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Unit-4 Dr.

Sudha Mathpal

DNA Diagnostics

Genetic analysis of human diseases


Genetic analysis is the overall process of studying and researching in fields of science that
involve genetics and molecular biology.
Genetic analysis may be done to identify genetic/inherited disorders and also to make
a differential diagnosis in certain somatic diseases such as cancer. Genetic analyses of cancer
include detection of mutations, fusion genes, and DNA copy number changes.
Genetic disorders occur when a mutation affects your genes or when you have the wrong amount
of genetic material. Genes are made of DNA (deoxyribonucleic acid), which contain instructions
for cell functioning and the characteristics that make you unique.
You receive half your genes from each biological parent and may inherit a gene mutation from
one parent or both. Sometimes genes change due to issues within the DNA (mutations). This can
raise your risk of having a genetic disorder. Some cause symptoms at birth, while others develop
over time.
Genetic disorders can be:
 Chromosomal: This type affects the structures that hold your genes/DNA within each
cell (chromosomes). With these conditions, people are missing or have duplicated
chromosome material.
 Complex (multifactorial): These disorders stem from a combination of gene mutations
and other factors. They include chemical exposure, diet, certain medications and tobacco
or alcohol use.
 Single-gene (monogenic): This group of conditions occurs from a single gene mutation.
Chromosomal disorders
 Down syndrome (Trisomy 21).
 FragileX syndrome.
 Klinefelter syndrome.
 Triple-X syndrome.
 Turner syndrome.
 Trisomy 18.
 Trisomy 13.
Multifactorial disorders
 Late-onset Alzheimer’s disease.
 Arthritis.
 Autism spectrum disorder, in most cases.
 Cancer, in most cases.
 Coronary artery disease.
 Diabetes.
 Migraine headaches.
 Spina bifida.
Unit-4 Dr. Sudha Mathpal

 Isolated congenital heart defects.


Monogenic disorders
 Cystic fibrosis.
 Deafness that’s present at birth (congenital).
 Duchenne muscular dystrophy.
 Familial hypercholesterolemia, a type of high cholesterol disease.
 Hemochromatosis (iron overload).
 Neurofibromatosis type 1 (NF1).
 Sickle cell disease.
 Tay-Sachs disease.
Rare genetic disorders include:
 AA amyloidosis.
 Adrenoleukodystrophy (ALD).
 Ehlers-Danlos syndrome.
 Mitochondrial diseases.
 Usher syndrome.
Causes of genetic disorders
To understand genetic disorder causes, it’s helpful to learn more about how your genes and DNA
work. Most of the DNA in your genes instructs the body to make proteins. These proteins start
complex cell interactions that help you stay healthy.
When a mutation occurs, it affects the genes’ protein-making instructions. There could be
missing proteins. Or the ones you have do not function properly. Environmental factors (also
called mutagens) that could lead to a genetic mutation include:
 Chemical exposure.
 Radiation exposure.
 Smoking.
 UV exposure from the sun.
Symptoms of genetic disorders
Symptoms vary depending on the type of disorder, organs affected and how severe it is. You
may experience:
 Behavioral changes or disturbances.
 Breathing problems.
 Cognitive deficits, when the brain can’t process information as it should.
 Developmental delays that include challenges with speech or social skills.
 Eating and digestive issues, such as difficulty swallowing or an inability to process
nutrients.
 Limb or facial anomalies, which include missing fingers or a cleft lip and palate.
 Movement disorders due to muscle stiffness or weakness.
 Neurological issues such as seizures or stroke.
 Poor growth or short stature.
Unit-4 Dr. Sudha Mathpal

 Vision or hearing loss.

How are genetic disorders identified


If you have a family history of a genetic disorder, you may wish to consider genetic counselling
to see if genetic testing is appropriate for you. Lab tests can typically show whether you have
gene mutations responsible for that condition. In many cases, carrying the mutation does not
always mean you’ll end up with it. Genetic counsellors can explain your risk and if there are
steps you can take to protect your health.
If there’s a family history, DNA testing for genetic disorders can be an important part of starting
a family. Options include:
 Carrier testing: This blood test shows whether you or your partner carry a mutation
linked to genetic disorders. This is recommended for everyone considering pregnancy,
even if there is no family history.
 Prenatal screening: This testing usually involves blood testing from a pregnant person
that tells them how likely it is that a foetus could have a common chromosome condition.
 Prenatal diagnostic testing: You can find out whether the developing foetus faces a
higher risk for certain genetic disorders. Prenatal testing uses a sample of fluid from your
uterus (amniocentesis).
 Newborn screening: This test uses a sample of your newborn baby’s blood and is
performed on all babies born in Ohio. Detecting genetic disorders early in life can help
your child receive timely care if needed.

Mutational Analysis
The search for unknown mutations in genomic DNA is important for a broad spectrum of
research studies, including fundamental research on gene structure and function, the study of
genetic diseases and disorders, and species identification. In any living cell, mutations occur
when there is a failure in DNA repair.
Different Types of Mutations

Several different types of mutations have been identified:


1. Single Base Substitution (single nucleotide polymorphism [SNP]). A base is exchanged for
another (for example, C to T) in the DNA sequence. Depending on the substitution, this type of
mutation can change the encoded amino acid (missense mutation) to produce a different protein
or an incomplete protein (nonsense mutation) which can lead to a diseased state. An example of
this type of mutation is sickle cell disease.
2. Insertions and Deletions
Single or multiple base-pairs are incorporated or deleted from a DNA sequence. This type of
mutation can create frameshifts that cause the mRNA sequence to be not read properly by the
Unit-4 Dr. Sudha Mathpal

translational machinery. Frameshifts can have devastating consequences. An example of this


type of mutation is Huntington disease.
3. Duplications
A section of the genome is doubled. This type of mutation can create overexpression. An
example of this type of mutation is high blood pressure.
4. Translocations
A piece of one chromosome is transferred to a nonhomologous chromosome. An example of this
type of mutation is Burkitt lymphoma.
These mutations can be beneficial (or detrimental) depending on which gene they affect.
Mutational analysis assists with identifying unknown mutations as well as identifying precursors
to diseases and disorders by allowing researchers to study these mutations in a control
environment.

Mutation Detection Methods


Over the past couple of decades, mutation detection techniques, such as denaturing gradient gel
electrophoresis (DGGE), constant denaturing gel electrophoresis (CDGE), temporal temperature
gradient gel electrophoresis (TTGE), single-strand conformation polymorphism (SSCP),
and protein truncation test (PTT), have assisted researchers with analyzing mutations. More
recently, high resolution melt (HRM) analysis has also become a technique of choice for
mutation detection. All of these techniques require DNA amplification via PCR before the
technique can be used.

Denaturing Gradient Gel Electrophoresis


Denaturing gradient gel electrophoresis (DGGE) is an electrophoretic method to identify single
base changes in a segment of DNA. In a denaturing gradient acrylamide gel, double-stranded
DNA is subjected to an increasing denaturing environment and will melt in discrete segments
called "melting domains." The melting temperature (Tm) of these domains is sequence specific.
When the Tm of the lowest melting domain is reached, the DNA will become partially melted,
creating branched molecules. Partial melting of the DNA reduces its mobility in a
polyacrylamide gel. Since the Tm of a particular melting domain is sequence specific, the
presence of a mutation will alter the melting profile of that DNA when compared to wild type.
DNA containing mutations will encounter mobility shifts at different positions in the gel than the
wild type. If the fragment completely denatures, then migration again becomes a function of size.
Unit
Unit-4 Dr. Sudha Mathpal

Constant Denaturing Gel Electrophoresis (CDGE)


Constant denaturing gel electrophoresis (CDGE) is a modification of DGGE. In CDGE, the
denaturant concentration that gives optimal resolution from a parallel or perpendicular DGGE
gel is held constant. The optimal concentration of denaturant to use for a CDGE is determined
from the maximum split between
tween wild-type
wild and mutant DNA.

Temporal Temperature Gradient Gel Electrophoresis (TTGE)


Temporal Temperature Gradient Gel Electrophoresis (TTGE) exploits the principle on which
DGGE is based, without requiring a chemical denaturing gradient. Amplified mutant and wild wild-
type DNA from the gene of interest is loaded onto a polyacrylamide gel containing a constant
concentration of urea. During electrophoresis, the temperature is increased gradually and
uniformly. The result is a linear temperature gradient over the length of the electrophoresis run.
Thus, a denaturing environment is formed by the constant concentration of urea in the gel in
combination with the temporal temperature gradient. With no chemical gradient required, rapid,
high-throughput
throughput screening is possible.

Single-Strand
Strand Conformation Polymorphism
strand conformation polymorphism (SSCP) technique is based on the fact that single
Single-strand single-
sequence specific secondary structure. Sequence differences as small as a
stranded DNA has a sequence-specific
Unit-4 Dr. Sudha Mathpal

single base change can affect this secondary structure and can be detected by electrophoresis in a
nondenaturing polyacrylamide gel. Double-stranded mutant and wild-type samples are first
denatured into single strands and then loaded onto the gel. Differences in mobility of the single
strands between the control wild-type DNA and the other samples indicate a mutation. SSCP is a
widely used mutation screening method because of its simplicity. However, since experimental
conditions cannot be predicted for a particular DNA, it is important to optimize gel
electrophoresis conditions. The ability to detect single base changes rests on several factors
which optimize band resolution.

1. Fragment size — the estimated efficiency for detecting single base changes is 90–95% for
fragments less than 350 bp, but the efficiency will decrease as the length of fragment increases.
2. Gel temperature — migration differences due to a single mutation are observed at buffer
temperatures between 4–25°C. Optimal temperature must be determined empirically.
3. Gel additives — in some cases, 5–10% glycerol can be added to the gel to improve the
mobility differences in fragments. Since glycerol can reduce the mobility of single-stranded
DNA fragments at low temperatures, it is typically used with gels run near room temperature.
4. Crosslinking ratio — the acrylamide/bis ratio determines the percent of crosslinking. SSCP
gels generally use 1–2 % crosslinking. Acrylamide concentrations will vary from 5 to 10%.
5. Buffer concentration — gels are run with TBE buffer at concentrations of 0.5x or 1.0x. In
some cases, 0.5x TBE appears to give slightly better results than 1.0x TBE.

Protein Truncation Test (PTT)


Protein Truncation Test (PTT) is a mutation screening method that detects truncated proteins
after translation of the coding sequence.

High Resolution Melt


High Resolution Melt (HRM) analysis uses DNA melt curve profiles that are both specific and
sensitive enough to distinguish nucleic acid species based on small sequence differences for
mutation scanning, methylation analysis, and genotyping. The use of HRM for mutation
scanning is becoming a more popular technique for high-throughput environments because
HRM:
 Decreases the risk of contamination
 Increases throughput
 Saves on cost and turnaround time

DNA fingerprinting
Satellite DNA regions are stretches of repetitive DNA which do not code for any specific
protein. These non-coding sequences form a major chunk of the DNA profile of humans. They
depict a high level of polymorphism and are the basis of DNA fingerprinting. These genes show
a high level of polymorphism in all kind of tissues as a result of which they prove to be very
Unit
Unit-4 Dr. Sudha Mathpal

useful in forensic studies. Alec Jeffreys developed this technique in which he used satellite
DNAs also called VNTRs (Variable Number of Tandem Repeats) as a probe because it showed
the high level of polymorphism.
Principle of DNA fingerprinting
The human genome consists of innumerable small noncoding sequences which are inheritable
and repeatedly present. They can be separated from the bulk DNA as satellite upon performing
density gradient centrifugation and thus known as satellite DNA. They can be categorized into
depending on the length, base composition and tandemly
either microsatellites or microsatellites depending
repetitive units. These satellite DNAs show polymorphism and this polymorphism is the basis of
DNA fingerprinting. The repeat regions can be divided into two groups based on the size of the
le number tandem repeats (VNTRs) and short tandem repeats. These repeats act as
repeat - variable
genetic markers and every individual inherits these repeats from their parents. Thus, every
individual has a particular composition of VNTRs and this is the main principle of the t DNA
fingerprinting technique.
DNA Fingerprinting Steps:
Isolating the DNA.

Digesting the DNA with the help of restriction endonuclease enzymes.

Separating the digested fragments as per the fragment size by the process of electrophoresis.

Blotting the separated fragments onto synthetic membranes like nylon.

Hybridising the fragments using labelled VNTR probes.

Analysing the hybrid fragments using autoradiography.
Unit
Unit-4 Dr. Sudha Mathpal

DNA Fingerprinting Applications


1. In forensic science, it is used to identify prospective criminal suspects.
prospective
2. To prove paternity and establish familial ties.
3. To identify and protect the commercial crop and livestock types.
4. To figure out an organism's evolutionary history and the relationships between different
groupings of species.
Concept
cept of pharmacogenomics and pharmacogenetics
Pharmacogenetics is the study of genetic causes of individual variations in drug response
whereas pharmacogenomics deals with the simultaneous impact of multiple mutations in the
genome that may determine the patient's
p response to drug therapy.
Unit-4
Unit Dr. Sudha Mathpal
Unit-4
Unit Dr. Sudha Mathpal

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