FKUNISBA2011

Intro Lab
Thalassemia
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Intro Lab Thalassemia
Bu Lely
Introduction Lab Act FBS 4
Thalassemia
Heterogeneous group of inherited disorders hemoglobin synthesis
Characterized by the absence or reduced output of one or more of
globin chains of hemoglobin

Hemoglobin (Hb)
Normal Adult :
Hb A (22) 97 %
Hb A2 (22) 1-2 %
Hb F (22) 0,5 1 %

-thalasemia deficiency of chains

-thalasemia deficiency of chains
Variation in the clinical manifestations
Thalasemia mayor homozygous state
Thalasemia minor heterozygous form
Thalasemia intermedia

Pathophysiology
Deficient hemoglobin (Hb) synthesis resulting from deficient
production of one or another of the polypeptide chains of globulin
Red cell membran demage
Shortened red cell survival

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Animal Cell

Isolation of DNA

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Distruption of the cell membrane
Disosiasi of the protein DNA complexes

Separation of the DNA from other solube cellular components

Polymerase Chain Reaction

An in vitro technique, which allows the amplification of a specific
DNA
Tube system for DNA replication that allows a target DNA
sequence to be selectively amplified, or enriced several million-
fold iin just a few hours

PCR

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 Specific DAN sequence amplification techique to enable further
DNA analysis
Principle : - DNA replication proses
DNA template
Primer (20-25 nucleotida)
Polymerase Enzyme (Taq Polimerase)
Substrate (dNTP
Difference PCR : DNA unwind through
temperature change (high temperature)
Replication : DNA unwind by enzyme

The cycling reactions/ replication cycles :

Denaturation
Denaturation at 94C :

During the denaturation, the double strand melts open to single

stranded DNA, all enzymatic reactions stop (for example : the extension
from a previous cycle).

Extension
extension at 72C :

Extension of nucleotide chain catalyzed by Taq plymerase using dNTPs

as substrates to form a new DNA chain as complement of DNA template
in temperature optimal for Taq polymerase

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 1.8E+09

1.6E+09

1.4E+09

1.2E+09

DNA copy number

1.0E+09

8.0E+08

6.0E+08

4.0E+08

2.0E+08

0.0E+00
0 5 10 15 20 25 30 35
PCR cycle
10

7
DNA copy number (log)

0
0 5 10 15 20 25 30 35
PCR cycle
Electrophoresis DNA Resolution of DNA fragments On agarose gel

Electrophoresis Hemoglobin Identification of hemoglobin variants

Qualitative
evaluation & Quantitative evaluation

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