Detection and Measurement of Genetic Variation 1. Blood Groups

1. Blood Groups:
Each of the blood group system is determined by a
different gene or set of genes. The various antigens that
can be expressed within a system are the result of
different DNA sequences in these genes.
Two blood systems that have special medical
significance, the ABO and Rh systems.
The ABO system consists of two major antigens, labeled
A and B located on the surface of erythrocytes.
Individuals can have one of four blood types depends on
presence or absence of one or both antigens.
The ABO system is encoded by a single gene on
chromosome 9, consists of three primary alleles, labeled IA,
IB , and IO.
 Individuals with IA allele have the A antigen on their
erythrocyte surface (blood type A),
 Those with IB have the B antigen on their cell surface
(blood type B).
 Those with both alleles express both antigens (blood type
AB), and
 those with two copies of IO allele have neither antigen (type
O blood), because the IO allele produces no antigens.
Relationship between ABO Genotype and blood type
Blood type
1. ABO blood system used in studies of genetic
variation among individuals and populations.
2. Determining the compatibility of blood
transfusions and tissue grafts.
3. Some combinations of these systems can produce
maternal- fetal incompatibility with serous results for
the fetus (Rh- system)
The principle of this technique is to detect variations in
DNA, RNA, and variations in serum proteins that
encoded by certain genes.
These variations are observable because all (DNA, RNA,
Protein) can be separated by means of an electric field.
Clinical Application:
To determine whether an individual has normal hemoglobin
(HbA) or the mutation that causes Sickle cell disease
(HbS). The replacement of glutamic acid with valine in the
ß- globin chain produces a difference in electrical charge.
The hemoglobin is placed in an
electrically charged gel composed of
starch or agarose. The slight
difference in charge resulting from
amino acid replacement causes the
HbA and HbS forms to migrate at
different rates through the gel.
After several hours of migration, the
protein then stained with chemical
solutions so that their positions can be
So polymorphism can detected if the
HbA is homozygote or HbSS
homozygote, or having a
heterozygote HbA on one
chromosome and HbS on the other.
The rate of variation in human DNA occurs at an
average of 1 in every 300 to 500 base pair (bp).
Thus, approximately 10 million polymorphisms may
exist among the 3 billion bp that comprise the human
Fortunately, molecular techniques developed during
the past 30 years enable the detection of thousands of
new polymorphisms at the DNA level.
These techniques includes:
PCR making millions of copies of a short, specific DNA sequence
very quickly.
 Heating- cooling cycles are used to denature DNA and then
build new copies of a specific, primer- bounded sequence.
 Clinical Application:
Because of its speed and ease of use, this technique is now widely
used for:
 Assessing genetic variation for diagnosis genetic diseases
 forensic purpose
 detection and diagnosis of infectious diseases
 used as fingerprints to identify genetic relationship between
individuals, such as parent- child or between siblings, and
 are used in paternity testing
PCR instruments includes:
Thermocycler PCR
Agarose gel electrophoresis
PCR process requires four components:
1. Two primers: each consisting of 15-20 bases of
DNA, containing sequences complementary to the
3’ end of target region of DNA that contains the
polymorphism or a mutation that causes disease.
2. Heat- stable DNA polymerase enzyme: originally
isolated from the bacterium Thermus aquaticus
with a temperature optimum at round 70 C.
3. A large number of free DNA nucleotides (dNTPs).
4. Small quantity of Genomic DNA from an
individual act as a template.
 Typically,
PCR consists of a series of 20- 40 repeated
temperature changes called cycles, with each cycle
commonly consisting of 2-3 discrete temperature steps
usually three.
1. Denaturation step:
The genomic DNA is first heated to a temperature of
94-98 C for 20-30 seconds. It causes melting of the
DNA template by disrupting the hydrogen bonds
between complementary bases, yielding singlestranded DNA molecules.
2. Annealing step:
The reaction temperature is
lowered to 50-65 C for 20-40
seconds allowing annealing
of the primers to the single –
stranded DNA template.
Stable DNA-DNA hydrogen
bonds are formed when
primer sequence closely
matches the template
sequence. The polymerase
enzyme binds to the primertemplate hybrid and begins
DNA synthesis
3. Extension/ elongation
Taq polymerase has its
optimum activity temperature
at 75- 80 C, and commonly a
temperature of 72 C is used
with this enzyme. At this step
the DNA polymerase
synthsizes a new strand
complementary to the DNA
template strand by adding
dNTPs in 5’ to 3’ direction.
The DNA polymerase will
polymerize a thousand bases
per minute.
Finally agarose gel
electrophoresis is
employed for size
separation of the PCR
products. The size(s)
of PCR products is
determined by
comparison with a
DNA ladder (a
molecular weight
marker) which
contains fragments of
known size, run on a
gel alongside the PCR
Is a technique that exploits variations in homologous DNA
sequences. It refers to a difference between samples
of homologous DNA molecules that come from differing
locations of restriction enzyme sites.
It took advantage of the existence of bacterial enzymes
known as restriction endonucleases or restriction enzymes.
These enzymes are produced by various bacterial species to
“restrict” the entry of foreign DNA into the bacterium by
cutting or cleaving the DNA at specifically recognized
sequences. These sequences are called restriction sites.
For example, Escherichia coli produces a restriction
enzyme called EcoR1, that recognizes the DNA sequence
GAATTC so this enzyme cleaves the sequence between the
G and the A, this produces DNA restriction fragments.
 The
RFLP process:
First DNA is extracted from blood samples
then digested by a restriction enzyme
and then loaded on a gel.
Electrophoresis separate the DNA fragments according to
their size.
The DNA is denaturated and transferred to a solid
and hybridized with a radioactive probe.
Exposure to x-ray film appears specific DNA fragments
(bands) of different sizes in individuals.
The cloning of a gene produce many identical copies.
Recombinant DNA technology is used when a very
large quantity of the gene is required.
Recombinant DNA (rDNA) contains DNA from two
or more different sources. It required a vector to
introduce the rDNA into a host cell. One common type
of vector is plasmid. Plasmids are small accessory
rings of DNA from bacteria. The ring is not part of the
bacterial chromosome and replicates on its own.
 Two
enzymes are needed to introduce foreign DNA
into vector DNA.
The first enzyme, called a restriction enzyme, cleaves
the vector’s DNA,
and the second, called DNA ligase, seals foreign DNA
into the opening created by the restriction enzyme.
The single-stranded, but complementary, ends of the
two DNA molecules are called “sticky ends” because
they can bind a piece of foreign DNA by
complementary base pairing
Bacterial cells take up recombinant plasmids.
Thereafter, if the inserted foreign gene is replicated and
actively expressed, the investigator can recover either
the cloned gene or a protein product.
Example: Cloning of the human Insulin gene in
bacterial cell.

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