Steps and strategies of gene cloning & DNA libraries

2. Agene is a basic physical
and functional unit of
heredity. Genes which are
made up of DNA, acts as
instructions to molecules
called proteins.

3. Cloning is the process
of generating an
identical copy of a cell
or an organism. Clones
are those organisms
that have identical
genes.

4. “Gene cloning is genetic engineering
technique in which a gene of interest is
fused into a plasmid and introduced into a
suitable host, self replicates and generates a
large number of identical copies of the
particular gene.”

5. Requirements for Gene Cloning
(Cell-based)
1.DNA fragment containing the desired genes to be
cloned.
2.Restriction enzymes and ligase enzymes.
3.Vectors – to carry, maintain and replicate cloned
gene in host cell.
4.Host cell– in which recombinant DNA can replicate.
DNA cloning can be achieved by two different
methods:
1.Cell based DNA cloning
2.Cell-free DNA cloning (PCR)

6. The basic 9 steps involved in gene cloning are:
• Choice of host organism and cloning vector
• Preparation of vector DNA
• Isolation of DNA [gene of interest] fragments to be cloned.
• Insertion of isolated DNA into a suitable vector to form recombinant
DNA.
• Introduction of recombinant DNA into a suitable organism known as
host.
• Selection of transformed host cells and identification of the clone
containing the gene of interest.
• Multiplication/Expression of the introduced Gene in the host.
• Isolation of multiple gene copies/Protein expressed by the gene.
• Purification of the isolated gene copy/protein
GENE CLONING:
STEPS INVOLVED IN

7. GENE CLONING:
STEPS INVOLVED IN
Generation of foreign DNAmolecules
Selection of suitable vector
Insertion of foreign DNAinto vector
Recombinant DNAintroduced into host cells
Selection and screening methods of
recombinant clones

8. Specialized applications may call for specialized host-vector systems.
• For example, if the experimentalists wish to harvest a particular
protein from the recombinant organism, then an expression vector is
chosen that contains appropriate signals for transcription and
translation in the desired host organism.
• Alternatively, if replication of the DNA in different species is desired
(for example, transfer of DNA from bacteria to plants), then a multiple
host range vector (also termed shuttle vector) may be selected.
• In practice, however, specialized molecular cloning experiments
usually begin with cloning into a bacterial plasmid, followed
by subcloning into a specialized vector.
Choice of host organism and cloning vector

9. Whatever combination of host and vector are used, the vector
almost always contains four DNA segments that are critically
important to its function and experimental utility:[3]
• DNA replication origin is necessary for the vector (and its
linked recombinant sequences) to replicate inside the host
organism
• One or more unique restriction endonuclease recognition
sites to serves as sites where foreign DNA may be
introduced
• A selectable genetic marker gene that can be used to enable
the survival of cells that have taken up vector sequences
• A tag gene that can be used to screen for cells containing
the foreign DNA

10. Preparation of vector DNA
• The cloning vector is treated with a restriction
endonuclease to cleave the DNA at the site where foreign
DNA will be inserted.
• The restriction enzyme is chosen to generate a
configuration at the cleavage site that is compatible with
the ends of the foreign DNA (see DNA end).
• Typically, this is done by cleaving the vector DNA and
foreign DNA with the same restriction enzyme, for
example EcoRI.
• Most modern vectors contain a variety of convenient
cleavage sites that are unique within the vector molecule

11. • (so that the vector can only be cleaved at a single site)
and are located within a gene (frequently beta-
galactosidase) whose inactivation can be used to
distinguish recombinant from non-recombinant
organisms at a later step in the process.
• To improve the ratio of recombinant to non-recombinant
organisms, the cleaved vector may be treated with an
enzyme (alkaline phosphatase) that dephosphorylates
the vector ends.
• Vector molecules with dephosphorylated ends are
unable to replicate, and replication can only be restored
if foreign DNA is integrated into the cleavage site.

12. Isolation of the DNA fragment or gene
• The target DNA or gene to be cloned must be first
isolated. A gene of interest is a fragment of gene
whose product (a protein, enzyme or a hormone)
interests us. For example, gene encoding for the
hormone insulin.
• The desired gene may be isolated by using restriction
endonuclease (RE) enzyme, which cut DNA at specific
recognition nucleotide sequences known as restriction
sites towards the inner region (hence endonuclease)
producing blunt or sticky ends.
• Sometimes, reverse transcriptase enzyme may also be
used which synthesizes complementary DNA strand of
the desired gene using its mRNA.

13. Selection of suitable cloning vector
• The vector is a carrier molecule which can carry the
gene of interest (GI) into a host, replicate there along
with the GI making its multiple copies.
• The cloning vectors are limited to the size of insert that
they can carry. Depending on the size and the
application of the insert the suitable vector is selected.
• The different types of vectors available for cloning are
plasmids, bacteriophages, bacterial artificial
chromosomes (BACs), yeast artificial
chromosomes (YACs) and mammalian artificial
chromosomes (MACs).
• However, the most commonly used cloning vectors
include plasmids and bacteriophages (phage λ) beside
all the other available vectors.

14. Essential Characteristics of Cloning Vectors
All cloning vectors are carrier DNA molecules. These
carrier molecules should have few common features in
general such as:
• It must be self-replicating inside host cell.
• It must possess a unique restriction site for RE
enzymes.
• Introduction of donor DNA fragment must not interfere
with replication property of the vector.
• It must possess some marker gene such that it can be
used for later identification of recombinant cell
(usually an antibiotic resistance gene that is absent in
the host cell).
• They should be easily isolated from host cell.

15. Formation of Recombinant DNA
• The plasmid vector is cut open by the same RE
enzyme used for isolation of donor DNA fragment.
• The mixture of donor DNA fragment and plasmid
vector are mixed together.
• In the presence of DNA ligase, base pairing of donor
DNA fragment and plasmid vector occurs.
• The resulting DNA molecule is a hybrid of two DNA
molecules – the GI and the vector. In the terminology
of genetics this intermixing of different DNA strands is
called recombination.
• Hence, this new hybrid DNA molecule is also called a
recombinant DNA molecule and the technology is
referred to as the recombinant DNA technology.

16. Transformation of recombinant vector into suitable host
The recombinant vector is transformed into suitable host cell mostly, a
bacterial cell.
This is done either for one or both of the following reasons:
To replicate the recombinant DNA molecule in order to get the
multiple copies of the GI.
To allow the expression of the GI such that it produces its needed
protein product.
Some bacteria are naturally transformable; they take up the
recombinant vector automatically.
For example: Bacillus, Haemophillus, Helicobacter pylori, which are
naturally competent.
Some other bacteria, on the other hand require the incorporation by
artificial methods such as Ca++ ion treatment, electroporation, etc.

17. Isolation of Recombinant Cells
• The transformation process generates a mixed
population of transformed and non-trans- formed host
cells.
• The selection process involves filtering the transformed
host cells only.
• For isolation of recombinant cell from non-recombinant
cell, marker gene of plasmid vector is employed.
• For examples, PBR322 plasmid vector contains different
marker gene (Ampicillin resistant gene and Tetracycline
resistant gene. When pst1 RE is used it knock out
Ampicillin resistant gene from the plasmid, so that the
recombinant cell become sensitive to Ampicillin.

18. Multiplication of Selected Host Cells
• Once transformed host cells are separated by the
screening process; becomes necessary to provide them
optimum parameters to grow and multiply.
• In this step the transformed host cells are introduced
into fresh culture media .
• At this stage the host cells divide and re-divide along
with the replication of the recombinant DNA carried by
them.
• If the aim is obtaining numerous copies of GI, then
simply replication of the host cell is allowed. But for
obtaining the product of interest, favourable conditions
must be provided such that the GI in the vector
expresses the product of interest.

19. Isolation and Purification of the Product
• The next step involves isolation of the multiplied GI attached with
the vector or of the protein encoded by it.
• This is followed by purification of the isolated gene copy/protein.

23. Strategies to generate
molecules:
foreign DNA
Using restriction enzymes
cDNA synthesis
Using gene machine

24. USING RESTRICTION ENZYMES:
 Restriction enzyme is a dna cutting enzyme
that recognizes specific sites in DNA, they
often called as restriction endonuclease.
 It cleaves the dna in two forms. They are
are
i.
ii.
Blunt end
cohesive end

26. Eg:
Eco
Eco
Eg:
Eco R1
Bam H1
Hind III
Eg:
Eco P1
Hinf III
EcoP15i
K1
A1
CfrA1

27. cDNA SYNTHESIS:
 Reverse transcription is the process of making
a double stranded DNAmolecule from a single
stranded RNAtemplate catalyzed by an
enzyme reverse transcriptase.
 it is also called as RNA-dependent DNA
polymerase.

29. USING GENE MACHINE:
“Gene machine is a fully automated
commercial instrument, also called as
automated polynucleotide synthesizer
which synthesizes predetermined
polynucleotide sequence.”

30. Working principle:
“Development of insoluble silica-based
support in the form of beads which provides
support for solid phase synthesis of DNA
chain.”
“Development of stable deoxyribonucleoside
phosphoramides as synthons which are stable
to oxidation and hydrolysis, and ideal for
DNAsynthesis.”

32. Library construction
screening
and

33. • A gene library is a collection
of different DNA sequences
from an organism,
which has beenAlso
called genomic
libraries or gene banks.
cloned into a vector for ease
of purification, storage and
analysis.
•
•

34. Uses of gene libraries
• To obtain the sequences of genes for analysis,
amplification, cloning, and expression.
Once the sequence is known probes, primers,
etc. can be synthesized for further diagnostic
work using, for example, hybridization reactions,
blots and PCR.
Knowledge of a gene sequence also offers the
possibility of gene therapy.
Also, gene expression can be used to synthesize a
product in particular host cells, e.g. synthesis of
human gene products in prokaryotic cells.
•
•
•

35. two types of gene library
depending upon the source of the DNA used.
genomic library contains DNA fragments
representing the entire genome of an
organism.
cDNA library contains only complementary
DNA molecules synthesized from mRNA
molecules in a cell.

36. Genomic Library :
Made from nuclear DNA of an organism
species.
DNA is cut into clonable size pieces as
randomly possible using restriction
endonuclease
• or
•
• Genomic libraries contain whole genomic
fragments including gene exons and introns,
gene promoters, intragenic DNA,origins of
replication, etc

37. Construction of Genomic Libraries
1. Isolation of genomic DNA and vector.
2. Cleavage of Genomic DNA and vector
by Restriction Endonucleases.
3. Ligation of fragmented DNA with the
vector.
4. Transformation of r-DNA in the
bacterial cell.
5. Amplification of the r-DNA in bacterial
cells.

38. • Step1:- Extraction of genomic DNA

39. • Step2:- Cut with a restriction endonuclease
enzyme
• Physical shearing (agitation or sonication )
• endonuclease enzymes (2 or more can be
used)

40. • Step3:-Ligate the gene with the vector which
cleaves by same enzyme

41. Step4:-transformatiom
• Transfer (transform) into
bacteria Cells which are
able to undergo this
treatment are termed
competent cells
CaCl 2 causes DNA to
precipitate on the
as
•
outside walls of bacterial
cells.

42. Step5:- Amplification
E.coli cells are grown in an agar medium
containing ampicillin or tetracyclin at 37°C.
•

43. Screening of genomic library
• Once the genomic library has been created,
is screened to identify the genes of interest.
One of the most common library screening
technique is called colony hybridization.
In the process of library construction, phage
vectors are used then the process of
it
•
identification of genes of interest involved is
the plaques hybridization

44. colony hybridization
• Colony Hybridization is the screening of ibrary
with a labeled probe (radioactive, etc.) to
identify a specific sequence of DNA, RNA,
enzyme, protein, or antibody.

46. Plaque hybridization :
• The plaques are screened by a
technique ,based on the hybridization
of oligonucleotide probe to target
DNA.
DNA is transferred directly from the
Petri dish to the filter,
which is then incubated with labeled
probes
•
•

48. Applications of Genomic Library
• 1. Genomic library construction is the first
step in any DNA sequencing projects
2. Genomic library helps in identification of
the novel pharmaceutically important genes.
3. Genomic library helps in identification of new genes
which were silent in the host.
4. It helps us in understanding the complexity of genomes.
•
•
•
• 5. Serving as a source of genomic sequence for generation
of transgenic animals through genetic engineering .
6. Study of the function of regulatory sequences in vitro.
7. Study of genetic mutations in cancer tissues.
•
•

49. cDNA library -
• complementary DNA molecules synthesized
from mRNA molecules in a cell. ,

50. • constitute some portion of the transcriptome of the
organism.
cDNA is produced from fully transcribed mRNA
found in the nucleus therefore contains only the
expressed genes of an organism. So ,tissue specific
cDNA libraries can be produced
•
• cDNA is created from a mature mRNA from a
eukaryotic cell with the use of an enzyme known as
reverse transcriptase.
• In eukaryotes, apoly-(A) tail distinguishes mRNA
from tRNA and rRNA and can therefore be used as a
primer site for reverse transcription

51. Steps
1. mRNA extraction
2
•
•
methods
trizol extraction
column purification.
 by using oligomeric dT nucleotide coated resins
where only the mRNA having the poly-A tail will bind.
 The rest of the RNAs are eluted out
The mRNA is eluted by using eluting buffer and some
heat to separate the mRNA strands from oligo-dT.

52. Once mRNA is purified,
oligo-dT is tagged as a complementary primer which
binds to the poly-A tail providing a free 3'-OH end that
can be extended by reverse transcriptase to create the
complementary DNA strand.
Now, the mRNA is removed by using a RNAse enzyme
leaving a single stranded cDNA (sscDNA).
This sscDNA is converted into a double stranded DNA
with the help of DNA polymerase
•
•
•
• However, for DNA polymerase to synthesize a
complementary strand a free 3'-OH end is needed

53. • This is provided by the sscDNA itself by generating a
hairpin loop at the 3' end by coiling on itself.
• polymerase extends the 3'-OH end and later the loop
at 3' end is opened by the scissoring action of S1
nuclease.
Restriction endonucleases and DNA ligase are then
•
used to clone the sequences into bacterial plasmids.
The cloned bacteria are then selected, commonly
through the use of antibiotic selection.
Once selected, stocks of the bacteria are created which
can later be grown and sequenced to compile the
cDNA library
•
•

55. cDNA Library uses
• cDNA libraries are commonly used when
reproducing eukaryotic genomes, as the amount
of information is reduced to remove the large
numbers of non-coding regions from the library.
cDNA libraries are used to express eukaryotic
genes in prokaryotes.
cDNA libraries are most useful in reverse
genetics where the additional genomic
information is of less use.
Also, it is useful for subsequently isolating the
gene that codes for that mRNA.
•
•
•

56. cDNA Library vs. Genomic DNA
Library
• cDNA library lacks the non-coding and
regulatory elements found in genomic DNA.
Genomic DNA libraries provide more detailed
•
information about the organism, but are more
resource-intensive to generate and maintain.