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Techniques in Molecular
Biology
Naglaa Alhusseini
RECOMBINANT DNA
TECHNOLOGY
Definition:

Recombinant DNA technology is genetic engineering
which effects artificial modifications of genetic
constitution of a living cell by introduction of foreign
DNA through experimental techniques.
The technique involves:
1.
Splicing of DNA by restriction endonucleases
2.
Preparation of chimeric molecules
3.
Cloning of large number of identical target DNA
molecules.
Tools of Recombinant DNA
technology:
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Enzymes : Restriction endonucleases- DNA
polymerases- DNA ligase – Reverse transcriptase
Vectors or vehicle DNA : DNA which act as a
carrier which includes : Bacterial plasmid –
Bacteriophages – Cosmids
Passenger DNA( foreign DNA ): DNA which
insert into the vector DNA , they are; Synthetic
DNA- Random DNA – cDNA (complementary DNA)
Hosts :
1. Restriction Endonucleases:
 These
are bacterial enzymes which have
the ability to cleave DNA molecules at
certain sites with high specific sequence
at the cleavage site.
It is called endonucleases because
they cut the polynucleotide in the
middle.
Most regions that are recognized by
the enzymes are Palindromes, the
two strands have the same sequence
in the 5` to 3` direction.
2-Reverse transcriptase:
It is an enzyme derived from viruses, oncogenic
(tumor producing) viruses or retroviruses.
 Its name derived from its action of reversing the
usual procedure i.e. instead of producing mRNA
from DNA as a usual process, reverse
transcriptase enzyme reverse the procedure
producing cDNA from RNA, while the last act as
a template.

3-DNA ligase:
 DNA ligase is an important tool for joining the
sticky ends in the preparation of recombinant
DNA.
4-DNA polymerases:
 They make complementary copies of DNA
templates . there are several types of DNA
polymerases . Examples, DNA polymerase I and
Taq polymerase (thermostable DNA polymerase)
2. Vectors:
Plasmids
 It is a segment of DNA to which the fragment of
DNA to be cloned is attached.
 Plasmids are the most popular vectors .
 It occurs in certain microorganisms (e.g.
bacteria).
 It is often contains antibiotic resistance genes; a
method of selection of cells containing recombinant DNA molecule (e.g. growth in
presence of antibiotic). Only the bacteria
containing the plasmids will grow.
 It is extra chromosomal, intra cytoplasmic
circular DNA found in bacteria.
It is much smaller than bacteria DNA produced
few thousand of nucleotide pairs, so it easy to
separate them from each other.
 Plasmids multiply independently from bacterial
host cells (autonomous replication).

Bacteriophages:
 They are usually having linear DNA molecule
into which foreign DNA can be inserted at
several restriction enzyme sites.
 They can accept DNA fragment 10-20kb long( one
kb =1000nucleotides base sequence)
Cosmids
 They are plasmid that also contain some portion
of bacteriophage DNA
 They are offer advantages of both plasmid and
bacteriophage vectors, as can be used to clone
DNA insert as long as 45kb.
Passenger DNA(foreign DNA)
Synthetic DNA:

Synthetic DNA can be produced purely by chemical
means

They are short segments of DNA(10-15 nucleotides)
Random DNA:

It is random pieces produced by a shot gun experiment
using restriction endonuclease enzymes
cDNA (complementary DNA):

Reverse trascriptase ( RNA dependant DNA polymerase)
makes complementary copies (cDNA) of mRNA templates

The cDNA does not contain introns which are present in
the genomic DNA sequence and removed from mRNA
during splicing process( post-transcription modification )
described before.
General Applications of
Recombinant DNA Technology:
1.
Manufacture of proteins / hormones :

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2.
3.
4.
5.
Proteins: such as plasminogen, interferon and blood
clotting factors.
Hormones: e.g. insulin and growth hormones…….
AIDS test : by using recombinant DNA
technology , the diagnosis of diseases like AIDS
by laboratory has become simple and rapid
Diagnosis of molecular diseases: for
example Sickle cell anemia , thalassaemias ,
familial hypercholesterolemia , cystic fibrosis
…..etc
Prenatal diagnosis of the genetic lesions
Gene therapy
6.
7.
8.
9.
Applications in agriculture: Genetically
engineered plants have been developed to resist
diseases, good quality of food and increased
yield in crops could be possible by applications
of this technique.
Industrial applications: Recombinant DNA
technology used for synthesis of enzymes which
are used to produce sugar, cheese and
detergents. Beside certain proteins products
used as food additives, to increase the nutritive
value beside flavor.
Applications in forensic medicine : genetic
engineering have greatly helped to identify
criminals and settle the doubtful of parenthood
of children
Transgenesis :The somatic gene replacement
therapy will not pass to the offspring ,
transgenesis refers to the transfer of gene into
TECHNIQUES IN MOLECULAR
BIOLOGY
1.
2.
3.
4.
5.
6.
Isolation of DNA
Electrophoresis of DNA
Hybridization and Blotting
Techniques
Techniques for DNA
Amplification
Restriction Fragment Length
Polymorphisms (RFLP)
DNA Finger Printing (DNA
Typing):
Techniques for DNA Amplification
I-DNA cloning (Gene Cloning)
 DNA cloning allows production for large number of
identical DNA (genes) which can be used for
another purpose.
 It is in vivo DNA amplification
The process consists of:1. Obtaining the gene.
2. Insertion of the gene into a vector (Plasmid or
phages).
3. Multiplication of the vector in a host cell
(bacteria).
4. Separation of the vector from the host cell.
5. Separation of the gene from the vector.
6. Hybridization i.e. specific fragment of cloned
gene can be identified with complementary
1) Obtaining copies of the gene of
interest:


a) Restriction enzymes (large No. of genes),
these enzymes are isolated from
microorganism's cellular genome and cut
DNA only at the site of the nucleotide
sequence recognized by these enzymes.
b) From mRNA by the reverse transcriptase
enzyme, where mRNA in a reverse process
act as a template to form a single strand of
DNA (cDNA) that completed to form double
strand DNA.
2-Digestion by restriction
enzyme:
1.
2.
The DNA to be cloned is cleaved with a
restriction endonuclease:- Restriction
endonuclease recognizes short sequences
in DNA and cleaves both strands within
this region. Most of the DNA regions
recognized by these enzymes are
palindromes, i.e. the two strands of
DNA have the same base sequence in the
5` - 3` direction.
Cleavage of vector DNA with the same
restriction enzyme that is used for
cleaving the target DNA
3-Production of recombinant DNA
molecule :
 The
plasmid and foreign DNA (inserted DNA)
both cleaved by the same restriction enzyme
are mixed together and treated with ligase
enzyme.

DNA ligase enzyme used to join (re-annealing)
the fragment to the DNA vector produced
chimeric plasmid contain the DNA insert
into the plasmid (recombinant DNA).
4-Multiplication of the vector in a
host cell

The chimeric plasmid are introduced into bacterial
host cell the process of introducing foreign DNA into
bacteria is termed “transformation”, Alternatively,
introducing foreign (recombinant DNA) into viral
genome is termed “Transfection” ,i.e. the virus is
infected and then infects the host cells, introducing
the recombinant DNA into the host cell genome. Only
5% of bacteria colonies contain the desired vector, so
we have to select the desired colonies

The bacterial host cell containing the recombinant
vector can be selected if the vector contains an
antibiotic resistance gene. Bacteria without vector die
in the presence of antibiotic.
5- Isolation of cloned foreign DNA or
its protein product:

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Cells containing an appropriate chimeric plasmid
are cultured.To obtain large quantities of the
foreign DNA, the plasmid are isolated from host
cells (the bacteria are lysed and the hybrid
plasmids are isolated) then treated with the
restriction enzyme to release the foreign DNA,
the plasmids are cut with the same restriction
enzyme releasing many copies of the DNA of
interest.
If the host cells are grown under conditions that
permit the production of protein produced from
target DNA, then the protein of interest can be
isolated.
6-Detection of either cloning DNA or
RNA:
 By
using labeled polynucleotide which
complementary to the target DNA or RNA.
It can be used of
for Gene
production
of viral coat
Importance
Cloning
1.
2.
3.
4.
5.
protein, which could be used for immunization of
human against e.g. Rabis or hepatitis (B. or C.),
used for synthesis of vaccines.
Can be used for the production of interferon with
a value in the treatment of viral infection and
possibly in cancer.
It explain the molecular bases of genetic diseases
and treated it by gene replacement (Gene
therapy) i.e. correction of genetic defect.
It is used in the synthesis of large quantities of
human proteins (Multiple copies of the normal
genes) e.g. insulin and GH.
It is used pharmaceutically in the production of
large quantities of protein, i.e. certain antibodies
II-Polymerase Chain Reaction (PCR)
PCR is defined as an in vitro DNA
amplification procedure in which millions of
copies of a particular sequence of DNA can be
produced within a few hours. It is like Xerox
machine for gene copying.
 This is an amplification reaction in which a small
amount of a DNA template is amplified to
provide enough copies (up to 100,000 fold) to
perform analysis (prenatal diagnosis,
detection of an infectious organisms or the
presence of a mutated oncogene).
 It is an extremely sensitive technique than
does cloning technique.

Karry Mullis invented this method in
1989, who was awarded Nobel Prize in
1993.
Material required:
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Target DNA may be chosen from any source such as
leukocytes, tissues and single cells
Two synthetic oligonucleotide primers: These
primers are complementary to the end of each
strand of target DNA to be amplified. The selection
of the primer requires the knowledge of the flanking
sequences of the gene of interest. Two primers of
about 20-30 nucleotides with complementary
sequence of the flanking region can be synthesized.
Heat stable DNA polymerase: This enzyme is
derived from bacteria Thermus acquaticus that
are found in hot springs. Therefore the enzyme is
not denatured at high temperature. This polymerase
is not denatured even at temperature around 95oC.
Heat-stable polymerase is vital
to the ease of the process
How is this different from cloning?
In vitro amplification (in a test tube)
 Enzymatic: Taq polymerase

– Temperature-resistant DNA

polymerase ( Thermus aquaticus)
 Heat resistant
 Best for <2 kb target

Problems with Taq
Taq DNA polymerase - thermostable
 Lack of 3′-5′ exonuclease – proofreading

►Error rate = 2 × 10-4 nucleotdes/cycle
 Newer polymerases have high fidelity
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High fidelity polymerase - HiFi Taq
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Technique steps:
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Denaturation: mixture heated to up 95°C for
15 seconds to 2 minutes to separate the two
DNA strands . The two separate strands serve
as complementary strand for DNA elongation
reaction.
Annealing, The reaction mixture is cooled to
about 50 -65oC for about 1-3 minutes. The
temperature chosen for cooling is usually about
2-3 below Tm.(melting temperature).
Extension. The elongation of DNA from the
site of annealing of primers is catalyzed by Taq
DNA polymerase. The polymerase reaction is
allowed to take place at 72°C for 30 seconds in
the presence of dNTPs (all four deoxy
ribonucleotide triphosphates).
Identification of the PCR products:


After the amplification procedure, DNA
electrophoresis and Southern blot analysis with a
suitable probe shows the presence of the DNA in
the sample tissue
RT-PCR (Reverse Transcription PCR) refers
to utilization of mRNA for the formation of target
DNA using reverse transcriptase enzyme.
Various Types of PCR

Multiplex-PCR: consists of multiple primer
sets within a single PCR mixture to produce
amplicons of varying sizes that are specific to
different DNA sequences. By targeting multiple
genes at once, additional information may be
gained from a single test run that otherwise
would require several times the reagents and
more time to perform. Annealing temperatures
for each of the primer sets must be optimized to
work correctly within a single reaction, and
amplicon sizes, i.e., their base pair length,
should be different enough to form distinct
bands when visualized by gel electrophoresis.
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Simultaneously
modification of more
than
one locus in the same
reaction
Rapid and convenient
– screening
Included different set
of primers

Nested PCR: increases the specificity of DNA
amplification, by reducing background due to
non-specific amplification of DNA. Two sets
(instead of one pair) of primers are used in two
successive PCRs. In the first reaction, one pair
of primers is used to generate DNA products,
which besides the intended target, may still
consist of non-specifically amplified DNA
fragments. The product(s) are then used in a
second PCR with a set of primers whose binding
sites are completely or partially different from
and located 3' of each of the primers used in the
first reaction. Nested PCR is often more
successful in specifically amplifying long DNA
fragments than conventional PCR, but it
requires more detailed knowledge of the target
sequences.

Quantitative PCR (Q-PCR): used to measure the
quantity of a PCR product (commonly in real-time)
RT-PCR. It quantitatively measures starting amounts of
DNA, cDNA or RNA. Q-PCR is commonly used to
determine whether a DNA sequence is present in a
sample and the number of its copies in the sample.
Quantitative real-time PCR has a very high degree of
precision. QRT-PCR methods use fluorescent dyes, such
as Sybr Green, EvaGreen or fluorophore-containing DNA
probes, such as TaqMan, to measure the amount of
amplified product in real time. It is also sometimes
abbreviated to RT-PCR (Real Time PCR) or RQ-PCR.
QRT-PCR or RTQ-PCR are more appropriate
contractions, since RT-PCR commonly refers to reverse
transcription PCR (see below), often used in conjunction
with Q-PCR.
In situ PCR

The advantages of real time PCR are rapid,
very sensitive, reproducible , reduced risk of
contamination (sealed reactions), easy to perform
and allow for quantitation of result using
software driven operation.

Reverse Transcription PCR (RT-PCR): for
amplifying DNA from RNA. Reverse
transcriptase reverse transcribes RNA into
cDNA, which is then amplified by PCR. RT-PCR
is widely used in expression profiling, to
determine the expression of a gene or to identify
the sequence of an RNA transcript, including
transcription start and termination sites. If the
genomic DNA sequence of a gene is known, RTPCR can be used to map the location of exons and
introns in the gene. The 5' end of a gene
(corresponding to the transcription start site) is
typically identified by RACE-PCR (Rapid
Amplification of cDNA Ends)
Clinical Applications of PCR
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1. Diagnosis of bacterial and viral diseases:
In early phases of tuberculosis, the sputum may contain
only very few tubercle bacilli, so that usual acid fast
staining may be negative. But PCR could detect even one
bacillus present in the specimen.
Any other bacterial infection could also be detected
similarly.
The specific nucleotide sequences of the bacilli are
amplified by PCR and then detected by Southern blot
analysis.
If reverse PCR is done, living organisms could be detected.
This technique is widely used in the diagnosis of viral
infections like Hepatitis C, Cytomegalovirus and HIV.
3. Diagnosis of genetic disorders:
 The PCR technology has been widely used to
amplify the gene segments that contain known
mutations for diagnosis of inherited diseases
such as sickle cell anemia, beta thalassemia,
cystic fibrosis,
 4. PCR is especially useful for prenatal diagnosis
of inherited diseases, where cells obtained from
fetus by amniocentesis are very few.
 5. Cancer detection: PCR is widely used to
monitor residual abnormal cells present in
treated patients. Similarly identification of
mutations in oncosuppressor genes (e.g. p53) and
retinoblastoma gene, etc. can help identify
individuals at high risk of cancer (oncogenes and
oncosuppressor genes).

2. Medico-legal cases: PCR allows the DNA in
a single cell or in a hair follicle to be analyzed.
The restriction analysis of DNA from the hair
follicle from the crime scene is studied after PCR
amplification.
 This pattern is then compared with the
restriction analysis of DNA samples obtained
from various suspects; the culprit’s sample will
perfectly match with that of PCR amplified
sample. The restriction analysis pattern of DNA
of one individual will be very specific (DNA finger
printing); but the pattern will be different from
person to person. This is highly useful in forensic
medicine to identify the criminal.

6- Quantification of gene expression (mRNA
template is first reverse transcribed into a cDNA
equivalent before amplification (RT-PCR).
 7- Tissue typing for transplanting, by PCR
and detection of genetic variants, especially of
MHC (Major histocompatibility complex).
 8. Fossil studies: DNA isolated from fossils and
amplified by PCR is used to study evolution by
comparing the sequences in the extinct and living
organisms.
