molecular genetic plasmid and cloning DNA

Genetics

1. Definitions:Extra chromosomal DNA molecule
found in all types of bacteria
plasmids

2. Functions:
1- help in bacterial survival under
difficult conditions (unsuitable
temp., toxins)
2- play role in bacterial
adaptation
3- help in gene expression

3. •Plasmid replication
•Plasmid replication Occur by partitioning
method
•Use partioning protein help in replication
process
•https://youtu.be/FhcZLqvs5yg
Rolling circle replication
mechanism

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5. Plasmid transfer
(plasmid integration)
•There are two types of plasmid integration:
❑horizontal transfere (from cell to another)
into a host bacteria: Non-integrating plasmids
replicate as with the top instance,
❑vertical transfere (through the same cell
episomes, the lower example, can integrate into
the host chromosome.
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9. Classifications and types
•Plasmids may be classified in a number of ways.
1.conjugative plasmids and non-conjugative
plasmids.
Conjugative plasmids contain a set of transfer or tra genes which
promote sexual conjugation between different cells.
In the complex process of conjugation, plasmid may be transferred
from one bacterium to another via sex pili encoded by some of the
tra genes (see figure).
Non-conjugative plasmids are incapable of initiating conjugation,
hence they can be transferred only with the assistance of
conjugative plasmids.
mobilizable plasmids An intermediate class of plasmids, carry only
a subset of the genes required for transfer. They can parasitize a
conjugative plasmid, transferring at high frequency only in its
presence.
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10. bacterial conjugation
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11. 2. incompatibility groups
• A microbe can harbour different types of
plasmids, however, different plasmids can only
exist in a single bacterial cell if they are
compatible.
•If two plasmids are not compatible, one or the
other will be rapidly lost from the cell.
•Different plasmids may therefore be assigned to
different incompatibility groups depending on
whether they can coexist together. Incompatible
plasmids normally share the same replication or
partition mechanisms.
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12. Plasmid incompatability
Dark type open type
1- non compatible plasmid replication
one type lost i.e dilution occur for this type
during replication
2- compatible plasmid replication
two types found after replication

13. 3. Another way to classify plasmids is
by function
There are five main classes:
•FertilityF-plasmids, which contain tra genes. They are capable
of conjugation and result in the expression of sex pili.
•Resistance (R) plasmids, which contain genes that provide
resistance against antibiotics or poisons. Historically known as
R-factors, before the nature of plasmids was understood.
•Col plasmids, which contain genes that code for bacteriocins,
proteins that can kill other bacteria.
•Degradative plasmids, which enable the digestion of unusual
substances, e.g. toluene and salicylic acid.
•Virulence plasmids, which turn the bacterium into a pathogen.
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14. nomencleature
•p
•capital letters
•Number
•Ex. P COLE. 1
• PF

15. Plasmid applications
•Cloning
Plasmids are the most-commonly used bacterial cloning vectors.
[13]
These cloning
vectors contain a site that allows DNA fragments to be inserted, for example a
multiple cloning site or polylinker which has several commonly used restriction
sites to which DNA fragments may be ligated. After the gene of interest is inserted,
the plasmids are introduced into bacteria by a process called transformation. These
plasmids contain a selectable marker, usually an antibiotic resistance gene, which
confer on the bacteria an ability to survive and proliferate in a selective growth
medium containing the particular antibiotics. The cells after transformation are
exposed to the selective media, and only cells containing the plasmid may survive.
In this way, the antibiotics act as a filter to select only the bacteria containing the
plasmid DNA. The vector may also contain other marker genes or reporter genes to
facilitate selection of plasmid with cloned insert. Bacteria containing the plasmid
can then be grown in large amounts, harvested, and the plasmid of interest may
then be isolated using various methods of plasmid preparation.
A plasmid cloning vector is typically used to clone DNA fragments of up to 15
kbp.To clone longer lengths of DNA, lambda phage with lysogeny genes deleted,
cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are
used.
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16. Protein production
•Another major use of plasmids is to make large amounts of proteins. In this case,
researchers grow bacteria containing a plasmid harboring the gene of interest. Just
as the bacterium produces proteins to confer its antibiotic resistance, it can also be
induced to produce large amounts of proteins from the inserted gene. This is a
cheap and easy way of mass-producing the protein the gene codes for, for
example, insulin.
Gene therapy
•Plasmid may also be used for gene transfer into human cells as potential treatment
in gene therapy so that it may express the protein that is lacking in the cells. Some
strategies of gene therapy require the insertion of therapeutic genes at pre-
selected chromosomal target sites within the human genome. Plasmid vectors are
one of many approaches that could be used for this purpose. Zinc finger nucleases
(ZFNs) offer a way to cause a site-specific double-strand break to the DNA genome
and cause homologous recombination. Plasmids encoding ZFN could help deliver a
therapeutic gene to a specific site so that cell damage, cancer-causing mutations,
or an immune response is avoided.
Disease models
•Plasmids were historically used to genetically engineer the embryonic stem cells of
rats in order to create rat genetic disease models. The limited efficiency of
plasmid-based techniques precluded their use in the creation of more accurate
human cell models. However, developments in Adeno-associated virus
recombination techniques, and Zinc finger nucleases, have enabled the creation of
a new generation of isogenic human disease models.
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17. Episomes & cancer
•The term episome was proposed by François Jacob and Élie
Wollman in 1958 to describe extra-chromosomal genetic material
that may replicate autonomously or become integrated into the
chromosome.
•The use of the term, however, has diverged since it was first coined
as plasmid became the preferred word for autonomously
replicating extrachromosomal DNA as proposed in the symposium
in 1968 – it was suggested by some that the use of the term
episome be abandoned, although others continued to use the term
with a shift in meaning.
•In prokaryotes, episome is now used by some to refer to plasmid
that is capable of integrating into the chromosome. The integrative
plasmids may be replicated and stably maintained in a cell through
multiple generations, but always at some stage they exist as an
independent plasmid molecule.
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18. •In eukaryotes, episomes are used to mean non-integrated
extrachromosomal closed circular DNA molecule that may be replicated in
the nucleus.
[21][22]
Viruses are the most common examples of this, such as
herpesviruses, adenoviruses, and polyomaviruses, but some are plasmids.
Other examples include aberrant chromosomal fragments, such as double
minute chromosomes, that can arise during artificial gene amplifications
or in pathologic processes (e.g., cancer cell transformation). Episomes in
eukaryotes behave similarly to plasmids in prokaryotes in that the DNA is
stably maintained and replicated with the host cell. Cytoplasmic viral
episomes (as in poxvirus infections) can also occur. Some episomes, such
as herpesviruses, replicate in a rolling circle mechanism, similar to
bacterial phage viruses. Others replicate through a bidirectional
replication mechanism (Theta type plasmids). In either case, episomes
remain physically separate from host cell chromosomes. Several cancer
viruses, including Epstein-Barr virus and Kaposi's sarcoma-associated
herpesvirus, are maintained as latent, chromosomally distinct episomes in
cancer cells, where the viruses express oncogenes that promote cancer
cell proliferation. In cancers, these episomes passively replicate together
with host chromosomes when the cell divides. When these viral episomes
initiate lytic replication to generate multiple virus particles, they in general
activate cellular innate immunity defense mechanisms that kill the host
cell.
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19. Cloning in vivo can be done in
unicellular
microbes like E. coli
unicellular eukaryotes like yeast and
in mammalian cells grown in tissue culture.
STEPS
1- RECOMBINATION
2- TRANSFORMATION
3- SELECTIVE AMPLIFICATION
4- ISOLATION

21. Making Recombinant DNA (rDNA):

22. •Treat DNA from both sources with the same restriction
endonuclease (BamHI in this case).
•BamHI cuts the same site on both molecules
5' GGATCC 3'
3' CCTAGG 5'
•The ends of the cut have an overhanging piece of
single-stranded DNA.
•These are called "sticky ends" because they are able to
base pair with any DNA molecule containing the
complementary sticky end.
•In this case, both DNA preparations have
complementary sticky ends and thus can pair with
each other when mixed.
•a DNA ligase covalently links the two into a molecule
of recombinant DNA.

23. Transforming E. coli
•A suspension of E. coli is treated
with the mixture of religated DNA
molecules.
•The suspension is spread on the
surface of agar containing both
ampicillin and kanamycin.
•The next day, a few cells — resistant
to both antibiotics — will have
grown into visible colonies
containing billions of transformed
cells.
•Each colony represents a clone of
transformed cells.

24. An Example
•pAMP
•4539 base pairs
•a single replication origin
•a gene (amp
r
)conferring resistance to the antibiotic
ampicillin (a relative of penicillin)
•a single occurrence of the sequence 5' GGATCC 3'
3' CCTAGG 5' that, as we saw above, is cut by the restriction
enzyme BamHI
•a single occurrence of the sequence 5' AAGCTT 3'
3' TTCGAA 5' that is cut by the restriction enzyme HindIII
•Treatment of pAMP with a mixture of BamHI and HindIII
produces: a fragment of 3755 base pairs carrying both the
amp
r
gene and the replication origin
•a fragment of 784 base pairs
•both fragments have sticky ends

25. •pKAN
•4207 base pairs a single replication origin
•a gene (kan
r
) conferring resistance to the antibiotic
kanamycin.
•a single site cut by BamHI
•a single site cut by HindIII
•Treatment of pKAN with a mixture of BamHI and
HindIII produces: a fragment of 2332 base pairs
•a fragment of 1875 base pairs with the kan
r
gene
(but no origin of replication)
•both fragments have sticky ends
•These fragments can be visualized by subjecting the
digestion mixtures to electrophoresis in an agarose
gel. Because of its negatively-charged phosphate
groups, DNA migrates toward the positive electrode
(anode) when a direct current is applied. The smaller
the fragment, the farther it migrates in the gel.

26. Ligation Possibilities

28. Some recombinant DNA products
being used in human therapy
•insulin for diabetics
•factor VIII for males suffering from hemophilia A
•factor IX for hemophilia B
•human growth hormone (HGH)
•erythropoietin (EPO) for treating anemia
•several types of interferons
•several interleukins
•granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone
marrow after a bone marrow transplant
•granulocyte colony-stimulating factor (G-CSF) for stimulating neutrophil production (e.g.,
after chemotherapy) and for mobilizing hematopoietic stem cells from the bone marrow
into the blood.
•tissue plasminogen activator (TPA) for dissolving blood clots
•adenosine deaminase (ADA) for treating some forms of severe combined
immunodeficiency (SCID)
•parathyroid hormone
•many monoclonal antibodies
•hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus
•C1 inhibitor (C1INH) used to treat hereditary angioedema

29. DNA Sequencing
•Is the determination of the sequence of
nucleotides in sample of DNA
•The most popular method for doing this is called
the dideoxy method or sanger method or chain
termination method

30. Sanger sequencing
A method of DNA sequencing based on the
selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in
vitro DNA replication.
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32. Requirements
The classical chain-termination method requires
1.A single-stranded DNA template
2.A RNA primer
3.A DNA polymerase
4.Normal deoxynucleosidetriphosphates (dNTPs)
5.Modified di-deoxy nucleotide triphosphates
(ddNTPs),
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33. Procedure
•The DNA to be sequenced is prepared as a single strand.
•This template DNA is supplied with a mixture of all four
normal (deoxy) nucleotides in ample quantities
–dATP dGTP dCTP dttp
•a mixture of all four dideoxynucleotides, each
present in limiting quantities and each labeled
with a "tag" that fluoresces a different color:
–ddATP ddGTP ddCTP ddTTP
•DNA polymerase I
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34. •Because all four normal nucleotides are
present, chain elongation proceeds normally
until, by chance, DNA polymerase
inserts a dideoxy nucleotide (shown as colored
letters) instead of the normal deoxynucleotide
(shown as vertical lines).
• At the end of the incubation period, the
fragments are separated by length from longest
to shortest.
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35. •(`The resolution is so good that
a difference of one nucleotide
is enough to separate
that strand from the next
shorter and next longer strand).
•Each of the four
Dideoxynucleotides fluoresces
a different color when
illuminated by a laser beam
and an automatic scanner
provides a printout of
the sequence.
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