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Techniques in Molecular
Biology2
Naglaa Alhusseini
Hybridization and blotting techniques
Hybridization:
Depends
on the association
between two polynucleotide
chains, which may be of the
same or of different origin or
length provided that a base
complementary exists between
these chains.
Hybridization can take place between:
 *DNA chains.
 * Complementary RNA chains.
 * DNA-RNA combination.

Probe: is a single strand piece of
either DNA or RNA usually labeled
with radioisotope or non radioactive
isotope, the nucleotide sequence of
the probe is complementary of either
DNA or RNA of interest.
Blotting Techniques
 Visualization
and identification of a
specific DNA or RNA fragment or
protein among the many thousand of
molecules , requires a number of
techniques which are called
collectively a blot transfer
techniques
A- Southern Blotting:
 It is diagnostic technique to detect
specific sequences containing a DNA
fragment using labeled
polynucleotide (as a probe) with
complementary sequence to the
target DNA
Steps of Southern blotting
DNA is isolated
 DNA cleavage : cut with restriction enzyme at
specific sites.
 Electrophoresis :The DNA fragments are
separated on agarose gel or polyacrymide gel
electrophoresis.
 The DNA is denatured within the gel.
 Blotting : The denatured DNA is transferred
from the gel to a sheet of nitrocellulose paper by
blotting technique.
 Hybridization : The DNA is then hybridized
with a specific labeled probe.
 Autoradiography:

Uses
1- detection of genes in genome
 2- detection of mutation
 3- diagnosis of infectious diseases
 4- restriction fragment length
polymorphism
 5- DNA fingerprinting
 6- confirm the product of PCR

B- Northen Blotting:
 It
is similar to Southern blotting, the
difference being the northern
blotting detects RNA with
complementary sequence to the
target RNA.
 Uses: For detection and quantitation
of RNA of deferent tissues to study
changes of gene expression
Western Blotting:
It detects protein by using labeled antibodies
to the protein.
 Protein are first isolated from the tissues
 Electrophoresis of the whole protein is done and
transfer to the nitrocellulose membrane and
fixed.
 After fixation , the protein is probed by
radioactive antibodies
 The pattern of the bands that contained protein
are visualized by virtue( asset) of radiation from
the probe
 Uses: Western blot is very useful to identify the
production of a specific protein in a tissue.

In Situ Hybridization:
Is another variety of hybridization uses intact
DNA molecules within metaphase chromosomes.
Uses:
 It is used for cytogenetic mapping of cloned
genes
 Identification of chromosome aberration
 Localization of mRNA expression

DNA microarray :
A DNA mircroarray consists of an arrayed series
of thousands of microscopic spots of DNA
oligonucleotides of specific DNA sequence known
as probe .
 Sample containing labeled cDNA(target) is added
. This is followed by probe target hybridization.
 The probe target interaction is quantitation of
the labeled target (fluorescence detection).

DNA microarray :
The DNA microarray contains thousands of
immobilized DNA sequences organized in an
area no longer than a microscope slide.
 These microarrays formed from a known gene
(a few dozen to 100 of nucleotides in length) are
placed on a slide surface using robotic devices
that accurately deposit “nanolitres quantities”
of DNA solution.

Uses:
These- microarrays are used to
analyze a sample for the presence of
gene variation (polymorphism or
genotyping) and mutations.
 Measure changes in the expression
levels of many genes (gene
expression analysis).

Advantage
 Quantitation of many genes at one time.
 Disadvantage
 Decreased specificity

Gene library:
 It is a collection of recombinant DNA represent
the entire genome of an organism. Gene library
can be of two types:

Genomic Library, DNA library

cDNA library
Genomic liberary :
 A genomic library is prepared from the
total DNA of a cell line or tissue
 It is prepared by performing partial
digestion ot total DNA with restriction
enzyme that cuts the DNA frequently
to produce larger fragments that intact
gene can be obtained.
 Phage vectors are ideal and preferred
for this as they accept large pieces of
DNA up to 20kb.
cDNA library :
 cDNA library represents the population of
mRNA in a tissue
 cDNA libraries are prepared by first isolation
all mRNA in a tissue .
 mRNA serves a template to prepare the cDNA
using the enzymes '' reverse transcriptase and
DNA polymerase ''
 Plasmids are the ideal preferred vectors for
cDNA libraries as they are workable with
smaller fragments
Restriction Fragment Length
Polymorphisms (RFLP
Restriction Fragment Length
Polymorphisms (RFLP Definition: Restricted
fragment length polymorphisms (RFLP) refers
differences or polymorphisms (Greek; poly=many
, morphos= form) resulting from mutation that
alter the site of restriction fragmentation
catalyzed by a restriction enzyme.
 They affect the restriction enzymatic
cleavage sites, DNA fragments of different sizes
will result these variation are called Restriction
Fragment Length Polymorphism.

The genome variation includes both
polymorphism and mutation.
 Polymorphism is a variation in nucleotide
sequence from one individual to another. If they
occur in or near the gene of interest, they provide
the potential linkage marker for the following
mutant genes through families


RFLP, is genetic variant that can be examined
by cleaving the DNA into fragments (restriction
fragments) with restriction enzyme, the length
of restriction fragment is altered if the genetic
variant alters, so as to create or abolish a site of
restriction endonuclease cleavage ( restriction
site), RFLP can use to detect human genetic
defect.
Steps
 Restriction digestion of the isolated DNA:
 Southern blot of restriction enzyme digested
DNA:
Clinical Applications of RFLP
1- Diagnosis of genetic diseases: RFLP is used for the
diagnosis of genetic disease such as sickle cell
hemoglobin.
2- Cloning of mutant gene: The demonstration of
linkage between an RFLP and an inherited
disease can be used for cloning of mutant gene
that causes the disease.
3- Genetic analysis: The RFLP linked to a genetic
disease can be used for genetic analysis. It is
useful for prediction of genetic disorder in the
offspring.
4- RFLP is also useful in human population genetics,
geographical isolates and comparison of genetic
make up of related species.

Restriction Map: Is a diagram illustrating the
linear arrangement of restriction enzyme
cleavage sites on a piece of DNA and provides a
means of characterizing DNA.
DNA Finger Printing (DNA Typing):
Definition: DNA fingerprinting refers to a
variation of RFLP to establish a unique pattern
of DNA fragments for an individual.
 Technique:

Isolation and DNA amplification of region of target
DNA including repetitive sequences by using PCR
 Restriction enzyme digestion


Southern blotting
Application of DNA fingerprinting:
 Forensic uses: DNA fingerprinting is useful to
prove the identity of a person. This is done by
comparing the RFLPs from the unknown sample
and RFLPs of a known sample.
 Identity of parents: Science VNTR are
identical from generation to generation, DNA
fingerprinting can be used to establish the
identity of parent.
 Identification of linkage of a disease: the
micro-satellites have been found to be associated
with genes causing disease such as myotonic
dystrophy.

. Gene therapy:
Definition: It refers to delivering of normal
copy of defective gene to patients
 Types of gene therapy: Two strategies have
been described depending on the recipient:
Germ cell line gene therapy: refers to
introduction of foreign DNA into zygote or early
embryo, in which a goal is to pass the change to
the offspring (transgenesis or transgenic
animals)
Somatic cell therapy: refers to the introduction
of genetic material into somatic cell of the
affected individuals. The recipient's genome is
changed but the change is not passed along to


Technique: The principles of somatic cell gene
therapy protocols include the following:
Isolation of healthy gene along with the sequence
controlling its expression
 insertion of the target DNA into the appropriate
vector or delivery system
 Introduction of target DNA into patient's cells,
through ex vivo, in situ and in vivo methods

Methods of gene therapy:
 Ex vivo/in vitro method: Cells are taken from
the patients, cultured in the lab. This is followed
by introduction of defective gene into somatic
cells and modified cells are then administered to
the patients. Example: this methods is useful to
deliver the defective gene into bone marrow cells.
this therapy has been found to be successful for
the treatment adenosine deaminase deficiency


In vivo technique: in this approach, the gene
inserted into a vector is transfer direct to the
patient's cells. Example is introduction of
defective gene in cystic fibrosis through aerosol
containing the vector to the lungs.
Criteria to be satisfied prior to
initiation of gene therapy:
The disease should be aggressive and its
prognosis should be predictable.
 The gene should be isolated and its
regulatory regions defined.
 Target cells should be identified.
 Safe method for introducing the gene into
cells should be available.
 Evidence that the gene functions adequately
and produces no deleterious effects, should
available e.g. from cultured cells.
 The foreign material can be introduced into
affected cell by any of the following delivery
systems:

Viral vectors includes retroviruses ( RNA viruses ),
Adenoviruses (DNA viruses). The virus vectors used
in gene therapy are modified viruses. They are used
to integrate the target gene to chromosomal DNA,
but they will not cause infection.
 Non-viral delivery system includes liposome
containing plasmid vector ( liposome plasmid
complex) and DNA protein conjugate .

In theory, gene therapy can involve:
 Replacement: the mutant gene would be
removed & replaced with a normal gene.
 Correction: only the mutant area of the affected
gene would be corrected and the reminder left
unchanged.
 Augmentation: introduction of foreign genetic
material into cell to compensate for the defective
product of the mutant gene. This is the available
type of gene therapy at present.

Gene therapy application
Gene therapy are useful for the treatment of
inherited disorders such as:
 Cystic fibrosis
 Immune disorders such as adenosine deaminase
deficiency
 Cardiovascular disorder such as hypertension
 Infectious diseases such as viral infections
 Malignancies
 Neurodegenerative disorders