Artificial chromosomes are laboratory constructs that contain DNA sequences and that perform the critical functions of natural chromosomes. They are used to introduce and control new DNA in a cell, to study how chromosomes function, and to map genes in genomes. artificial chromosome A type of cloning vector that has some features of true chromosomes and is used to clone relatively large fragments of DNA. Bacterial artificial chromosomes (BACs) are based on the F (fertility) plasmid found naturally in E. coli bacteria (see sex factor). They can accommodate inserts of foreign DNA up to about 300 kilobase (kb) in length. Also included are several bacterial genes necessary for replication of the plasmid by the host cell and a gene (usually for resistance to an antibiotic) that allows selection of BAC-containing cells. Larger DNA fragments are cloned using yeast artificial chromosomes (YACs). These are linear vectors derived from a circular plasmid found naturally in baker's yeast (Saccharomyces cerevisiae) and capable of accommodating DNA inserts of up to 1000 kb. YACs have a centromere, enabling them to attach to the mitotic spindle of their yeast host and undergo normal segregation during cell division. They are also engineered with telomeres, the DNA sequences that cap either end of a chromosome. Thus YACs behave like mini-chromosomes. They are used for cloning eukaryotic genes or gene segments, for making DNA libraries of organisms with large genomes (e.g. mammals), and for studying gene function.
3. GENETIC
ENGINEERING
Also called genetic modification / genetic
manipulation / rDNA technology
Direct manipulation of an organism’s DNA using
biotechnology
Set of technologies used to change genetic makeup
of cells, including transfer of genes within and across
species boundaries to produce improved or novel
organisms
New DNA obtained by isolating & copying gene of
interest (GI) using rDNA methods or by artificially
synthesising DNA
4. SOME OF THE MOST
COMMON GENETIC
ENGINEERING TEST
SUBJECTS ARE MICE
AND ZEBRAFISH.
WHY?
PONDER ON THIS
6. STEPS IN GENE CLONING
The chosen piece of
DNA is ‘cut’ from the
source
organism using
restriction enzymes.
The piece of DNA is
‘pasted’ into a
vector and the
ends
of the DNA are
joined with the
vector DNA by
ligation.
The vector is
introduced into a host
cell, by a process
called transformation.
The host cells create
multiple
copies of the inserted
DNA.
The vector DNA is
isolated from the
host cells’ DNA
and purified.
7. Nucleic acid molecules capable of autonomous
replication, can carry a gene and deliver it into a cell
Different types of vectors includes :
Plasmids
Bacteriophages
Cosmids
Phagemids
Artificial Chromosomes (BAC, YAC, HAC, MAC, Plant
derived AC)
VECTORS
8. ARTIFICIAL CHROMOSOMES
SYNTHETIC CHROMOSOMES CONSISTING OF FRAGMENTS OF
USEFUL IN CLONING LARGER FRAGMENTS OF DNA, AS PLASMIDS
CONTAIN 300,000(BAC) TO 1,000,000(YAC) BASE PAIRSREDUCES
DNA INTEGRATED INTO A HOST CHROMOSOME- INTRODUCED
INTO HOST CELLS TO PROPAGATE AND USED TO TRANSFECT
OTHER CELLS, INTRODUCING NEW DNA.
CAN ONLY CONTAIN UP TO 10,000 BASE PAIRS AND PHAGES
ARE HARD TO WORK WITH.
AMOUNT OF RUNS NEEDED FOR A LARGE FRAGMENT
TO BE ANALYZED-EASIER AND QUICKER TO CLONE AND
TRANSFORM GENES
9. L I N E A R O R C I R C U L A R
D I F F E R E N T T Y P E S
B A C T E R I A L A R T I F I C I A L C H R O M O S O M E ( B A C )
Y E A S T A R T I F I C I A L C H R O M O S O M E ( Y A C )
P 1 D E R I V E D A R T I F I C I A L C H R O M O S O M E ( P A C )
M A M M A L I A N A R T I F I C I A L C H R O M O S O M E ( M A C )
H U M A N A R T I F I C I A L C H R O M O S O M E ( H A C )
Y A C – C L O N I N G I N Y E A S T
B A C & P A C – B A C T E R I A
M A C & H A C – M A M M A L I A N & H U M A N C E L L S
10. BACTERIAL ARTIFICIAL CHROMOSOME
DEVELOPED BY MEL SIMMONS AND COWORKERS IN 1992
1ST BAC VECTOR – PBAC108L
PLASMIDS CONSTRUCTED WITH REPLICATION ORIGIN OF
E.COLI F FACTOR, SO CAN BE MAINTAINED IN A SINGLE
COPY PER CELL
CAN HOLD DNA FRAGMENTS OF 75 TO 300 KB
RECOMBINANT BACS INTRODUCED INTO E.COLI BY
ELECTROPORATION
Then rBAC replicates like an F factor
11. ori : origin of replication
repE : for plasmid replication
parA and parB : for partitioning
Selectable marker for antibiotic
T7 & Sp6 phage promoters for
Components :
and regulation of copy number
F plasmids to daughter cells
during division and ensures
stable maintenance of the BAC
resistance. Some BACs also
have lacZ at cloning site
for blue/white selection
transcription of inserted genes.
13. ADVANTAGES
STABLE INSERTS AND DO NOT DELETE SEQUENCES
EASY TO MANIPULATE
TRANSFORMING EFFICIENCY HIGHER THAN THAT OF YAC
SPEEDY GROWTH OF E.COLI HOST
SIMPLER TO PURIFY
MORE USER FRIENDLY
HELPFUL IN DEVELOPMENT OF VACCINES
14. DISADVANTAGES
FRAGMENT DNA CONTAINS UNRELATED GENES MAY LEAD TO
GENERATION AND SCREENING OF RECOMBINANT BAC
OVERSIZED BAC DNA CONSTRUCTS ARE MORE EASILY SHEARED
RANDOM RECOMBINATION EVENTS MAY OCCUR.
INTRA MOLECULAR REARRANGEMENTS - REDUCE THE
INDIRECT, NON-SPECIFIC GENE EXPRESSION AND
UNANTICIPATED CHANGES IN THE CELL PHENOTYPE.
CONSTRUCTS CAN BE TIME-CONSUMING AND LABOR-INTENSIVE.
AND DEGRADED DURING MANIPULATION BEFORE
TRANSFECTION.
RECOMBINATION EFFICIENCY AND INCREASE THE RATE OF FALSE
POSITIVE CLONES IN SOME SELECTION/COUNTER-SELECTION
APPROACHES
15. YEAST ARTIFICIAL CHROMOSOME
DEVISED AND FIRST REPORTED IN 1987 BY DAVID BURK AND
PYAC3- FIRST YAC DEVELOPED.
SPECIAL LINEAR DNA VECTORS THAT RESEMBLE NORMAL YEAST
CIRCULAR DOUBLE STRANDED DNA - CONTAINS A REPLICATION
CLONE LARGE DNA SEGMENT (100 – 1000KB)
OLSON
CHROMOSOME
ORIGIN (COLE 1) COMPATIBLE WITH E. COLI IN ADDITION TO
YEAST REPLICATION ORIGIN OR AN YEAST ARS ELEMENT-USEFUL
FOR AMPLIFICATION OF THE VECTOR IN E. COLI.
16. BY INSERTING LARGE FRAGMENTS OF DNA, INSERTED
INITIALLY USED FOR THE HUMAN GENOME PROJECT- DUE TO
PRIMARY COMPONENTS - AUTONOMOUSLY REPLICATING
SEQUENCE (ARS), CENTROMERE (CEN) AND TELOMERES (TEL)
FROM S.CEREVISIAE.
SELECTABLE MARKER GENES, SUCH AS ANTIBIOTIC RESISTANCE
AND A VISIBLE MARKER - SELECT TRANSFORMED YEAST CELLS.
SEQUENCES CAN BE CLONED AND PHYSICALLY MAPPED USING
A PROCESS CALLED CHROMOSOME WALKING.
STABILITY ISSUES ABANDONED FOR THE USE OF BACTERIAL
ARTIFICIAL CHROMOSOME.
18. APPLICATIONS:
GENERATING WHOLE DNA LIBRARIES OF THE GENOMES OF
YAC CLONES USED AS HYBRIDIZATION PROBES FOR THE
STUDY OF REGULATION OF GENE EXPRESSION BY CIS-ACTING,
USED FOR ISOLATION OF FUNCTIONALLY ANALOGOUS
HIGHER ORGANISMS
SCREENING OF CDNA LIBRARIES
REGULATORY DNA ELEMENTS AFTER THE TRANSFER OF THESE
YACS FROM YEAST TO MAMMALIAN CELLS
MAMMALIAN DNA SEQUENCES IN ORDER TO DEVELOP MACS
19. LIMITATIONS:
VERY LARGE DNA MOLECULES ARE VERY FRAGILE AND PRONE TO
HIGH RATE OF LOSS OF THE ENTIRE YAC DURING MITOTIC
DIFFICULT TO SEPARATE YAC FROM THE OTHER HOST
SEPARATION REQUIRES SOPHISTICATED PULSE-FIELD GEL
YIELD OF DNA IS NOT HIGH WHEN YAC IS ISOLATED FROM
CLONES TEND TO BE UNSTABLE, WITH THEIR FOREIGN DNA
BREAKAGE, LEADING TO PROBLEM OF REARRANGEMENT.
GROWTH.
CHROMOSOMES BECAUSE OF THEIR SIMILAR SIZE.
ELECTROPHORESIS (PFGE).
YEAST CELLS.
INSERTS OFTEN BEING DELETED.
20. LAMBDA PHAGE
ENTEROBACTERIA PHAGE Λ (LAMBDA PHAGE, COLIPHAGE Λ, OFFICIALLY
ESCHERICHIA VIRUS LAMBDA) IS A BACTERIAL VIRUS, OR BACTERIOPHAGE, THAT
INFECTS THE BACTERIAL SPECIES ESCHERICHIA COLI (E. COLI). IT WAS DISCOVERED
BY ESTHER LEDERBERG IN 1950.
THE WILD TYPE OF THIS VIRUS HAS A TEMPERATE LIFE CYCLE THAT ALLOWS IT TO
EITHER RESIDE WITHIN THE GENOME OF ITS HOST THROUGH LYSOGENY OR ENTER
INTO A LYTIC PHASE, DURING WHICH IT KILLS AND LYSES THE CELL TO PRODUCE
OFFSPRING.
THE PHAGE PARTICLE CONSISTS OF A HEAD (ALSO KNOWN AS A CAPSID), A TAIL, AND
TAIL FIBERS .
DURING INFECTION, THE PHAGE PARTICLE RECOGNIZES AND BINDS TO ITS HOST, E.
COLI, CAUSING DNA IN THE HEAD OF THE PHAGE TO BE EJECTED THROUGH THE TAIL
INTO THE CYTOPLASM OF THE BACTERIAL CELL.
21. AS A GENETIC MATERIAL
a vector for the cloning
of recombinant DNA. The
48 kb DNA fragment of
lambda phage is not
essential for productive
infection and can be
replaced by foreign DNA.
the use of its site-specific
recombinase (int) for the
shuffling of cloned DNAs
by the gateway method;
and the application of its
Red operon, including the
proteins Red alpha (also
called 'exo'), beta and
gamma in the DNA
engineering method
called recombineering.
Lambda phage will enter
bacteria more easily than
plasmids making it a
useful vector that can
destroy or can become
part of the host's DNA.
Lambda phage can be
manipulated and used as
an anti-cancer vaccine,
nanoparticle, targeting
human aspartyl
(asparaginyl) β-
hydroxylase (ASPH,
HAAH).
