPs occur
approximately once every 100-300 bases
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SNPs can occur in both coding and non-coding regions of the
genome
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Genetic variation and Alzheiers disease
The APOE gene is located on chromosome 19 and comprises four exons and three introns,
totallying 3597 base pairs
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Both dysfunctional
cholesterol processing and amyloid beta aggregation have been implicated in Alzheimer;s
disease risk
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APOE 2 is relatively rare and may provide protection against AD, if AD occurs in an
individual with this allele, it usually develops later in life than it would in someone with the
allele APOE 4
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The POE 4
allele is one of the strongest genetic risk factors for AD, and is associated with an earlier age

of disease onset
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PAOE 4 is linked to episodic
memory-related dysfunction in the medial temporal lobe (MTL), one of the earliest brain
regions impacted by the progression of AD
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The
APOE risk gene is associated with inflammation/microglia related pathways
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The human genome project
The human genome project (HGP) was an international scientific research project that aimed
to; identify all the genes in the human DNA, sequence the 3 billion chemical base pairs of
the human DNA, store this information in databases, improve tools for data analysis, transfer
related technologies to the private sector, and address possible ethical, legal and social
issues
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The current genome sequence
contains 2
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It covers 99% of the
euchromatic genome and is accurate to an error rate of 1 event per 100 000 bases
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The near complete sequence, the first for a vertebrate,
greatly improves the precision of biological analyses of the human genome including studies
of gene number, birth and death
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The genome sequence serves as a firm foundation for biomedical
research
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A reference genome is often assembled from DNA sequences from several donors,
but reference genomes do not accurately represent the genetic code of any single individual
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Levy, et al
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The diploid genome sequence
was produced from approximately 32 million random DNA fragments, sequenced by sanger
dideoxy technology and assembled into 4528 scaffolds, comprising 2810 million bases (Mb)
of contiguous sequence with approximately 7
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Comparison of this genome with previous reference genomes sequences, which were
composites comprising multiple humans, revealed more than 4
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3 Mb
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non-SNP DNA variation accounts for 22% of all events identified in the
donor, however they involve 74% of all variant bases
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Moreover, 44% of
genes were heterozygous for one or more variants
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Next generation DNA sequencing
The major advance offered by next generation sequencing (NGS) technologies is the ability
to produce in some cases, in excess of one billion short reads prt instrument run, which
makes them useful for many biological applications
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The leading NGS platforms use clonally
amplified templates, which are not affected by the arbitrary losses of genomic sequences
that are inherent in bacterial cloning methods
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PXRr can create
mutations that masquerade as sequence variants and amplification bias that
underrepresents AT-rich and GC-rich regions in target sequences
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To call sequence variants in genomes, NGS
reads are aligned to a reference sequence using various bioinformatics mapping tools
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NGS costs will continue to drop in the foreseeable future, although cost
reduction should be weighed against the quality of the produced genome sequence
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In the first step, a
DNA polymerase, bound to the primed template, adds or incorporates just one fluorescently
modified nucleotide, which represents the complement of the template base
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following incorporation, the remaining unincorporated nucleotides are washed away
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This is followed by a
cleavage step, which removes the terminating/inhibiting group and the fluorescent dye
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Helicos
Biosciences uses a one colour-CRT cycle
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Sequencing by ligation (SBL) is another cyclic method that differs from CRT in its use of
DNA ligase and either one-base0 encoded probes or two-base-encoded probes
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DNA ligase is then added to join the dye-labelled probe to the primer
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The cycle can be repeated either by using cleavable probes to
remove the fluorescent dye and regenerate a 5’ phosphate group for subsequent ligation
cycles, or by removing and hybridizing a new primer to the template
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Pyrosequencing manipulates DNA polymerase by the single
addition of a dNTP in limiting amounts
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The order and intensity of

light peaks are recorded as flowgrams, which reveal the underlying DNA sequence
pyrosequencing is utilised by Roche-454
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Single DNA polymerase molecules are attached to the
bottom surface of individual zero-mode waveguide detectors that can obtain sequence
information while phospho linked nucleotides are being incorporated into the growing primer
strand
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Pharmacogenomics
In pharmacogenomics, genomic information is used to study an individual’s responses to
drugs
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Multigene analysis of whole genome SNP profiles can be used to
assess gene variants affecting an individual’s drug response
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Pharmoacokinetics encompassess
four processes; absorption (how a drug enters the bloodstream), distribution (where the drug
travels after absorption and how much drug reaches the target site), metabolism (how the
drug is broken down in the body), and excretion (how the drug leaves the body)
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Genetic variation in genes
encoding drug-metabolising enzymes, drug receptors, and drug transporters have been
associated with individual variability in the efficacy and toxicity of drugs
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Predicting
serious adverse drug reactions is a priority for pharmacogenomic research
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Molecular cloning and characterization studies of the gene that codes for this enzyme
have described more than 70 variant alleles
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Individuals who are
homozygous or heterozygous for the wild-type or normal activity enzymes (75-85% of the
population are called extensive metabolizers, intermediate (10-15%) or poor (5-10%)
metabolisers are carriers of two alleles that decrease enzyme activity and ultrarapid
metabolizers (1010%) are carriers of duplicated genes
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Polymerase chain reaction
PCR is the process of preparation of complementary DNA (cDNA) from RNA
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This cycle is
repeated 20-40 times in a thermal cycler
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PCR also
provides a method to identify bacteria, particularly clinically important pathogen species that

are proved to be not cultivable via standard diagnostic protocols, for example H
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Fluorescence in situ hybridization
The process of FISH is as follows; labelling DNA-probe complementary with the target gene
sequence, denaturation of both the probe and the target DNA sequence, annealing probe
with the target gene sequence, and then detection with a fluorescence detection system
uses the nucleotide probe is directly labelled with a fluorophore
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The Philadelphia chromosome
is present in over 95% of cases of chronic myeloid leukaemia (CML)
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This aberrant
fusion gene encodes the breakpoint cluster region-proto-oncogene tyrosine-protein kinase
(BCR-ABL1) oncogene protein with persistently enhanced tyrosine kinase activity
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Each BCR-ABL1 transcript is present in a distinct
leukemia phenotype, which predicts both response to therapy and clinical outcome
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FISH is also used to determine whether breast cancer is HER2 positive
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25% of breast
cancers are HER2 positive
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DNA microarray
DNA microarrays are a group of technologies in which specific DNA sequences are either
deposited or synthesised in a 2D or 3D array on a surface in such a sway that the DNA is
covalently or noncovalently attached to the surface
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By generalising to a very large number of spots of DNA, an
array can be used to quantify a large number of different nucleic acid sequences in solution
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Proteome- the complete set of proteins expressed by an organism, tissue, or cell
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The HPP aims to determine; protein
abundance, distribution, and subcellular localization; protein interactions with other
biomolecules; and protein functions at specific time points
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Immunohistochemistry

The method of detecting a primary antibody that is bound to the antigen of interest in an
immunohistochemistry (IHC) experiment can be either direct or indirect
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In direct detection methods, the
primary antibody is directly conjugated to a label
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For direct detection, the primary antibody can be conjugated to
an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphate (AP), or a
fluorochrome
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Another benefit of direct detection is increased flexibility
in the design of multi colour experiments, given the wide range of fluorochromes that are
available
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Signal amplification
occurs through the potential for two or more labelled secondary antibodies to bind to each
primary antibody
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Immunostaining of breast cancer tissues can detect HER2 presence
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Flow cytometry also
simultaneously measures the light that is scattered by each cell
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The amount of light scattered
[perpendicular to the laser light beam provides information about the presence or absence of
intracellular membranous structures
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In a FACS instrument, cells that bear the
desired combination of light-scattering properties and fluorescent markers are tagged with
an electrical charge, and can be deflected into a separate pot for further analysis
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when the number of CD4+ T-cells in the blood of a patient with AIDS falls below a
certain level, the patient is at high risk for opportunistic infections
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ELISAs can be used
qualitatively to detect the presence of antibody or antigen, alternatively ,a standard curve
based on known concentration of a sample
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Biomarker- a characteristic that is objectively measured and evaluated as an indicator of
normal biologic processes, pathogenic processes, or pharmacologic responses to a
therapeutic intervention
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PSA is secreted by the prostate epithelium in normal, benign, and cancerous
conditions
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PSA levels can
be used to monitor the progression of prostate cancer
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PSA
serum levels are elevated in patients with prostate cancer, benign prostatic hypertrophy and
inflammatory conditions, such as prostatitis
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31% of men with PSA levels 4-10 ng/ml go on to develop
prostate cancer
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Mass spectroscopy
Mass spectroscopy is an analytical tool useful for measuring the mass-to-charge ratio of one
or more molecules present in a sample
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Mass spectrometry-based proteomics allows quantitative
system-wide analysis of the proteome, including post-translational modifications,
protein-protein interactions and cellular localization
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With the availability of genomic
information the massive capacity for peptide identification by mass spectroscopy is being
used to annotate gene sequences and to find new protein-coding genes and splicing
variants
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Protein affinity strategies allow purification
of candidate proteins and their interacting partners, which are subsequently identified by MS
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The improvements in sensitivity, robustness and high-throughput of
MS based proteomics now permits applications in the clinical field, including the possibility of
discovering disease-related biomarkers and screening molecular targets of candidate drugs

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