Blue Illustrated Laboratory Presentation.pptx

1. MOLECULAR DETECTION
OF DISEASE DUE TO
GENETIC MUTATION
AIZA MALIK 2021-MLTE-001
HIRA RIAZ 2021-MLTE-002

2. INTRODUCTION
Genetic mutations refer to alterations in the DNA
sequence which affects the amount of gene product
produced. Molecular diagnostics map health accurately at
molecular level and detect biological molecules
(biomarkers) associated with genetic diseases in patient
samples. Biomarkers can be specific proteins, DNA or RNA
sequences, or metabolites that are present (or present at
altered levels) in body fluid or tissue samples of affected
individuals compared to healthy individuals.

3. • To detect and determine a
particular disease
• To identify cause of disease
• To predict the likelihood of
developing a disease
• To monitor the progress of a
disease.
OBJECTIVE
As these DNA-based approaches
can diagnose the disease at the
genetic level, so they are now
widely used in personalized
medicine for various purposes
such as

4. MOLECULAR DIAGNOSTIC
TECHNIQUES
Molecular diagnostics for genetic mutations involve various
techniques aimed at identifying specific mutations or variations
in the DNA sequence associated with diseases or conditions.
Since there is no single perfect method to screen for unknown
mutations so combinations of these methods may be necessary
for accurate genetic diagnosis.

5. Some commonly used molecular diagnostic methods for genetic
mutations are
• Polymerase Chain reaction (PCR)
• Multiplex Ligation-dependent Probe Amplification (MLPA)
• High-Resolution Melting Analysis (HRMA)
• Fluorescence In Situ Hybridization (FISH)
• DNA microarrays
• Sequencing
• Restriction Fragment Length polymorphism (RFLP)
• Southern Blotting

6. Polymerase Chain reaction (PCR)
The applications of polymerase chain
reaction (PR) technology to genomic
screening have made rapid and accurate
genetical diagnosis possible.It is a widely
used molecular biology technique involves
repeated cycles of DNA denaturation, primer
annealing, and DNA extension using a heat-
stable DNA polymerase enzyme.It amplifies
specific regions of DNA, allowing for the
detection of mutations in genes of interest.

7. Following PCR techniques are used to identify specific
mutations associated with various genetic disorders.
• real-time PCR
• allele-specific PCR
• digital PCR
• multiplex PCR

8. Real time PCR also known as quantitative
PCR allows for the quantification of DNA
during the amplification process in real-
time. It uses fluorescent probes or DNA-
binding dyes to measure the accumulation
of amplified DNA with each cycle of PCR.
qPCR is highly sensitive and accurate,
making it suitable for detection of genetic
mutations associated with diseases
Real time PCR

9. Allele-specific PCR (AS-PCR) is a
modification of standard PCR that
amplifies only the allele containing the
mutation of interest. This technique uses
allele-specific primers designed to
specifically amplify the wild-type or
mutant allele. AS-PCR is particularly useful
for the detection of specific genetic
variations associated with inherited
diseases or polymorphisms.
Allele-specific PCR

10. dPCR is a highly sensitive method for quantifying nucleic acids. It can be
used to detect and quantify rare mutations present at low levels in a
sample. dPCR is extensively used in detecting mutation status of
heteroplasmic mitochondrial DNA, which determines the manifestation
and progression of mtDNA-related diseases, as well as allows for the
prenatal diagnosis of monogenic diseases and the assessment of the
genome editing effects.
Limitations
• necessity of highly allele-specific probes
• a large sample volume.
Digital PCR

12. Multiplex PCR allows for the
simultaneous amplification of multiple
target sequences in a single reaction. It
uses multiple primer pairs targeting
different regions of the genome, enabling
the detection of multiple mutations or
targets in a single PCR assay. Multiplex
PCR is useful for its application in
genetic screening
Multiplex PCR

13. Multiplex Ligation-dependent Probe Amplification (MLPA) is used to detect
deletions, duplications, and other copy number variations in specific
genes.It can be particularly useful for identifying mutations associated with
disorders caused by structural variations in the genome.MLPA assay can
also be used in the molecular diagnosis of genetic diseases characterized
by the presence of abnormal DNA methylation. Due to the large number of
genes that can be analyzed by a single technique, MLPA assay represents
the gold standard for molecular analysis of all pathologies derived from the
presence of gene copy number variation.
MLPA

15. High-Resolution Melting Analysis (HRMA) is a molecular diagnostic
technique used to detect genetic mutations by analyzing the melting
behavior of PCR-amplified DNA fragments. It begins with the amplification
of the target DNA region using PCR, followed by a gradual increase in
temperature to denature the double-stranded DNA into single strands. As
the DNA strands separate, the fluorescence of a DNA-binding dye, such as
SYBR Green, decreases, generating a melting curve that reflects the melting
behavior of the amplified DNA fragments.
Mutations within the target DNA region alter the melting characteristics,
resulting in a shift in the shape or position of the melting curve compared
to the wild-type sequence.
HRMA

17. FISH is used to detect
chromosomal abnormalities or
gene rearrangements associated
with certain genetic disorders. It
involves the hybridization of
fluorescently labeled probes to
specific DNA sequences on
chromosomes.
FISH

18. In molecular diagnostics, sequencing is used to identify genetic mutations
by determining the precise order of nucleotides in a DNA sample. This can
involve techniques like
• Sanger sequencing
• Next-generation sequencing (NGS)
These can detect various types of mutations such as single nucleotide
polymorphisms (SNPs), insertions, deletions, and rearrangements. The
sequence data is then analyzed to pinpoint mutations associated with
diseases or genetic conditions.
Sequencing

19. Sanger sequencing is a
traditional method for
determining the nucleotide
sequence of DNA. It can be
used to directly sequence
genes and identify mutations
within them.
Sanger Sequencing

20. NGS technologies enable the rapid
and cost-effective sequencing of
large portions of the genome or
specific genes. Whole exome
sequencing (WES) and targeted gene
panel sequencing are used to
identify mutations associated with
inherited diseases.
NGS

22. DNA microarrays in molecular diagnostics utilize probes immobilized on a
solid surface to simultaneously analyze thousands of genes or genetic
variations. Patient DNA, labeled with a fluorescent dye, is hybridized to
these probes. If the patient's DNA contains mutations or variations
complementary to the probes, they bind to the corresponding probes on
the microarray. Through scanning the microarray, fluorescence signals
indicate successful hybridization. Data analysis of the intensity and
patterns of fluorescence helps identify genetic mutations or variations
present in the patient's sample.
DNA microarray

24. RFLP (Restriction Fragment Length Polymorphism) is
a technique used to detect genetic variations,
including mutations. It involves analyzing DNA
fragments that result from digesting a DNA sample
with specific restriction enzymes. By comparing the
lengths of these fragments between individuals,
researchers can identify variations such as
mutations associated with diseases. RFLP has been
particularly useful in identifying mutations linked to
genetic disorders like sickle cell anemia and cystic
fibrosis.
RFLP

26. Applications
• Disease study
• Parental testing
• Criminal suspects
Advantage
Tells either individual is homozygous or
heterozygous
Disadvantage
Costly

27. Southern blotting is a molecular technique used to detect specific DNA
sequences, including genetic mutations. It involves fragmenting DNA
samples, separating the fragments by size via gel electrophoresis,
transferring them onto a membrane, and hybridizing them with labeled
probes complementary to the target sequence. The presence of the target
sequence is then detected through probe binding. In mutation detection,
Southern blotting enables the identification of mutations by comparing the
presence or absence of specific DNA sequences between individuals or
between healthy and affected samples.
Southern Blotting

29. Cystic Fibrosis:
• Autosmal recessive disorder
• Due to abnormality in function and production of CFTR(Transmembrane
conductance regulator)
Methods of detection:
1. Hybridization
Genetic diseases
PCR HYBRIDIZATIO
N
DETECTIO
N
BLOTTING

32. Genetic Diseases

34. THANK YOU
VERY MUCH!