What is Anemia?

• Anemia is defined as a low number of red blood cells.

• In a routine blood test, anemia is reported as a low hemoglobin or hematocrit.

• Hemoglobin is the main protein in your red blood cells. It carries oxygen, and
delivers it throughout your body.
• If you have anemia (low number of red blood cells), your hemoglobin level will
be low too.
• If it is low enough, your tissues or organs may not get enough oxygen.

• Symptoms of anemia -- like fatigue or shortness of breath -- happen because your

organs aren't getting what they need to work the way they should.
 Thalassemia
• Thalassemia is an inherited (i.e., passed from parents to children through genes)
blood disorder caused when the body doesn’t make enough of a protein called
hemoglobin, an important part of red blood cells.
• When there isn’t enough hemoglobin, the body’s red blood cells don’t function
properly and they last shorter periods of time, so there are fewer healthy red
blood cells traveling in the bloodstream.
• Thalassemia is a blood disorder passed down through families (inherited) in
which the body makes an abnormal form or inadequate amount of hemoglobin.
Hemoglobin is the protein in red blood cells that carries oxygen. The disorder
results in large numbers of red blood cells being destroyed, which leads to
anemia.
• each red blood cell have 200-300 million molecules of hemoglobin (Hb).
Structure of hemoglobin
 Molecular basis of thalassemia
• Hemoglobin is made of two proteins:
1. Alpha globin--- 2 subunit (gene on chromosome 16)
2. Beta globin---2 subunit (gene on chromosome 11)
• Thalassemia occurs when there is a defect in a gene that helps control production of one of
these proteins.
• α-Thalassemia: α-Thalassemia are caused by a decreased synthesis or total absence of α-globin chain of
Hb.
• β-Thalassemia: Decreased synthesis or total lack of the formation of β-globin chain causes β-
thalassemia.
 Molecular basis of β-thalassemia
• β-Thalassemia: Decreased synthesis or total lack of the formation of β-globin chain causes β-thalassemia.
• Beta globin---2 subunit (gene on chromosome 11)
• There are mainly two types of β-thalassemia:
1. β-Thalassemia major (receive the faulty gene from both parents)
2. β-Thalassemia minor (receive the faulty gene from only one parent)
• Thalassemia minor occurs if you receive the faulty gene from only one parent. People with this form of
the disorder are carriers of the disease. Most of the time, they do not have symptoms.
• Thalassemia major occurs if you receive the faulty gene from both parents.

β-globin gene from Father Normal Normal Defective

β-globin gene from Mother Normal Defective Defective

 Molecularbasis
Molecular basisofofβ-thalassemia
thalassemia
• In beta thalassemia minor, the one allele is mutated and another is normal which produce a
small amount of normal mRNA and, thus, a low amount of beta globin chain is still synthesized.
The quantity of hemoglobin is reduced·
• In beta thalassemia major, beta globin gene of both alleles are unable to encode for any
functional mRNA and therefore there is no beta chain synthesize so no hemoglobin produced.
 Consequences of Beta-Thalassemia
The Pathophysiology of Beta-Thalassemia:

• Beta-thalassemia is caused by reduced or absent synthesis of the beta-globin chains of the adult hemoglobin
tetramer (HbA), which is made up of two α-globin and two β-globin chains (α2β2).

• When beta-globin chains are absent, alpha-globin chains and their degradation products precipitate, so there
isn’t enough hemoglobin.

• When there isn’t enough hemoglobin, the body’s red blood cells don’t function properly and they last
shorter periods of time, so there are fewer healthy red blood cells traveling in the bloodstream, which leads
to anemia.

• Anemia, in turn, stimulates erythropoietin synthesis, resulting in intense proliferation of the bone marrow,
skeletal deformities, and a variety of growth and metabolic abnormalities.

• Splenomegaly (Splenomegaly is defined as the enlargement of the spleen measured by weight or size) is
typically seen in patients with beta-thalassemia as a result of extramedullary hematopoiesis or as a response to
 Consequences of Beta-Thalassemia
• How Genetic
Defects in Beta-
Thalassemia Lead
to Ineffective
Erythropoiesis
and
Hemolysis????
 Beta-Thalassemia
Symptoms of Beta Thalassemia

• It is characterize by severe anemia that can begin months after birth Paleness
• Delays in growth and development
• Bone marrow expansion.
• Untreated Beta Thalassemia major can lead to child death due to heart failure.

Treatment of Beta Thalassemia

• Regular blood transfusion helps prevent severe anemia and allows for more normal growth
and development
• There are various medications that target the production of red blood cells (i.e.
erythropoietin)
• Iron chelation therapy
• Bone marrow transplant
 The amplification-refractory mutation system (ARMS)
• The amplification-refractory mutation system (ARMS) is a simple PCR method for detecting any mutation
involving single base changes or small deletions.
• ARMS is based on the use of sequence-specific PCR primers that allow amplification of test DNA only when the
target allele is contained within the sample.
• Following an ARMS reaction the presence or absence of a PCR product is diagnostic for the presence or
absence of the target allele.

What is ARMS-PCR? why it is called an amplification refractory mutation system.

The allele-specific PCR is also called as the (amplification refractory mutation system) ARMS-PCR because of the use
of two different primers for two different alleles.

Here the word “refractory” is very important (Refractory= resistant to something).

As we discussed, two sets of primers are designed, the mutant set of the primer is refractory (resistant) to the
normal PCR and the normal set of the primers are refractory to the mutant PCR reaction.
That is why it is called an amplification refractory mutation system.
 The amplification-refractory mutation system (ARMS)
What is ARMS or Allele-Specific PCR?
• Alleles are alternative gene forms. When a mutation occurs, it produces two different alleles
for a gene; one mutant one and another normal one. The Allele-specific PCR has the power
to detect a single specific allele.

• Meaning, If you wish to amplify only a mutant allele, design a primer set accordingly and
amplify it using this technique. Each set of specific primers is designed for each specific
allele.

• However, we need to do a minor change in primers to amplify each allele. PCR amplifies each
allele simultaneously, afterward. While gel electrophoresis prepares results to evaluate.

• Two different primer sets are prepared for separate alleles. The mutant allele-specific primers
are refractory (resistant) to normal allele and vice versa. Henceforth, known as Amplification
Refractory Mutation System. R. Newton had given the name ARMS- PCR.
 Principle of ARMS- PCR
• The principle of the present technique relies on the modification of primers to amplify a specific allele.

• The 3’ end of the primer is modified in such a way that one primer can amplify a mutant allele while the other
can amplify the normal allele. To do this, researchers modify a few bases from the primer’s 3’ OH end based on
the mismatch concept.

• Only a single set of primers can only amplify a specific allele present in a sample, during the reaction. Gel
electrophoresis helps to examine results.

The mismatch concept:

• By introducing a mismatch in a primer makes refractory amplification possible. Here the mismatch between the
primer and the template DNA plays a crucial role in achieving the amplification.

• Mismatch indeed alters the annealing temperature for different sets of primers. Here are examples of
several mismatches.

• Strong mismatch: G/A, C/T, T/T

• Medium mismatch: A/A, G/G, C/C,

• Weak mismatch: C/A, G/T

 Principle of ARMS- PCR
• See the image below so you can understand the concept easily.

Point mutation from A to G

The image represents the concept of a mismatch for ARMS-PCR.

 Principle of ARMS- PCR
• Primer preparation and designing play a crucial role here. Remember the primer must be allele-specific. A few points
to keep in mind while preparing are,

• See the image in previous slide first, suppose our DNA sequence has A-G point mutation viz, A in normal allele and G in
place of A in the mutant allele.

• We have to design a forward primer in such a manner that for the normal allele the primer contains T (complementary
to A) at 3’ end and the mutant primer contains C in place of T.

• Although it works magically when we add a mismatched base near our mutation at the 3’ end. You may wonder why
we need to add a mismatch? The mismatch is the key factor in achieving the amplification.

• We should add strong mismatch to avoid false-amplification near the 3’-OH end of the primer (at -2 position ideally).
Mismatch prevents binding to the non-complementary sequence and terminates amplification. Mismatch indeed
alters the annealing temperature for different sets of primers.

• C:T, G:A and T:T is the strong mismatch base pairs that reduce the amplification process up to 100 fold. We have to
modify only a single primer, either forward or reverse, the other primer usually remains unchanged and can work for
either allele.

• Besides, the primer should follow all the standard criteria for primer designing. It must have less GC content, less hairpin
structure and a length between 20 to 30 nucleotides.
 The amplification-refractory mutation system (ARMS)

• A standard ARMS PCR consists of two complementary reactions (two tubes) and utilizes 3
primers.

• One primer is constant and complementary to the template in both reactions,

• the other primers differ at their 3' terminal residues and are specific to either the wild type
DNA sequence or the mutated sequence at a given base - only one of these primers is used per
tube.

• PCR products are separated by electrophoresis through an agarose gel containing ethidium
bromide.

• If the sample is homozygous mutant or homozygous wild type amplification will only occur in
only one of the tubes, if the sample is heterozygous amplification will be seen in both tubes.
 Procedure and steps of ARMS-PCR
1. Primer designing
2. Collection of blood
3. DNA extraction
4. PCR Amplification by allele specific primers
5. Agarose gel electrophoresis
6. Results interpretations

• One of the common mutation in beta thalassemia : nucleotide 29 (A>G),

• Normal allele contain A in 29 nucleotide position of β-globin gene

• Mutant allele contain G in 29 nucleotide position of β-globin gene
 Procedure and steps of ARMS-PCR
1. Primer design:

• 3 primers are used.

• 2 forward primer have to design in such a manner that for the normal allele the primer
contains T (complementary to A) at 3’ end and the mutant primer contains C in place of T
with a strong mismatch at -2 position ideally.
• One internal control primer (amplify a larger portion of beta globin gene) is also used to
check that our reaction preparation, cycling conditions and other practices are fine.
• And we have to design a reverse Primer for the three forward primers.

2. Collection of blood

3. DNA extraction
 Procedure and steps of ARMS-PCR
4. PCR Amplification by allele specific primers:

• 2 tubes for each sample have to be prepared.

• The reactions for the mutant and the normal alleles are usually carried out in separate tubes. But these
may be done in the same tube after labeling the two primers with different fluorescent dyes.

• In order to get ‘correct’ amplification, we should have to maintain healthy amplification conditions.

• Set higher annealing temperature, depending upon the primer’s highest annealing temperature.

• If set lower, it increases the chances of non-specific amplification, remember, we are playing with
only a single base change primer.

• Set a lower PCR cycle to maintain the specificity of the reaction. Ideally set 22 to 25 cycles only. More
PCR cycles increase the chances of non-specific bindings, primer-dimer and redundant results. It
gives false-positive results.
ARMS-PCR Procedure
 Procedure and steps of ARMS-PCR
5. Agarose gel electrophoresis:

• The ARMS PCR has direct diagnostic use.

• The results are run on 2% agarose gel, ideally under proper agarose gel running conditions. Along
with, add a 1000 bp molecular marker.
• The gel electrophoresis technique is sufficient to separate different alleles and evaluate results. ARMS-
PCR results are interesting, go to the next section.

6. Results and interpretation:

• To examine results, the very first is to observe the internal control. The internal control band must be
present in all reactions. It shows that our reaction preparation, cycling conditions and other practices
are fine.
• Then analyze each band for the normal and mutant alleles.
• To understand the results let’s prepare a hypothetical situation.
• Suppose we have three samples; one normal, one heterozygous carrier and one homozygous
dominant. We have prepared 2 tubes for each sample, a total of 6 tubes for samples.
 Procedure and steps of ARMS-PCR
5. Agarose gel electrophoresis:

• The ARMS PCR has direct diagnostic use.

• The results are run on 2% agarose gel, ideally under proper agarose gel running conditions. Along
with, add a 1000 bp molecular marker.
• The gel electrophoresis technique is sufficient to separate different alleles and evaluate results. ARMS-
PCR results are interesting, go to the next section.

6. Results and interpretation:

• To examine results, the very first is to observe the internal control. The internal control band must be
present in all reactions. It shows that our reaction preparation, cycling conditions and other practices
are fine.
• Then analyze each band for the normal and mutant alleles.
• To understand the results let’s prepare a hypothetical situation.
• Suppose we have three samples; one normal, one heterozygous carrier and one homozygous
dominant. We have prepared 2 tubes for each sample, a total of 6 tubes for samples.
 Procedure and steps of ARMS-PCR
6. Results and interpretation:

• As shown in the figure above, homozygous normal shows a single DNA band, heterozygous carrier
shows two bands (one for normal and one for mutant allele) and homozygous disease conditions show
a single mutant band.

• It can also be stated like this, the mutant primer can’t amplify the normal homozygous allele, both
primers amplify both alleles in heterozygous and only a single mutant primer can amplify the mutant
allele.
 The amplification-refractory mutation system (ARMS)

Why aren’t we using high-fidelity Taq DNA polymerase in ARMS PCR?

In the conventional ARMS-PCR, we use only a simple Taq DNA polymerase. The high-fidelity DNA
polymerase has polymerase as well as exonuclease activity and hence will remove the
mismatches.

The Amplification Refractory Mutation System (ARMS) is an application of PCR in which DNA is
amplified by allele specific primers. In PCR mismatch at the 3’ end of the primer can dramatically
reduce the annealing and hence the amplification. This is due to the absence of 3’ to 5’
exonuclease proofreading activity of Taq polymerase. High fidelity DNA polymerases, that have
this activity, cannot be used in ARMS. It is an extremely useful method for identification of point
mutations or polymorphisms.
 Questions
• Write a short note on Anemia. (Very popular question)

• Write a short note thalassemia, beta thalassemia.

• Write down the molecular basis of thalassemia/ beta thalassemia.

• Describe pathology / clinical significances / consequences of Beta-Thalassemia.

• What is ARMS-PCR?

• How ARMS-PCR is used in diagnosis of Beta-Thalassemia: explain with illustration.

• “Amplification refractory mutation system can be used to detect point mutation in beta globin gene” – explain.

• What are the advantages of ARMS-PCR?

• Why aren’t we using high-fidelity Taq DNA polymerase in ARMS PCR?