The Advent of Molecular Diagnostic tools

Carranza, Maricarl
De Guzman, Liyah
De Leon, Ron Angelo
Derecho, Elaine
Sayson, Jaira

FISH technology

The FISH technology, an acronym for fluorescence in situ hybridization, describes a very powerful
technique that allows a direct visualization of genetic alterations on a cell-by-cell basis. This was
developed in the early 1980’s and its applications of the FISH assay have been on the rise since
the 1990’s. It also contributes on chromosome preparations of peripheral blood, uncultured blood
or bone marrow, blood or bone marrow smears, epithelial cells from buccal smears and urine, hair
root cells, human sperm cells, tissue sections, nucleus extractions, and even used on zoology and
virology. This is one of the laboratory techniques for detecting and locating a specific DNA
sequence on a chromosome. The technique relies on exposing chromosomes to a small DNA
sequence called a probe that has a fluorescent molecule attached to it.

Emphasize the principles of technology

This technology are either in the form of gains or losses of segments of chromosome materials,
but its aim is to demonstrate either imbalances and demonstrate specific breakpoints with or
without imbalance. Since fluorescence hybridization initially was introduced for chromosome
classification (Pinkel et al. 1986) the technique has been adopted in an ever-increasing range of
applications in both medicine and biology and almost 10,000 articles have now been published
utilizing FISH technology. The present review will give an outline of the principles of this
technique and its use in different areas of human disease and in research, with special emphasis on
FISH as a diagnostic tool in chromosome and genome analysis within clinical genetics and
oncology.

The technique of this technology, Fluorescence in situ hybridization (FISH), is altered in the aspect
of the useful genome research and molecular diagnostics. Any are detected, or can be detected, by
hybridizing probes to naked DNA fiber even the e.g. deletion of few kilobases. The techniques
exploiting FISH modifications and/or latest innovative techniques are Forward and Reverse
Chromosome Painting, Chromosome in situ suppression hybridization (CISS), Multicolor FISH,
Chromosomal bar coding, Micro-FISH, In situ Hybridization to mRNA, in-cell RT-PCR,
Fluorescence immunophonotyping and interphase cytogenetics as tool for investigation of
neoplasmas (FICTION), Primed in situ DNA synthesis (PRINS), Fiber-FISH, FISHES, and
Comparative genomic hybridization (CGH).

How is it done?

Fluorescence in situ hybridization (FISH) are considered as a new advent in the field of cytology,
specifically. Initially, it was developed as a physical mapping tool to delineate genes within
chromosomes. The accuracy and versatility of FISH were subsequently capitalized upon in
biological and medical research. This visually appealing technique provides an intermediate
degree of resolution between DNA analysis and chromosomal investigations. It advantages
medical science for it is applied to detect genetic abnormalities that include different characteristic
gene fusions or the presence of an abnormal number of chromosomes in a cell or loss of a
chromosomal region or a whole chromosome. It is also applied in different research applications,
such as gene mapping or the identification of novel oncogenes.

The FISH technology detects specific gene sequences on the chromosome, or either its presence
or absence, is the central concern of cytogenetic technique in diagnosing as well as enumerating a
genetic disorder or abnormalities. And FISH is a less time-consuming technique in comparison to
the conventional method of cytogenetic metaphase karyotype analysis. By using this technique,
specific cytogenetic abnormalities, as well as a copy of aberrations numbers, can be enumerated
and sketched. For example, chromosomal microdeletion, amplification, and subsequently,
translocation can easily be detected through FISH. The improved diagnostic techniques have made
the treatment easier and increased the life expectancy of people for it plays a huge role on medical
science now since the 1990’s. Therefore, FISH is becoming a more vital tool to detect and monitor
the specific therapy with regards to the gene abnormalities; for example, the detection of the
BCR/ALB1 translocation in chronic myeloid leukemia, human epidermal growth factor receptor
2 (HER2) augmentation in breast cancer, and anaplastic lymphoma kinase (ALK) rearrangement
in adenocarcinoma.

What is the procedure?

For a specific implementation of the FISH technology, we will discuss The Basic Elements of
FISH: DNA Probe and a Target Sequence.
The first step, is to prepare probes. Probes are a shirt sequences of single-stranded DNA that match
a portion of the gene that we are looking for. These DNA probe is labelled in various ways, such
as nick translation, random primed labelling, and PCR. And in here, the two labelling strategies
are used which are the indirect labelling (probes are being labelled with nucleotides containing a
fluorophore) and direct labelling (the modified nucleotides that contain a hapten at which probes
are being labelled). Then, the labeled probe and the target DNA are denatured. The annealing of
complementary DNA sequences happens due to the combining consequences of denatured probe
and target DNA. In the case of indirect labeling, an extra step is needed for visualization of the
non-fluorescent hapten that uses an enzymatic or immunological detection system.

The basic steps of the FISH procedure depicted.

Other Findings of the Studies that Used the Fluorescence In Situ Hybridization (FISH) Technique
CARD-FISH: catalyzed reporter deposition FISH; CAT-FISH: capture antibody targeted detection
FISH; CB-FISH: cytochalasin B FISH; CO-FISH: cyotochrome orientation FISH; COD-FISH:
chromosome orientation and direction FISH; D-FISH: dual color FISH; DBD-FISH:
deoxyribonucleic acid breakage detection FISH; DNA: deoxyribonucleic acid; M-FISH: multiple
spectral karyotyping FISH; ML-FISH: multilocus FISH; mRNA: messenger ribonucleic acid;
PCC-FISH: premature chromosome condensation FISH; Q-FISH: quantitative FISH; QD-FISH:
quantum dots FISH; RNA: ribonucleic acid; T-FISH: tissue FISH
However, the selections of the FISH probe are dependent upon the diseases, anomalies, or
anomalies under the field of interest.

Samples and equipment needed

If you would compare the regular and/or standard application of clinical diagnosis, the FISH is
much more accurate and straightforward, as well as reliable, than the other molecular profiling
techniques, e.g., array-based comparative genomic hybridization, single nucleotide polymorphism
(SNP), etc. In addition to that, the FISH has a recognition of being a physical mapping technique
to support large-scale mapping and sequencing efforts related to the human genome project.

You will just need the DNA sequence, the fluorescence microscope, and the slide denaturation for
FISH slides.

1. Slide denaturation for FISH slides (ThermoBrite)- this is a programmable temperature-

controlled slide processing system. This is ideal for the denaturation and hybridization
steps in slide-based FISH procedures.

2.
Fluorescent miscroscope- same as a conventional light microscope with added features to
enhance its capabilities It uses a much higher intensity light source which excites a fluorescent
 species in a sample of interest. As a summary, it is used to image specific features of small
specimens.

Using these, we can visualize and conceptualize genes, chromosomes, transcription, and nucleic
acid movements through FISH. FISH also provides reliable biomarker information. In terms of
using the FISH technology around the world, we found that the FISH technique has very limited
use in developing countries because of unavailability and lack of expert knowledge. Hence, FISH
should be the preferred approach in anticipating the complex components of gene expression
leading to any disease.

Microarray Principle and Technology

• The principle of DNA microarray technology is based on the fact that the complementary
sequences of DNA can be used to hybridize, immobilized DNA molecules
• Samples are labeled using fluorescent dyes
• At least two samples are hybridized to chip
• Complementary nuclei acid sequences get pair via hydrogen bonds
• There are four major steps in performing a typical microarray experiment.
1. Sample preparation and labeling
2. Hybridisation
3. Washing
4. Image acquisition and Data analysis
How The Test is Done?
- A blood sample is typically used for microarray analysis. The DNA (chromosome content)
from the cells in the sample is taken in the lab and compared to a standard DNA sample to
see if any incomplete or extra pieces of chromosome material exist.

Types of Results from This Test

1. Normal Result - no missing or extra pieces of chromosome material were found.
2. Likely Pathogenic - a missing or extra piece of genetic material was found.
3. Likely benign - a missing or extra piece of chromosome material was found
4. Variant of unknown significance (VUS or VOUS) - a missing or extra piece of
chromosome material was found, but it is not clear if this causes any health or learning
problems. Sometimes testing the parents may be recommended to see if this missing or
extra piece came from a parent.
5. Regions of Homozygosity (ROH) - one or many chromosome parts were found to be
genetically identical.

DNA Microarray measurement of gene expression

A basic protocol for a DNA Microarray is as follow as:
1. Isolate and purify mRNA from samples of interest
2. Reverse transcribe and label the mRNA
3. Hybridize the labeled target to the microarray
4. Scan the microarray and quantitate the signal

Samples and Equipment needed

Sample required?
A blood sample drawn from a vein in the arm. Sometimes cells obtained through amniocentesis or
chorionic villus sampling from a pregnant woman.

Microarray Equipment

Microarray - Microarrays, also known as chips or biochips, consist of a

substrate of interest fixed to a solid matrix such as a glass slide, used for
genotyping, antibody detection and comparative genome analysis.
 Genepix 4000B Microarray Scanner - The Genepix 4000B Microarray
scanner is currently used to scan gene expression microarrays and spotted
protein arrays slides at high resolution (up to 5 μm/pixel).

NimbleGen MS 200 Microarray Scanner - Our newest acquisition, the

MS 200 Microarray Scanner has a 2μm pixel resolution and is uniquely
capable of scanning 2.1 and 4.2 million feature high-density NimbleGen
microarrays.

What is MALDI-TOF MS?

- “MALDI” means Matrix-Assisted Laser Desorption/Ionization. An ionization
technique where the matrix absorbs ultraviolet light and converts into energy.
- “TOF MS” means Time of Flight Mass Spectrometry
- To rapidly and accurately detect/identify pathogenic microorganisms
- Also used for antimicrobial susceptibility testing (AST)
- Widely used diagnostic technique in microbiology laboratories.
Equipments/Instruments
- Bacterial Sample
- Solid agar plate
- Sterile tip
- MALDI Target Plate
- TOF System Machines
- Pipet
- Analyte
Procedure
Preparation
- Clean the MALDI Target Plate using tissue and alcohol.
- Rinse the plate with ultra-purified water and solvent.
- (Optional) Use ultrasonic bath for more thorough cleaning.
- Completely dry the MALDI target plate.
- Use gloves and handle only the edges. Seal in a storage container until ready for use.

Sample Preparation
- Bacterial samples are separated and cultivated on a solid agar medium.
- Selected single colony is directly smeared as a thin film on a MALDI target. It is
handpicked using a sterile tip and then smeared onto a ground steel MALDI target plate
- The samples are dried and a small amount of matrix is added on MALDI target, then
the sample is introduced into the mass spectrometer for the acquisition of results or
data.
(Preparation and other workflow may vary based on the biological sample and needs to be
optimized for MALDI-TOF MS analysis)
MALDI Matrix Preparation
- UV-absorbing matrix is used in MALDI-TOF MS so that it will co-crystallize with the
sample and be ionized by ion source (e.g. ultraviolet laser)
- Common matrix compounds, such as CHCA, are small organic molecules that can
absorb UV Light. By this, it allows the matrix molecules to physically ablate from the
surface, carrying the analyte molecules into gas phase along with them. In addition to
this, matrix molecules are usually acidic for the ionization to happen.
- Matrix choice depends on the sample you will use.

Analyte Solution
- The common dilutions are 0.1 – 10 µM
- Several dilutions should be tried. Too dilute or too concentrated may result into
difficulties.
- Choose a solvent that has perfect volatility
Loading the MALDI Target Plate
- Method depends on the compound; however, several methods can be used to know the
suitable and best method.
1. Dried Droplet Method: the original method
- Analyte and matrix solutions are mixed.
- A single 1-2µL droplet is applied to the target
- The solvent will evaporate while the matrix and analyte will co-crystallize.
-On-plate mixing: faster way of dried droplet method wherein the solutions are mixed
directly on the plate by using pipet tip
2. Thin-layer (fast evaporation) method
 - The sample and analyte are mixed into volatile solvents.
- The matrix are applied first then dried into a thin layer. Then, the analyte solution is
applied on top of the dried matrix.

Other loading methods:

-Sandwich/dual-layer (SA Matrix)
-Crushed crystal (non-volatile solvents)

DNA sequencing
a laboratory technique used to determine the exact sequence of bases (A, C, G, and T) in a DNA
molecule. The DNA base sequence carries the information a cell needs to assemble protein and
RNA molecules.
DNA sequence information is important to scientists investigating the functions of genes. The
technology of DNA sequencing was made faster and less expensive as a part of the Human
Genome Project.
The principles of DNA Sequencing
The process of determining the order of the nucleotide bases along a DNA strand is called
sequencing. In 1977, twenty-four years after the discovery of the structure of DNA, two separate
methods for sequencing DNA were developed: the chain termination method and the chemical
degradation method. Both methods were equally popular to begin with, but, for many reasons, the
chain termination method is the method more commonly used today. This method is based on the
principle that single-stranded DNA molecules that differ in length by just a single nucleotide can
be separated from one another using polyacrylamide gel electrophoresis, described earlier.
The DNA to be sequenced, called the template DNA, is first prepared as a single-stranded DNA.
Next, a short oligonucleotide is annealed, or joined, to the same position on each template strand.
The oligonucleotide acts as a primer for the synthesis of a new DNA strand that will be
complimentary to the template DNA. This technique requires that four nucleotide-specific
reactions--one each for G, A, C, and T--be performed on four identical samples of DNA. The four
sequencing reactions require the addition of all the components necessary to synthesize and label
new DNA, including:
• A DNA template;
• A primer tagged with a mildly radioactive molecule or a light-emitting chemical;
• DNA polymerase--an enzyme that drives the synthesis of DNA;
• Four deoxynucleotides (G, A, C, T); and
• One dideoxynucleoside, either ddG, ddA, ddC, or ddT.

After the first deoxynucleotide is added to the growing complementary sequence, DNA
polymerase moves along the template and continues to add base after base. The strand synthesis
reaction continues until a dideoxynucleoside is added, blocking further elongation. This is because
dideoxynucleosides are missing a special group of molecules, called a 3'-hydroxyl group, needed
to form a connection with the next nucleotide. Only a small amount of a dideoxynucleoside is
added to each reaction, allowing different reactions to proceed for various lengths of time, unit, by
chance, DNA polymerase inserts a dideoxynucleoside, terminating the reaction. Therefore, the
result is a set of new chains, all of different lengths
DNA Sequencing Technologies
Early DNA Sequencing Technologies
- In 1973 Gilbert and Maxam reported the sequence of 24 base pairs using a method known
as wandering-spot analysis (Gilbert & Maxam, 1973)
- 1970s, when researcher Frederick Sanger developed several faster, more efficient
techniques to sequence DNA (Sanger et al., 1977).
- Indeed, Sanger's work in this area was so groundbreaking that it led to his receipt of the
Nobel Prize in Chemistry in 1980.

The Sanger Method

In the Sanger method, which became the more commonly employed of the two approaches,
DNA chains were synthesized on a template strand, but chain growth was stopped when
one of four possible dideoxy nucleotides, which lack a 3’ hydroxyl group, became
incorporated, thereby preventing the addition of another nucleotide. A population of
nested, truncated DNA molecules was produced that represented each of the sites of that
particular nucleotide in the template DNA. The molecules were separated according to size
in a procedure called electrophoresis, and the inferred nucleotide sequence was deduced
by a computer. Later, the method was performed by using automated sequencing machines,
in which the truncated DNA molecules, labeled with fluorescent tags, were separated by
size within thin glass capillaries and detected by laser excitation.

Next-Generation Sequencing Technology

Next-generation (massively parallel, or second-generation) sequencing technologies have
largely supplanted first-generation technologies. These newer approaches enable many
 DNA fragments (sometimes on the order of millions of fragments) to be sequenced at one
time and are more cost-efficient and much faster than first-generation technologies. The
utility of next-generation technologies was improved significantly by advances in
bioinformatics that allowed for increased data storage and facilitated the analysis and
manipulation of very large data sets, often in the gigabase range (1 gigabase =
1,000,000,000 base pairs of DNA).

Applications Of DNA Sequencing Technologies

- used to find genes, segments of DNA that code for a specific protein or phenotype
- it can be screened for characteristic features of genes.
- a gene sequence can be screened for functional regions. In order to determine the function
of a gene, various domains can be identified that are common to proteins of similar function
-
- discover genes involved in disease, and to better understand genomic structure and
diversity among species generally.

Steps in DNA Sequencing

- Sample preparation (DNA extraction)

- PCR amplification of target sequence
- Amplicon’s purification
- Sequencing pre-prep
- DNA Sequencing
- Data analysis

Sample prep:
Here the DNA is our starting material, therefore we need to isolated DNA first. Animal,
plant, bacterial, plasmid, or environmental DNA can be used for it.

As the quality of DNA is the major concern in sequencing, we recommend using proteinase
K or spin-column DNA extraction methods which has a high yield.

With the quantity, good quality of DNA also reduces the chance of reaction failure.
Conclusively, a DNA sample with a 260/280 ratio of nearly ~1.80 (or 1.7 to 1.88) is
considered as pure DNA. The quantity of DNA should be ~100ng for the present assay.

PCR amplification of target sequence:

Amplification is the pivotal step in sequencing. here only the gene of interest or the DNA
sequence we wish to study is first amplified, the rest of the DNA is discarded. Nonetheless,
in the whole-genome sequencing, the entire genome of an organism can be sequenced.

A set of flanking primers anneal at the outer regions of the DNA sequence of interest,
therefore, unwanted DNA can’t amplify. Using the standard PCR conditions, the
amplification process is done; 35 cycles, denaturation at 94°C, annealing at 55°C to 65°C
and extension at 72°C.

After the amplification, the PCR product is run on 2% agarose gel along with the DNA
ladder. Once the amplification achieved, DNA purification is performed.

Read more:

A Complete Guide of the Polymerase Chain Reaction

Agarose gel electrophoresis
Amplicon purification:
Why purification is needed?

This information is very interesting, no one will tell you about it.

If you check the purity of the PCR product at 260 and 280nm wavelength, it will always
be 1.80, exactly! because the amplicon is the pure DNA fragment. If any contaminants are
present in the DNA, amplification will not happen. Then why amplicon purification
required? the answer is here,

Unbound primers, primer-dimers, unused Taq DNA polymerase, unused DNA templates,
and other unused PCR reaction buffer components can abort DNA sequencing. That is why
amplicon purification is required.

Here, the alcohol purification can’t work, we have to purify our PCR product with the spin
column PCR amplicon purification kit to achieve maximum purification. After removing
debris, the PCR amplicons send to the sequencing lab.

Sequencing pre-preparation:
Sample pre-preparation is a very crucial step during DNA sequencing. During this step,
adaptor DNA sequences are ligated on both DNA ends. To the adaptor, the primer anneals
for doing amplification. The amplification process in sequencing is similar to PCR,
however, here the nucleotides are either radio or fluorescent-labeled.

With it, high fidelity DNA polymerase and other key ingredients are added in the reaction.

DNA sequencing:
 The prepared reaction tubes are placed into the sequencer machine. During, the reaction in
the sequencer, denaturation, annealing, and extension occurs, simultaneously. Here, as we
are using the labeled nucleotides, the signals produced during the reaction is recorded.

That signals of the addition of each complementary nucleotides are recorded by the
machine and the data is sent to the computer.

Data analysis:
Once the whole DNA is sequenced, the result is saved into one unique file formate. The
inbuilt software (provided by the manufacturer) processed the data and compared it with
the available data.

Different methods of DNA sequencing:

Various methods of DNA sequencing are explained here.

• Maxam and Gilbert method

• Chain termination method
• semiautomated method
• automated method
• Pyrosequencing
• The whole-genome shotgun sequencing method
• Clone by the clone sequencing method
• Next-generation sequencing method

Samples and Equipment for DNA Sequencing

ABI 3730 DNA Analyzer

The ABI 3730 DNA Analyzer is a 48-capillary DNA sequencer and genotyper that provides
automated high throughput genetic analysis of DNA fragments. The 3730 can sequence 48 samples
in parallel in two hours providing the capacity for analyzing hundreds of DNA samples per day.
The instrument is fully automated from sample loading through data analysis. The 3730 is used
for DNA fragment analysis such as microsatellites, SNP analysis, mutation detection, and Sanger
Sequencing of plasmids and PCR products.
Turner BioSystems TBS-380 Fluorometer
The TBS-380 Fluorometer is a mini-fluorometer that is capable of accurately measuring extremely
low concentrations of nucleic acids – dsDNA as low as 1 ng/ml in a PicoGreen assay, RNA as low
as 1 ng/ml in a RiboGreen assay. Unlike spectrophotometers, the TBS-380 when used in
conjunction with PicoGreen or RiboGreen is relatively insensitive to proteins, free nucleotides,
and reagent contaminants generally found in DNA and RNA preparations. When used with
minicell cuvettes, measurements can be carried out in as little as 50 ul.
 -

The following list of subgroups of individuals are considered at-risk and are eligible for
testing, arranged from greatest to lowest need for testing:

● Subgroup A: patients or healthcare workers with severe/critical symptoms, and relevant history
of travel or close contact;

● Subgroup B: patients or healthcare workers with mild symptoms, with relevant history of
travel or close contact and who are considered vulnerable. These vulnerable populations include
the elderly and those with pre-existing medical conditions that predispose them to severe
presentation and complications of COVID-19;

● Subgroup C: patients or healthcare workers with mild symptoms, and relevant history of travel
or close contact;

● Subgroup D: patients or healthcare workers with no symptoms but with relevant travel history
and close contact;

● Subgroup E: frontliners indirectly involved in healthcare provision in the response against

COVID 19; and

● Subgroup F: other vulnerable patients such as those with comorbidities, those who will
undergo high-risk, elective surgical procedures, those living in confined spaces, and others

Covid-19 PCR Assay

Definition: It is a test used to diagnose people who are currently infected with SARS-CoV-2,
which is the coronavirus that causes COVID-19.
-The PCR test is considered the “gold standard test” for diagnosing COVID-19 because
it is so far the most accurate and reliable test.
Samples and Equipment:
Equipment: Definition / Uses
Nasal Swabs Is a test that checks for viruses and bacteria that
cause respiratory infections. A nasal swab test
is done by taking a sample of cells from the
nostrils.

Nasopharyngeal Swabs The swab is inserted into the patient’s nose and
is aimed in a parallel direction to the nasal and
septum floor. As long as there are no
obstructions present within the nasal cavity,
the swab will continue to move in this direction
until it reaches the nasopharynx, at which point
resistance will be felt by the testing personnel.

Swab Kit The Swab Kit is ideal for determining the

relative degree and type of biological
contamination in an area.
How to Use:
• Remove the sterile swab from its
package aseptically.
• Remove the sealing cap from the tube
containing the sponge and moisten the
swab tip.
• Using a rolling motion, gently swab the
desired area thoroughly. Templates are
included in the kit for defined area
sampling.
• Insert the swab into the tube, replace
the sealing cap, and prepare for
transport to a laboratory.

Thermal Cycler The Thermal Cycler (also known as a

Thermocycler, PCR Machine or DNA
Amplifier) is a laboratory apparatus used to
 amplify segments of DNA via the Polymerase
Chain Reaction (PCR).

How is it done: There are three key steps to the Covid-19 PCR Test
1. Sample Collection.
2. Extraction.
3. PCR.
What is the Procedure:
1. Sample Collection
• Sample collection is done using a swab to collect respiratory material found in your nose.
• A swab contains a soft tip on a long, flexible stick that is inserted into the nose.
• Nasal Swabs – collect samples inside the nostrils.
• Nasopharyngeal Swabs – collect samples that goes further the nasal cavity.
• After collection, the swab is sealed in a tube and then sent to a laboratory.
2. Extraction
• This method isolates the genetic materials from the sample including genetic material from
any virus that may be present.
3. PCR.
• This method uses special chemicals and a PCR machine called a thermal cycler, which
cause a reaction to occur that make millions of copies of small portions of the SARS-CoV-
2 virus genetic material.
• One of chemicals that is present produces a fluorescent light, if SARS-CoV-2 is present in
the sample.
• The fluorescent light is a signal that can be detected by the PCR machine and a special
software is used to interpret if there is a positive result
What do Covid-19 PCR test results mean?
A positive test result: means that it is very likely that you have COVID-19. Most people have
mild illness and can recover safely at home without medical care
A negative test result: means you probably didn't have COVID-19 at the time you took your
test. However, it is possible to be infected with SARS-CoV-2 but not have enough virus in
your body to be detected by the test.
Example: For example, this may happen if you recently became infected but you don’t have
symptoms, or it could happen if you've had COVID-19 for more than a week before being
tested. Keep in mind that a negative test doesn’t mean you are safe for any length of time.
How soon are results of a COVID-19 PCR test available?
You should receive the results of your test as early as 24 hours after sample collection, but
sometime it can take a few days depending on long it takes the sample to reach the laboratory
and how many other samples are in the queue to be tested.
Advantages: The main advantages of COVID-19 PCR test are its accuracy and reliability. It
is so far the most accurate available test for COVID-19 detection.
Disadvantages: Because the test is able to detect very small amounts of the virus genetic
material, it can continue to detect fragments of SARS-CoV-2 virus even after the patient has
recovered from COVID-19 and no longer contagious. In conclusion, there is a possibility that
you may continue to test positive if you have had COVID-19 in the past, even though you can’t
spread the SARS-CoV-2 virus to people.

DNA Fingerprinting (Paternity Testing)

Definition: It is a chemical test that shows the genetic makeup of a person or other living
things. It’s been widely used in evidence in courts, identify bodies, track down blood
(biological parent, relatives, etc.) and to look for cures for disease.
Samples and Equipment:
Equipment:
Microscope is an instrument that can be used to observe
small objects, even cells. It is commonly
used in DNA Fingerprinting by laboratory
workers.
 Polymerase Chain Reaction Machine The Thermal Cycler (also known as a
Thermocycler, PCR Machine or DNA
Amplifier) is a laboratory apparatus used to
amplify segments of DNA via the
Polymerase Chain Reaction (PCR).

Gel Electrophoresis Machine instruments are used to separate nucleic

acids and proteins based on their size and
charge. Can be used forensic, molecular
biology, genetics, and microbiology labs.
Gel electrophoresis instrument were used to
run and compare DNA samples.

Samples:
Saliva Hair
Nails Body Fluids
Buccal Swabs Blood Samples

Molecular Methods:
Plasmid Fingerprinting provides a rapid and dependable means of
identifying bacterial isolates of the same
strain. The stability, wide distribution, and
diverse nature and size of extrachromosomal
elements make it suitable for virtually all
bacterial genera.
Ribotyping is a molecular technique for bacterial
identification and characterization that uses
information from rRNA-based phylogenetic
analysis. It is a rapid and specific method
widely used in clinical diagnostics and
analysis
Amplified Fragments Length is a PCR-based technique that uses
Polymorphism selective amplification of a subset of
digested DNA fragments to generate and
 compare unique fingerprints for genomes of
interest.
Random Amplified Polymorphic DBA is a PCR-based technique which uses
(RAPD) arbitrary primers which bind to the
nonspecific sites on the DNA and amplify the
DNA. These amplified fragments are then
migrated on agarose gel and difference in the
band pattern is observed.
Pulsed-Field Gel Electrophoresis (PFGE) It is used in the separation of DNA fragments
for DNA fingerprinting to investigate crime
scenes, findings of biological parent, analyze
genes for a particular illness, and it is used to
study evolutionary relationships by
analyzing genetic similarities among
populations or species.

How is it done:
• To get your DNA finger print, you would give a sample of cells from your body. This
can come from a swab inside the mouth, from the skin, hair, saliva, sweat and other
fluids.
• Blood is usually the easiest way, because lab workers treat the samples with chemicals
to separate the DNA, which is then dissolved in water.
What is the procedure:
• The DNA that is gathered are cut into smaller segments with another chemical process
to get sections of 5 to 10 base pairs.
• The lab workers take the strips of DNA and mix into a gel. Then they run an electric
current through the gel, which separates smaller strands of DNA from the larger ones.
• A dye will be added to the gel to make the DNA strip stand out when they are placed
against an ultraviolet light or lit up with a laser
• The more the short segments are tested, the more accurate the DNA profile.
• The strips will show a barcode-like pattern that can then be compared to the results
from another sample of DNA to find a match.

Laws and Program

- Bayanihan to Recover as One Act
R.A 11494
- The Department of Health (DOH) has updated its interim guidelines on expanded risk-
based testing of COVID-19 cases. Department Memorandum No. 2020-0258
- in view of the increased RT-PCR (Reverse Transcription Polymerase Chain Reaction) testing
capacity of the country.
he active participation of all our laboratories, we have significantly increased our testing
capacity. We have thus updated our guidelines to be attuned to the needs of all those needing
to be tested. With adequate testing,” Health Secretary Francisco T. Duque said.

References:
Luke, S., & Shepelsky, M. (1998). FISH: recent advances and diagnostic aspects. Cell vision: the
journal of analytical morphology, 5(1), 49-53. Retrieved from
https://europepmc.org/article/med/9660726

Ratan, Z. A., Zaman, S. B., Mehta, V., Haidere, M. F., Runa, N. J., & Akter, N. (2017). Application
of fluorescence in situ hybridization (FISH) technique for the detection of genetic aberration in
medical science. Cureus, 9(6). Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501716/
Biocompare Lab Equipment. (n.d.). Retrieved from https://www.biocompare.com/Lab-
Equipment/6580-Microarray-Equipment/4
Biology Microarray-Equipment. (2018, December 3). Retrieved from
https://biology.uiowa.edu/ccg/microarray-center/microarray-equipment
Nationwide Children Organization. (n.d.). Retrieved from
https://www.nationwidechildrens.org/family-resources-education/health-wellness-and-
safety-resources/helping-hands/microarray-analysis-test

Feucherolles, M. (2019, May 17). MALDI-TOF mass spectrometry as a diagnostic tool in human

and veterinary helminthology: a systematic review. Parasites & Vectors. Retrieved from

https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-019-3493-9

Principle of MALDI/TOFMS. (n.d.). Shimadzu. Retrieved from

https://www.shimadzu.com/an/products/maldi/ms-applications/principle-of-

malditofms/index.html
CHINESE GENERAL HOSPITAL COLLEGES

SCIENCE, TECHNOLOGY, AND SOCIETY

THE ADVENT OF MOLECULAR

DIAGNOSTICS TOOLS

Cristobal, Nicole Angela

de Guzman, Ma. Ynna
dela Sierra, Cesar Ian
Glorioso, Gio

APRIL 28, 2021

MOLECULAR BIOLOGY
Molecular biology, field of science concerned with studying the chemical structures and
processes of biological phenomena that involve the basic units of life, molecules. The
field of molecular biology is focused especially on nucleic acids (e.g., DNA and RNA)
and proteins—macromolecules that are essential to life processes—and how these
molecules interact and behave within cells. Molecular biology emerged in the 1930s
when scientists focused on explaining the phenomena of life by studying the
macromolecules that generate life, and was developed out of the related fields of
biochemistry, genetics, and biophysics; today it remains closely associated with those
fields

SUBDISCIPLINES OF MOLECULAR BIOLOGY

1. Comparative Genomics
- This is the study of human genetics by comparisons with model organisms
such as mice, the fruit fly and the bacterium E. coli.
2. DNA Forensics
- This is the use of DNA for identification. Some examples of DNA use are
to establish paternity in child support cases, to establish the presence of a
suspect at a crime scene and to identify accident victims.
3. Functional Genomics
- This is the study of genes, their resulting proteins and the role played by
the proteins in the body’s biochemical processes.
4. Gene Therapy
- This is an experimental procedure aimed at replacing, manipulating or
supplementing non functioning or malfunctioning genes with healthy
genes.
5. Genomics
- This is the study of genes and their functions.
6. Molecular Genetics
- This is the study of macromolecules important in biological inheritance.
7. Pharmacogenomics
- This is the study of the interaction of an individual’s genetic makeup and
response to a drug.
8. Proteomics
- This is the study of the full set of proteins encoded by a genome.
9. Structural Genomics
- This is the effort to determine the three-dimensional structures of large
numbers of proteins using both experimental techniques and computer
simulation.
10. Toxicogenomics
- This is the study of how genomes respond to environmental stressors or
toxicants.
FISH

Fluorescence in situ hybridization (FISH) is the most convincing technique for locating
the specific DNA sequences, diagnosis of genetic diseases, gene mapping, and
identification of novel oncogenes or genetic aberrations contributing to various types of
cancers. FISH involves annealing of DNA or RNA probes attached to a fluorescent
reporter molecule with specific target sequence of sample DNA, which can be followed
under fluorescence microscopy. In medicine, FISH can be used to form a diagnosis, to
evaluate prognosis, or to evaluate remission of a disease, such as cancer. Treatment
can then be specifically tailored. A traditional exam involving metaphase chromosome
analysis is often unable to identify features that distinguish one disease from another,
due to subtle chromosomal features; FISH can elucidate these differences. FISH can
also be used to detect diseased cells more easily. Examples of diseases that are
diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, 22q13
deletion syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia,
Cri-du-chat, Velocardiofacial syndrome, and Down syndrome.

Purpose and application

FISH is useful, for example, to help a researcher or clinician identify where a particular
gene falls within an individual's chromosomes. Scientists use three different types of
FISH probes, a single strand of DNA or RNA that is complementary to a nucleotide
sequence of interest, each of which has a different application:

Locus specific probes bind to a particular region of a chromosome. This type of probe is
useful when scientists have isolated a small portion of a gene and want to determine on
which chromosome the gene is located, or how many copies of a gene exist within a
particular genome.

Alphoid or centromeric repeat probes are generated from repetitive sequences found in
the middle of each chromosome. Researchers use these probes to determine whether
an individual has the correct number of chromosomes. These probes can also be used
in combination with "locus specific probes" to determine whether an individual is missing
genetic material from a particular chromosome.

Whole chromosome probes are actually collections of smaller probes, each of which
binds to a different sequence along the length of a given chromosome. Using multiple
probes labeled with a mixture of different fluorescent dyes, scientists are able to label
each chromosome in its own unique color. The resulting full-color map of the
chromosome is known as a spectral karyotype. Whole chromosome probes are
particularly useful for examining chromosomal abnormalities, for example, when a piece
of one chromosome is attached to the end of another chromosome.
For many applications, FISH has largely been replaced by the use of microarrays.
However, FISH remains useful for some tests. FISH may also be used to study
comparisons among the chromosomal arrangements of genes across related species.

Method

(a) The basic elements of FISH are a DNA probe and a target sequence.

(b) Before hybridization, the DNA probe is labeled by various means, such as nick
translation, random primed labeling, and PCR. Two labeling strategies are commonly
used: indirect labeling (left panel) and direct labeling (right panel). For indirect labeling,
probes are labeled with modified nucleotides that contain a hapten, whereas direct
labeling uses nucleotides that have been directly modified to contain a fluorophore.

(c) The labeled probe and the target DNA are denatured.

(d) Combining the denatured probe and target allows the annealing of complementary
DNA sequences.

(e) If the probe has been labeled indirectly, an extra step is required for visualization of
the nonfluorescent hapten that uses an enzymatic or immunological detection system.
Whereas FISH is faster with directly labeled probes, indirect labeling offers the
advantage of signal amplification by using several layers of antibodies, and it might
therefore produce a signal that is brighter compared with background levels.

How are the data reported?

Depending on the method, FISH results can be presented in two different ways:
• If FISH is evaluated using a microscope and manual counting of labeled cells, the
results are presented as cells per unit (liter of liquid or gram of solid) analyzed.
• If FISH is evaluated with advanced microscopy techniques and digital image
processing, the results are usually presented on a relative volume or area basis, which
can be converted by the laboratory to cells per unit of liquid or solid.

Materials
Slide denaturation for FISH slide- This programmable system automates the
denaturation and hybridization steps in slide-based FISH procedures, and provides
walk-away convenience for clinical and personnel.

Fluorescent microscopes- Usually come equipped with a standard set of filters. The
common filters include: DAPI filter (blue), FITC filter (green), and a Texas red filter. it
uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or
absorption, to study the properties of organic or inorganic substances.

MICROARRAY
Purpose

Microarray is one of the most recent advances being used for cancer
research; it provides assistance in pharmacological approach to treat various
diseases. Microarray helps in analyzing large amounts of samples which have
either been recorded previously or new samples; it even helps to test the
incidence of a particular marker in tumors.

Application

Medical diagnosis and treatment

a. The discovery of target: in this application, the microarray is used to
compare diseased tissues/cells with healthy tissues/cells to find the
characteristics of a particular disease. This helps in finding the genes
responsible for that disease.
b. The discovery of drugs and leads: after the target has been discovered,
microarrays can be used to screen potential compounds and identify the
toxicity of the lead compound that will help in deciding proper medication
for the patient. The study of antibodies, as well as microorganisms like
bacteria and viruses, also helps in the discovery of more effective
antibiotics and vaccines.
c. Diagnostics and prognostics: the microarray is widely used to know the
state of disease, type of tumor and other factors important for the patient.
As mentioned above, it is used to diagnose a number of diseases and
infections, most notably cancer.
d. Pharmacogenomics and theranostics: the microarray technique can be
used to decide a patient’s treatment and therapy on the basis of his/her
genetic makeup. Thus, it helps in carrying out personalized treatments
rather than using generalized ones. It can also help in controlling side
effects of medications.

Crime and security

Microarray has a prominent role in detecting “Biological Warfare Agents”
(BWAs): BWAs are microorganisms or toxins produced by them that are
intentionally dispersed by terrorists to spread diseases in man and other
organisms. The microarray provides a platform that provides fast, sensitive, and
simultaneous identification of these agents. This is very much useful for national
security and protection of life.

Early detection of oral precancerous lesions

Leukoplakia or white lesions of the oral cavity may result from a myriad of
reversible conditions. Currently, microscopic examination fails to identify the
small subset of these lesions that progress to oral cancer. Identification of gene
expression profiles or “genomic fingerprints” will allow clinicians to differentiate
harmless white lesions from precancerous lesions or from very early cancer.
Recent studies have illustrated the effectiveness of microarrays in oral cancer.
Early diagnosis and management of oral cancer is correlated with increased
survival.

Equipment

Microarrays consist of a substrate of interest fixed to a solid matrix such

as a glass slide, used for genotyping, antibody detection and comparative
genome analysis. Microarrays can be produced in house with specialized
equipment, or purchased from a manufacturer. Microarray slides or chips are
spotted with arrayers, hybridized in ovens, and refined by washers and strainers.

Procedure

To perform a microarray analysis, mRNA molecules are typically collected

from both an experimental sample and a reference sample. For example, the
reference sample could be collected from a healthy individual, and the
experimental sample could be collected from an individual with a disease like
cancer. The two mRNA samples are then converted into complementary DNA
(cDNA), and each sample is labeled with a fluorescent probe of a different color.
For instance, the experimental cDNA sample may be labeled with a red
fluorescent dye, whereas the reference cDNA may be labeled with a green
fluorescent dye. The two samples are then mixed together and allowed to bind to
the microarray slide. The process in which the cDNA molecules bind to the DNA
probes on the slide is called hybridization. Following hybridization, the microarray
is scanned to measure the expression of each gene printed on the slide. If the
expression of a particular gene is higher in the experimental sample than in the
reference sample, then the corresponding spot on the microarray appears red. In
contrast, if the expression in the experimental sample is lower than in the
reference sample, then the spot appears green. Finally, if there is equal
expression in the two samples, then the spot appears yellow. The data gathered
through microarrays can be used to create gene expression profiles, which show
simultaneous changes in the expression of many genes in response to a
particular condition or treatment.
MALDI-TOF

Purpose

The Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass

Spectrometry (MALDI-TOF MS) is a rapid, accurate, and cost-effective method of
microbial characterization and identification. In the majority of clinical
laboratories, this technology is currently being used mainly for bacterial
diagnosis, but several approaches in the field of virology have been investigated.
The introduction of this technology in clinical virology will improve the diagnosis
of infections produced by viruses but also the discovery of mutations and variants
of these microorganisms as well as the detection of antiviral resistance.

Application

Viral diagnosis
A recent study performed a MALDI-TOF MS method that can successfully
detect human herpes viruses from a wide variety of archival biological
specimens. The concordance rate between the MALDI-TOF MS technique and
reference methods was high and the detection limits of the MS methods were
comparable to the previous reports of multiplex herpes virus detection using an
oligonucleotide microarray and multiplex PCR techniques. Low viral loads near
the detection limit of the method and weak cross-reactivity might contribute to the
few discrepant results with reference methods. Thus, this multiplex approach can
be very useful for large-scale epidemiological research studies in which broad
virus detection is necessary and may also become highly useful for multiplex
clinical diagnostic testing.

Diagnosis of mutations and identification of viral genotypes

MassARRAY systems based on nucleic acid analysis by MALDI-TOF MS
technology provide an alternative approach to HBV and HCV genotyping, being
accurate to identify all eight HBV genotypes. MassARRAY system generates MS
patterns that are compared to sequences derived from known HBV genotypes.
 The main advantages of this assay are its rapidity, cost-effectiveness, and
versatility. The assay produces automatic data reports and does not require any
additional data interpretation.

Identification of drug resistance

The application of MALDI-TOF MS technique could be useful for detecting
drug resistance against some antivirals. A recent study has developed a
sensitive and rapid method for the detection of ganciclovir resistance by
PCR-based MALDI-TOF analysis.

Equipment

The Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass

Spectrometer has three major components: the ionization source, the analyzer
and the detector. In addition there is an inlet used for loading the sample into the
source and a computer hooked up to the detector to record and analyze the data
produced during the analysis.

Procedure

In MALDI-TOF mass spectrometry, protein or peptide samples are mixed

with a matrix compound and dried onto a metal sample plate. After the plate is
placed in a high vacuum source chamber in the mass spectrometer, a small
portion of the sample is vaporized by blasts from a nitrogen laser. The ions
produced ‘fly’ up a tube to the mass analyzer and their masses (actually their
mass-to-charge ratio) are determined by their ‘time-of-flight’.

DNA SEQUENCING
Technique used to determine the nucleotide base sequence of DNA. The
nucleotide sequence is the most fundamental level of knowledge of a gene or
genome. It is the blueprint that contains the instructions for building an organism,
and no understanding of genetic function or evolution could be complete without
obtaining this information.
Components and equipment:
a. DNA template - strand used by DNA polymerase or RNA polymerase to
attach complementary bases
b. DNA primer - short nucleic acid sequence that provides a starting point for
DNA synthesis
c. DNA polymerase - responsible for synthesizing DNA
d. Deoxynucleotides (A, G, C, T)
e. Dideoxynucleotides (ddA, ddG, ddC, ddT)
f. Capillary Gel Electrophoresis - allows the short fragments move quickly
through the pores of the gel, while long fragments move more slowly. As
each fragment crosses the “finish line” at the end of the tube, it is
illuminated by a laser, allowing the attached dye to be detected.
g. Chromatograms - record the series of peaks in fluorescence intensity.

Evolution of DNA Sequencing

1. First Generation DNA Sequencing – Sanger Sequencing
- Also known as chain termination method
- Developed by English biochemist Frederick Sanger and his
colleagues in 1977
- It is considered a gold standard for validating DNA sequences
- Used to sequence individual pieces of DNA, such as bacterial
plasmids or DNA copied in PCR
- Designed for determining the sequence of nucleotide bases in a
piece of DNA with less than 1,000 base pairs
Procedure:
 Data Analysis:

DNA migrates from the negative pole towards the positive pole. The smaller,
lighter DNA migrates the furthest. The sequence is read from bottom to the top of
the plate. The generated result is a complementary sequence of the DNA
sample.
Advantages:
- Gives high quality sequence for DNA with less than 1000 base
pairs
- With 99.99% base accuracy
Disadvantages:
- Expensive and inefficient for larger-scale projects, such as the
sequencing of an entire genome
Video reference: https://www.youtube.com/watch?v=FvHRio1yyhQ

2. Next Generation Sequencing - Illumina/Solexa Technology

- Also known as Sequencing by Synthesis (SBS)
 - Invented by Bruno Canard and Simon Safarti
- Newer approaches enable many DNA fragments (sometimes on
the order of millions of fragments) to be sequenced at one time
- More cost-efficient and much faster than first-generation
technologies
Procedure:

Data Analysis:

Millions and billions of reads are generated throughout the process. These reads
are overlaid and compared to a reference genome. A whole genome sequence
can be generated from the overlay.
Advantages:
- Cost Effective
- Comprehensive genomic coverage
- Faster turnaround time for high sample volumes
Disadvantages:
- Many of the identified abnormalities has unknown clinical significance
- Requires sophisticated bioinformatics systems
- Lack in staffing to analyze and clinically interpret the data.
Video Reference: https://www.youtube.com/watch?v=CZeN-IgjYCo

3. Third Generation Sequencing – MinION, Oxford Nanopore Technology

- unique, scalable technology that enables direct, real-time analysis of
long DNA or RNA fragments
- works by monitoring changes to an electrical current as nucleic acids
are passed through a protein nanopore
- the resulting signal is decoded to provide the specific DNA or RNA
sequence.
Procedure:
Check out the video source: http://youtube.com/watch?v=RcP85JHLmnI
Advantages:
- Providing immediate access to results
- Rapid access to time critical information
- Generation of early sample insights
- Facility to stop sequencing once a result has been achieved
Disadvantages:
- Error rates in nanopore sequencing can be as high as 15%

Application of DNA Sequencing:

1. Finding genes or segments of DNA that code for a specific protein or
phenotype
2. Homologous DNA sequences of different organisms can be compared to
plot evolutionary relationships both within and between species.
3. Gene sequence can be screened for functional regions
4. Discover genes involved in disease
5. Understand genomic structure and diversity among species

Human Genome Project

HGP was initiated in 1990 under the leadership of American geneticist

Francis Collins, with support from the U.S. Department of Energy and the
National Institutes of Health (NIH). The effort was soon joined by scientists from
around the world.
 The primary goal of the Human Genome Project is to generate detailed
maps of the human genome. These maps will aid in determining the location of
genes within the human genome; more specifically, it will assign genes to their
chromosomes. There are two types of maps that are being developed, genetic
linkage maps and physical maps. Genetic linkage maps determine the relative
arrangement and approximate distances between genes and markers on the
chromosomes, whereas physical maps specify the physical location and distance
between genes or DNA fragments.
After mapping is complete, the DNA must be sequenced to determine the
order of all the nucleotide bases of the chromosomes, and the genes in the DNA
sequence must be identified. In all aspects of the project, a major focus has been
developing instrumentation to increase the speed of data collection and analysis.

PCR TESTING
A polymerase chain reaction (PCR) test is a standard method to detect
genetic material from a specific organism, such as a virus. The test detects the
presence of a virus if you are infected at the time of the test. The test could also
detect fragments of the virus even after you are no longer infected.

COVID-19 and PCR Testing

- PCR Test is for COVID-19 to diagnose people infected with SARS-CoV-2, which
is the coronavirus that causes COVID-19. The PCR test is the “Gold standard”
test for diagnosing COVID-19 because it’s the most accurate and reliable result
for COVID-19.

How does a COVID-19 PCR test work?

There are three steps to the COVID-19 PCR test

● sample collection
● extraction
● PCR

PROCEDURE

● Sample collection
- Sample collection is done using a swab to collect respiratory material
found in your nose.
- A swab contains a soft tip on a long, flexible stick that is inserted into your
nose

Different types of nose swab

- nasal swabs that collected a sample immediately inside your nostrils.

- nasopharyngeal swabs that go further into the nasal cavity for collection.
- After collection, the swab is sealed in a tube and then sent to a laboratory.
● Extraction
 - This method isolates genetic material from the sample, including genetic
material from any virus that may be present.
● PCR
- This method uses special chemicals and a PCR machine, called a thermal
cycler, which causes a reaction to occur that makes millions of copies of a
small portion of the SARS-CoV-2 virus’s genetic material.
- During this process, one of the chemicals produces a fluorescent light if
SARS-CoV-2 is present in the sample
- This fluorescent light is a “signal” that is detected by the PCR machine
and special software is used to interpret the signal as a positive test result.

Assay Techniques and Test Development for COVID-19 Diagnosis

1. Diagnostic test - tests that will detect the presence of the virus
- Molecular test
- Antigen test
2. Antibodies test - Detection of the immune response to the virus
- Serologies test

MOLECULAR/GENOMIC TEST

● Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

- RT-PCR is a diagnostic technique to detect viral genetic material (viral
RNA) in a biological sample after amplifying it to allow for its detection. It is
the current reference for detecting the presence of the virus in the
respiratory tract, i.e., identifying active infections.
- This technique has excellent sensitivity and specificity, meaning that it is
very reliable.
● Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)
- RT-LAMP is a technique similar to conventional RT-PCR tests, with the
exception that the nucleic acid amplification occurs at a constant
temperature, and thus equipment such as expensive thermal cyclers used
to regulate sample temperature in RT-PCR are not required
● CRISPR-Based Assays
- CRISPR-based tests work by identifying a sequence of viral COVID‑19
RNA and cutting apart any nearby single-stranded RNA. These cuts
release a separately introduced fluorescent particle in the test solution.
When the sample is then hit with a burst of laser light, the released
fluorescent particles light up, signalling the presence of the viral genetic
material.

ANTIGEN TEST

- Antigen tests detect another portion of the SARS‑CoV‑2 virus, the protein coat
that surrounds the RNA genome. Like molecular tests, antigen tests are intended
 to detect the viral presence in symptomatic or asymptomatic individuals and are
performed on samples obtained from the respiratory tract.

SEROLOGY

- Serologic tests look for the presence of disease-specific antibodies in

someone’s biological fluids (usually blood)
- This tests determine whether a person has developed antibodies against a
given pathogen as a result of exposure or infection.

Molecular/genomic tests Antigen tests Serologic tests

Objects Detection of the virus presence in the organism Detection of the immune
of the responsible in the virus
test

Techniq RT-PCR RT- LAMP CRISPR-based Rapid antigen ELISA test Immunochromat
ue Test test tests o-graphic assays
(rapid tests)

What Looks for the Looks for the Looks for the Looks for the Looks for the presence of an
does it presence of presence of presence of presence of immune response (antibodies)
look viral genetic viral genetic viral genetic viral genetic against the virus in the patients’
for? material material material (RNA) material fluids (usually blood)
(RNA) in a (RNA) in a in a sample (RNA) in a
sample taken sample taken taken from the sample taken
from the from the patient (usually from the
patient patient a patient
(usually a (usually a nasopharyngea (usually a
nasopharyng nasopharynge l swab) nasopharynge
eal swab) al swab) al swab)

What The virus is present in the patient The patient has been exposed
does a to the virus and is either
positive recovering or has already
test recovered
mean?

What is To know whether a patient has

the test To know whether an individual is currently infected with been exposed to SARS‑CoV‑2
used SARS‑CoV‑2 and could therefore be
for? protected against new
To know whether SARS‑CoV‑2 circulates in a given group or infections (and potentially not
spread the disease anymore)
population
Pros - High
sensitivity - Performance - Reported - Simple to - More - Less resource
and close to performance process reliable than intensive than
specificity
RT-PCR close to immunochro ELISA tests
- Cheap
RT-PCR mato-graphi
- Can be used - Can be
- Give result in c assays
at - Can be used performed at
15 to 30
point–of-care at point-of-care - Provides a point–of-care
minutes
quantitative
- Give result - Can quantify - Rapid (15 to 30
- Can be used information
in less than viral load minutes)
at (concentrati
one hour
- Give result in point-of-care on in
usually
15 to 30 antibodies)
- Good
minutes
specificity

Cons - Lower
-Labour-inten - Not yet - Not yet sensitivity - Possible errors of
sive; widely available at compared to interpretation if performed too
RT-PCR
available at point-of-care early in the infection process as
- Majority of
point-of-care. antibodies have not yet been
tests still - Price still
produced
need to be - Price still uncertain
processed in uncertain - Possible false positives
a lab (interaction with other diseases)

- Takes time
to give results
(several
hours at
least)

- Testing
materials in
short supply
DNA FINGERPRINTING
- Also called DNA typing, DNA profiling, genetic fingerprinting, genotyping, or
identity testing
- Invented in 1984 by Professor Sir Alec Jeffreys
- Although more than 99.9% of the genome is the same throughout the human
population, the remaining 0.1% of human DNA shows variations between
individuals.
- Method used to identify an individual from a sample of DNA by looking at unique
patterns in their DNA
Samples obtained:
- swab inside the mouth
- Skin
- roots of the hair
- saliva, sweat, or other body fluids

Different Methods of DNA Fingerprinting

1. Restriction Fragment Length Polymorphism (RFLP) Analysis
- type of polymorphism that results from variation in the DNA
sequence recognized by restriction enzymes.
Procedure:
a. Restriction digest - extraction of desired fragments of DNA using
restriction endonuclease (RE).
b. Gel electrophoresis - digested fragment are run in polyacrylamide gel
electrophoresis or Agarose gel electrophoresis to separate the
fragments based on length or size
c. Denaturation - gel is placed in sodium hydroxide (NaOH) solution for
denaturation so that single stranded DNA are formed.
d. Blotting - single stranded DNA obtained are transferred into charge
membrane
e. Baking and blocking - nitrocellulose paper transferred with DNA is fixed
by autoclaving. Then, membrane is blocked by using bovine serum
albumin
f. Hybridization and visualization - labelled RFLP probe is hybridized with
DNA on the nitrocellulose paper. RFLP probes are complimentary as
well as labelled with radioactive isotopes so they form color bands
under visualization by autoradiography.
2. Short Tandem Repeat (STR) Analysis
- most common type of DNA profiling today for criminal cases and
other types of forensic uses
- repetitive sequence elements 3–7 base pairs in length scattered
throughout the human genome
Procedure:
 a. Thousands of copies of a particular variable region are amplified by PCR
b. STR with a known repeat sequence is amplified and separated using
gel-electrophoresis
c. Primers are specially designed to attach to a highly conserved common
nonvariable region of DNA that flanks the variable region of the DNA.
d. STR sequence size amplified by PCR with the other known samples, will
give the final result of the DNA fingerprinting
Video Reference: https://www.youtube.com/watch?v=ijps41MkSzw

Application of DNA Fingerprinting:

1. Personal Identification
2. Diagnosis of Inherited Disorders
3. Development of Cures for Inherited Disorders
4. Paternity and Maternity Determination

Paternity Testing - uses DNA, usually taken from a cheek swab, to determine whether
a man is the child’s biological father
Samples obtained:
1. Blood
2. Buccal swabs
Except in the case of identical multiple births, everyone’s DNA is unique. A child
receives half of his or her genetic material (DNA) from the biological mother, and half
from the biological father. During DNA testing, the genetic characteristics of the child are
compared to those of the mother. Characteristics that cannot be found in the mother
must have been inherited from the father
DNA paternity testing is the most accurate form of paternity testing possible. If
DNA patterns between the child and the alleged father do not match on two or more
DNA probes, then the alleged father can be totally ruled out. If the DNA patterns
between mother, child, and the alleged father match on every DNA probe, the likelihood
of paternity is 99.9 percent.

DNA Paternity Test during pregnancy:

1. Noninvasive prenatal paternity test (NIPP) - This test analyzes fetal DNA
found in a pregnant woman’s blood during the first trimester.
2. Chorionic villus sampling (CVS) – A small sample of tissue from placenta is
taken to compare the DNA from the mother’s and the potential father’s DNA.
This takes place between 10 to 13 weeks after a woman’s last menstrual
period. The procedure carries a slight risk of miscarriage or pregnancy loss
3. Amniocentesis – a small amount of amniotic fluid is compared to the DNA
from the mother and the potential father. Takes place between the 15th and
20th weeks of pregnancy. The test slightly increases the risk of miscarriage.
References
Molecular Biology and FISH
Prepared by: Nicole Angela Cristobal
https://www.britannica.com/science/molecular-biology
https://www.aboutbioscience.org/topics/molecular-biology/
https://www.genome.gov/about-genomics/fact-sheets/Fluorescence-In-Situ-Hybridizatio
n
https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327/

Microarray and MALDI-TOF

Prepared by: Gio Glorioso
https://www.nature.com/scitable/definition/microarray-202/#:~:text=A%20microarray%20
is%20a%20laboratory,known%20DNA%20sequence%20or%20gene
https://www.future-science.com/doi/10.2144/000112046
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467903/
https://academic.oup.com/femsre/article/36/2/380/565595#:~:text=The%20capability%2
0of%20MALDI%2DTOF,monitoring%2C%20and%20food%20quality%20control
https://journals.lww.com/oncology-times/fulltext/2003/10100/maldi_tof_mass_spectrome
try__getting_a_feel_for.11.aspx
https://pcl.tamu.edu/proteinpeptide-mass-determination/procedure-for-maldi-tof-analysis
/#:~:text=In%20MALDI%2DTOF%20mass%20spectrometry,onto%20a%20metal%20sa
mple%20plate.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150354/
https://www.news-medical.net/life-sciences/Microarray-Aplications.aspx?fbclid=IwAR3X
qISmrxMYLmFevSjJn-XW3emXIFGDoN5knp4DCf67HtCNSchKcnxinE8

DNA Sequencing
Prepared by: Ma. Ynna de Guzman
Griffitghs, A. (n.d.) DNA Sequencing. Retrieved from
https://www.britannica.com/science/DNA-sequencing
Nanopore DNA Sequencing (n.d.) Retrieved from
https://nanoporetech.com/applications/dna-nanopore-sequencing
Sanger Sequencing: Introduction, Principle, and Protocol (February 21,
2020) Retrieved from
https://www.cd-genomics.com/blog/sanger-sequencing-introduction-principle-and
-protocol/
The Human Genome Project (n.d.) Retrieved from
https://www.ndsu.edu/pubweb/~mcclean/plsc431/students99/leno.htm#:~:text=Th
e%20Human%20Genome%20Project%20is,other%20components%20of%20the
%20genome
What is DNA Sequencing (n.d.) Retrieved from
https://modmedmicro.nsms.ox.ac.uk/the-dna-sequencing-technologies-we-use/

PCR Testing
Prepared by: Cesar Ian dela Sierra
https://my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing#test-de
tails
https://www.oecd.org/coronavirus/policy-responses/testing-for-covid-19-how-to-best-use
-the-various-tests-c76df201/#back-fnotea0z4
DNA Fingerprinting
Prepared by: Ma. Ynna de Guzman
Aryal, S. (2021, February 04) DNA Fingerprinting – Principle, Methods,
Applications. Retrieved from
https://microbenotes.com/dna-fingerprinting-principle-methods-applications/
DNA Paternity Test (n.d.) Retrieved from
https://my.clevelandclinic.org/health/diagnostics/10119-dna-paternity-test
Karki, G. (2017, November 24) Restriction fragment length polymorphism
(RFLP): principle, procedure and application. Retrieved from
https://www.onlinebiologynotes.com/restriction-fragment-length-polymorphism-rfl
p-principle-procedure-application/
Paternity Blood Tests and DNA (2018, October 02) Retrieved from
https://www.findlaw.com/family/paternity/paternity-tests-blood-tests-and-dna.html