The ppt discusses about gene cloning experiment.

2. The procedure in a gene cloning experiment is:
1. To place a foreign gene into a bacterial cell;
2. To grow a clone of those modified bacteria.
The principle factors for gene cloning experiment:
 Restriction endonucleases
 Vectors
 Specific probe

3.  The main advantage of restriction enzyme is there
ability to cut a DNA reproducibly in the same place; this
is the basis of many techniques used to analyze genes.
 Many restriction enzymes make staggered cut in the
two DNA strands, leaving a sticky ends, that can base-
pair together briefly.

4.  Restriction endonucleases recognize specific sequences
in DNA molecules and make cuts in both strands. This
allows very specific cutting of DNAs. Also, because the
cuts in the two strands are frequently staggered,
restriction enzymes can create sticky ends that help
link together two DNAs to form a recombinant DNA in
vitro.

5. Step 1: DNA ligase reacts with an AMP donor—either ATP or
NAD(nicotinamide adenine dinucleotide), depending on
the type of ligase. This produces an activated
enzyme(ligase-AMP).
Step 2: The activated enzyme donates a phosphate to the
free 5’-phosphate at the nick in the lower strand of the
DNA duplex, creating a high-energy diphosphate group on
one side of the nick.
Step 3: With energy provided by cleavage of the diphosphate,
a new phosphodiester bond is created, sealing the nick in
the DNA. This reaction can also occur in both DNA strands
at once, so two independent DNAs can be joined together
by DNA ligase.

6. Alkaline phosphatase prevents vector re-ligation.
 Step 1: Cut the vector with BamHI, This produces
sticky ends with 5’-phosphates.
 Step 2: Remove the phosphates with alkaline
phosphatase, making it impossible for the vector to
re-ligate with itself.
 Step 3: Also cut the insert with BamHI, producing
sticky ends with phosphates that we do not remove.
 Step 4: Ligate the vector and insert together. The
phosphates on the insert allow two phosphodiester
bonds to form, but leave two unformed bonds, or
nicks, These will be completed once the DNA is
transformed in the bacterial cell.

7.  Vectors serve as carriers to allow replication of
recombinant DNAs.
Origin of replication
Multiple cloning site(MCS)
Selection gene
Plasmids pBR322 pUC
Phages λphage cosmids M13
Phagemids

8.  The first generations of plasmid cloning vectors were
pBR322 and the pUC plasmids. The former has two
antibiotic resistance genes and a variety of unique
restriction sites into which one can introduce foreign
DNA. Most of these sites interrupt one of the antibiotic
resistance genes, making screening straightforward.
Screening is even easier with the pUC plasmids. These
have an ampicillin resistance gene and a multiple
cloning site that interrupts a partial β-galactosidase
gene. One screens for ampicillin-resistant clones that do
not make active β-galactosidase and therefore do not
turn the indicator, X-gal, blue. The multiple cloning site
also makes it convenient to carry out directional cloning
into two different restriction sites.

10.  Different kinds of phages have been especially popular
as cloning vectors.
 Phage Replacement Vector: The first of these is λ, from
which certain nonessential genes have been removed to
make room for inserts. Some of these engineered
phages can accommodate inserts up to 20 kb, which
makes them useful for building genomic libraries, in
which it is important to have large pieces of genomic
DNA in each clone.


12.  They can accept even larger inserts—up to 50 kb—
making them a favorite choice for genomic libraries.
Behave both as plasmids and as phages;
 Contain the cos sites of λ and plasmid origin of
replication;
 Have room for 40-50 kb inserts.

14. Plasmids called phagemids have also been engineered to
produce single-stranded DNA in the presence of helper
phages.
 Single-stranded;
 Both phage and plasmid characteristics;
 Help phage
 Two RNA polymerase promoters (T7and T3)

16.  The second major class of phage vector is composed of
the M13 phages. These vector have the convenience of a
multiple cloning site and the further advantage of
producing single-stranded recombinant DNA, which can
be used for DNA sequencing and for site-direct
mutagenesis:
 β –galactosidease gene fragment
 pUC family MCS
 Single stranded DNA genome

17.  Polynucleotide Probes : Specific clones can be
identified using polynucleotide probes that bind to the
gene itself. Knowing the amino acid sequence of a gene
product, one can design a set of oligonucleotides that
encode part of this amino acid sequence. This can be
one of the quickest and most accurate means of
identifying a particular clone.
 High stringency
 Low stringency

18.  Yeast Artificial chromosomes (YACs)
• Centromers (CEN), telomeres (TEL) and autonomous
replicating sequence (ARS) for proliferation in the
host cell.
• ampr for selective amplification and markers such as
TRP1 and URA3 for identifying cells containing the
YAC vector.
• Recognition sites of restriction enzymes (e.g., EcoRI
and BamHI).

20.  Bacterial artificial chromosomes are based on the F
factor of E. coli and can be used to clone up to 350 kb of
genomic DNA in a conveniently handled E. coli host.
They are a more stable and easier to use alternative to
YAC.

21. › Plant parasite that causes Crown Gall Disease
› Encodes a large (~250kbp) plasmid called Tumor-
inducing (Ti) plasmid
 Portion of the Ti plasmid is transferred between
bacterial cells and plant cells  T-DNA (Tumor
DNA)
 T-DNA integrates stably into plant genome.
 T-DNA ss DNA fragment is converted to dsDNA
fragment by plant cell.
 Then integrated into plant genome.
 2 x 23bp direct repeats play an important role
in the excision and integration process.

23.  What is naturally encoded in T-DNA?
› Enzymes for auxin and cytokinin synthesis
 Causing hormone imbalance  tumor
formation/undifferentiated callus
 Mutants in enzymes have been characterized
› Opine synthesis genes (e.g. octopine or nopaline)
 Carbon and nitrogen source for A. tumefaciens
growth
 Insertion genes
 Virulence (vir) genes
 Allow excision and integration into plant genome

24.  Techniques to transform plant cells by A. tumefaciens:
› Wounding and direct inoculation
› Inoculation of explants in vitro
› Transformation of leaf-disks
› Co-cultivation of Agrobacterium with protoplasts