TECHNIQUES IN MOLECULAR
BIOLOGY- RECOMBINANT
DNA & CLONING, GENE
LIBRARY, GENE THERAPY
Dr. Pinaki Saha, MD(Biochemistry)
DIGESTING DNA MOLECULE
Enzymes termed restriction
endonucleases
fragment/cut the double
stranded DNA into smaller
easily manageable
fragments
The obstacle of huge size of
human genomic DNA could
be overcome by restriction
endonucleases
Key to molecular cloning
because of the specificity
they have for particular
DNA sequences
Over 600 enzymes,
recognising more than 200
different restriction sites,
have been characterized
PROPERTIES OF RESTRICTION
ENDONUCLEASES
Specificity: recognize short stretches of DNA
(four to eight bp) that contain specific
nucleotide sequences; sequences which differ
for each restriction enzyme are palindromic
Every copy of a given DNA molecule from a
specific organism will give the same set of
fragments when digested with a particular
enzyme .
DNA from different organisms will, in general,
give different sets of fragments when treated
with the same enzyme
PALINDROME
Exhibit twofold
rotational symmetry
=within a short region
of the double helix, the
nucleotide sequence on
the two strands
identical if each is read
in the 5'→3' direction.
If turned the page
upside down (rotate it
180 degrees around its
axis of symmetry)—the
sequence remains the
same.
NOMENCLATURE OF RESTRICTION
ENZYMES
Named according to the organism from which
isolated.
The first letter of the name from the genus of
the bacterium.
The next two letters from the name of the
species.
An additional letter indicates the type or strain
Final number appended to indicate the order in
which the enzyme was discovered in that
particular organism.
For example, Hae III is the third restriction
endonuclease isolated from the bacterium
Haemophilus aegyptius.
“STICKY” AND “BLUNT” ENDS
Cleave dsDNA to produce a 3'-hydroxyl group on
one end and a 5'-phosphate group on the other.
Taq I, form staggered cuts that produce “sticky” or
cohesive ends—the resulting DNA fragments have
single-stranded (ss) sequences that are
complementary to each other
Others such as Hae III, produce fragments that
have “blunt” ends and double stranded and
therefore do not form hydrogen bonds with each
other.
Using DNA ligase sticky ends of a DNA fragment of
interest can be covalently joined with other DNA
fragments that have sticky ends produced by
cleavage with the same restriction endonuclease
Another ligase , encoded by bacteriophage T4, can
covalently join blunt-ended fragments. D
RESTRICTION SITES
DNA sequence that is recognized
and cut by a restriction enzyme
called a restriction site.
Restriction endonuclease that
recognizes a specific four-base-
pair sequence produces many
cuts in the DNA molecule, one
every 4⁴ bp.
Enzyme requiring a unique
sequence of six base pairs
produces fewer cuts (one every
4⁶ bp) longer pieces.
Hundreds of these enzymes,
having different cleavage
specificities (varying in both
nucleotide sequences and length
of recognition sites),
commercially available as
analytic reagents.
LIGATING DNA MOLECULES
BIOLOGICAL FUNCTIONS OF RESTRICTION ENZYMES
Bacterial cells have restriction enzymes to destroy
invading foreign DNA, e.g. lambda phage which
injects its DNA into an E. coli cell. The bacterial
enzymes recognize the restriction sites and promptly
cut the DNA to prevent phage infections: it restricts
the ability of foreign DNA to infect the cell.
Why the restriction enzyme does not recognize the
same restriction site on the bacterial DNA and cleave
at that site? The cell guards against cutting its own
DNA by methylation: it adds a methyl group to one of
the bases in all of the recognition sequences on each
new DNA strand soon after it is synthesized. This does
not interfere with the base pairing or gene function,
but the restriction enzyme no longer recognizes the
methylated sequence and the cell’s own DNA,
therefore, protected from attack by that enzyme.
TERMINAL TRANSFERASES
They catalyze the addition of homo-
oligonucleotide sequences (poly dN) to the 3’
end of double-stranded DNA molecule No
template is required for these reactions.
The 3 ’ ends of DNA serve as primers. A given
enzyme can catalyze addition of many copies
of a particular nucleotide only, and there are
separate enzymes for each of the four deoxy-
ribonucleotides e.g. the enzyme catalyzing
addition of the poly-dA sequence different
from that catalyzing addition of the poly-dT
sequence.
DNA
CLONING
DNA CLONING
Introduction of a foreign DNA molecule into a
replicating cell termed cloning or amplification
(the production of many identical copies) of that
DNA.
Sometimes single DNA fragment isolated and
purified prior to cloning. More commonly, to
clone a nucleotide sequence of interest, the total
cellular DNA first cleaved with a specific
restriction enzyme, creating hundreds of
thousands of fragments.
Each of the resulting DNA fragments is joined to
a DNA vector molecule (cloning vector) to form a
hybrid or recombinant molecule.
Each recombinant DNA molecule conveys its
inserted DNA fragment into a single host cell,
e.g. a bacterium, where replicated
DNA CLONING
The process of introducing foreign DNA into a
cell is called transformation for bacteria and
yeast and transfection for higher eukaryotes.
As the host cell multiplies, it forms a clone in
which every bacterium carries copies of the
same inserted DNA fragment, hence the
name “cloning.”
The cloned DNA eventually released from its
vector by cleavage (using the appropriate
restriction endonuclease ) and isolated.
By this mechanism, many identical copies of
the DNA of interest can be produced
VECTORS
A molecule of DNA to which the fragment of
DNA to be cloned is joined.
Essential properties of a vector include:
1) must be capable of autonomous replication
within a host cell
2) must contain at least one specific nucleotide
sequence recognized by a restriction
endonuclease
3) must carry at least one gene that confers
the ability to select for the vector, such as an
antibiotic resistance gene.
Commonly used vectors include plasmids and
viruses.
PROKARYOTIC PLASMIDS
Prokaryotic cells contain single, large,
circular chromosome.
Also most species of bacteria normally
contain small, circular,
extrachromosomal DNA molecules called
plasmids
Plasmid DNA undergoes replication that
may or may not be synchronized to
chromosomal division.
Plasmids may carry genes that convey
antibiotic resistance to the host
bacterium, and may facilitate the
transfer of genetic information from one
bacterium to another.
Plasmids can be readily isolated from
bacterial cells, their circular DNA cleaved
at specific sites by restriction
endonucleases , and up to 10 kb of
foreign DNA (cut with the same
restriction enzyme) inserted.
PLASMIDS
Recombinant plasmid
can be introduced into a
bacterium, and large
numbers of copies
produced.
The bacteria grown in
the presence of
antibiotics, thus
selecting for cells [The experiment is
containing the hybrid conducted to favor
plasmids, which provide only one DNA
fragment being
antibiotic resistance inserted into each
plasmid and only one
plasmid being taken
up by each
bacterium.]
OTHER VECTORS
Improved vectors developed that can
accommodate larger DNA segments, or
express the passenger genes in different cell
types
Naturally occurring viruses that infect
bacteria (e. g. bacteriophage λ) or
mammalian cells (e.g. retroviruses), as well
as artificial constructs such as cosmids and
bacterial or yeast artificial chromosomes
(BACs or YACs, respectively), are currently in
wide use as cloning vectors.
BACs and YACs can accept DNA inserts of
100-200 kilobases (kb) and 200-500 kb
DNA LIBRARIES
A collection of cloned restriction fragments of
the DNA of an organism.
Two kinds of libraries commonly used:
genomic libraries and complementary DNA
(cDNA) libraries.
Genomic libraries ideally contain a copy of
every DNA nucleotide sequence in the
genome.
cDNA libraries contain those DNA sequences
that only appear as processed mRNA
molecules, and these differ from one cell
type to another.
[cDNA lacks introns and the control regions
of the genes, whereas these are present in
genomic DNA.]
GENOMIC DNA LIBRARIES
After the restriction fragments ligated to vectors,
recombinant DNA molecules replicate within host
bacteria. The amplified DNA fragments thus
represent the entire genome of the organism and
called a genomic library.
If digestion allowed to go to completion, the gene
of interest fragmented—that is, not contained in
any one clone in the library. To avoid this result, a
partial digestion is performed in which either the
amount or the time of action of the enzyme
limited. This results in cleavage occurring at only a
fraction of the restriction sites on any one DNA
molecule, thus producing fragments of about 20
kb.
CDNA LIBRARIES
If a protein-coding gene of interest expressed
at a high level in a particular tissue, then the
messenger RNA (mRNA) transcribed from
that gene is also present at high
concentrations in the cell.
mRNA can be used as a template to make a
complementary DNA (cDNA) molecule using
the enzyme reverse transcriptase
The resulting cDNA = double-stranded copy
of mRNA.
cDNA can be amplified by cloning or by the
polymerase chain reaction.
CDNA LIBRARIES
Can be used as a probe to locate the gene
that coded for the original mRNA (or
fragments of the gene) in mixtures containing
many unrelated DNA fragments
If the mRNA used as a template is a mixture of
many different size species, the resulting
cDNA is heterogeneous
Because cDNA has no intervening sequences,
it can be cloned into an expression vector for
the synthesis of eukaryotic proteins by
bacteria
GENE THERAPY
The goal of gene therapy is to insert the
normal, cloned DNA for a gene into the
somatic cells of a patient who has a defect in
that gene as a result of some disease-
causing mutation
The DNA must become permanently
integrated into the patient’s chromosomes in
such a way as to be properly expressed to
produce the correct protein.
e.g.- gene therapy in SCID (X-linked severe
combined immunodeficiency, or SCIDX1)
THANK YOU FOR YOUR KIND
ATTENTION
