Laboratory Techniques Answer 2022

Running head: LABORATORY TECHNIQUES
LABORATORY TECHNIQUES
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LABORATORY TECHNIQUES1
Answer1
Genes causing Chronic myeloid leukaemia (CML) can be identified through FISH
(Fluorescence in situ hybridization). It is another way to look at the chromosome. Fluorescent
dye is used, which binds specifically to the genes or particular areas in the chromosome. In the
case of CML, FISH techniques is used to identify the specific part of BCR-ABL gene on
chromosome (5). The dye tagging makes the defective gene easily visible and hence diagnosis
can be done. It is mainly used in the bone marrow or blood samples without culturing in the cells
first.
In case of Acute Myeloid leukaemia (AML), genes can be identified with various
techniques/ The commonly used technique is flow cytometry. The progenitors are having a
common percentage of CD45-positive and non-erythroid events. Various denominator
determines the morphologic enumeration (8). Morphologic as well as differentiation dependant
arrangements of leukaemia are restricted in their prognostic importance; cytogenetics in addition
to molecular genetics look to be significant for classifying entities having distinct forecasts and
clinical behaviour. Hence, the genes can easily be classified.
RFLP techniques is used in identifying the genes for sickle cell anaemia. Person suffering
from the sickle cell mutation do not have restriction site for normal β-globin genes. The absence
of this gene causes sickle cell anemia. RLFP helps in identifying the missing genes. In RFLP
technique, uses radioactive labelled β-globin probe for analysing sickle cell anaemia. The
mutation that reasons this disease corresponds with a Cvn I site (6). Chromosomes which is
lacking or having sickle mutation do not have the site where the chromosomes normal β-globin
genes. The process of digestion with is over with Cvn I in addition to hybridization with a β-
globin probe, DNA from the patient with sickle-cell anaemia produces fragment of size 1.3 kb,

LABORATORY TECHNIQUES2
DNA received from individuals with two normal β-globin genes produces 1.1 kb fragment, in
addition to transporter having 1.1- and 1.3-kb fragments.
ARMS technique is used in gene identification causing B-Thalassemia. The process
depends on the theory of allele-specific priming of the PCR (polymerase chain reaction) method,
which is a detailed primer that allows amplification at 3’ terminal nucleotide matches with its
target sequence. β-thalassaemia occurring can be identified my mutation in gene IVSI-5 (G→C),
here the 3’ nucleotide of the ARMS primer is G to base pair through the substituted C in mutant
DNA. The primer lead to form G-G mismatch in the normal DNA however it is the discrepancy
occurring for a short time and hence would not cause any extension of the primer by itself (2).
This mismatching mutation would help in the identification of β-thalassaemia. The strong
mismatches such as C-C, G-A and A-A were observed for reducing the efficiency to zero or less
than 5% (8). Hence, it prevents further amplification, mismatch with target sequence that needs
to be added in the second, third and fourth nucleotide from 3’ end of the primer. Therefore, as
there is no extension in the gene amplification easy identification can be done.
Gene causing α-Thalassemia can be identified by gap- PCR. Most of the α-thalassemia’s
occurs due to obliteration of one or more HBA genes, besides these deletions are also common in
the population affected with the disease. The molecular diagnostic techniques mainly focused on
explaining and identifying the cause of the common deletion process (1). Gap-PCR are the
chosen technique in this discussion. PCR primers which can flank the mutual breakpoints have
the ability to demonstrate the deletion process by generation of the PCR products of particular
size. The certain primers are used in laboratories depending on the common deletion of the
population that are being served. Gap-PCR easily then detects deletion of the HBA gene which is
missed during DNA sequencing. It has also been noticed that the Gap-PCR that helps in the

LABORATORY TECHNIQUES3
diagnosis of α-thalassaemia are associated with the rare kind of mutations. Hence, different other
technique is being employed such as next generation sequencing (NGS) as well as Multiple
ligation-dependent probe amplification (MLPA) technologies that are used in order to
characterise the mutation that is occurring in an individual at the molecular level and causing the
disease. Research study have shown that the basic principal of this gap-pcr depends on the
inability of the primers for generating PCR product until and unless the deletions gets back to the
flanking sequence together.
Answer 2
FISH Positive result for CML
FISH analysis denatures DNA. The florescent labelled DNA then identifies the control
and another one the target cell. In case of CML the target gene is BCR-ABL. First probes serve
as control that hybridises the target gene however outside the target location. In this picture we
can see that the target gene is detected along with normal, and hence further diagnosis can be
done.