VECTOR

Vector are use to transfer desirable genes that are located in one
chromosomes to another.
Vector play major role in recombinant DNA techniques for inserting
selected genes from one kind of organism into cell of another kind.
Vector can be transferred into bacterial cell.
To clone pieces of DNA in the laboratory:

METHOD IN GENE CLONING:

Gene cloning refer to procedures that lead to the formation of many copies of
particular gene.
Gene cloning involves:

I.
II.
III.
IV.
V.

Isolating vector from bacterial cell and DNA from region cell
Cutting vector and foreign DNA with the same restriction enzymes
Creating recombinant vector
Inserting the recombinant vector into the host cell through transformation
Screening and identify clones carrying the gene of interest

ISOLATING OF GENE:

isolate DNA from foreign cell contains gene of interest and plasmid from
bacterial cell

CUTTING VECTOR AND FOREIGN DNA WITH THE SAME RESTRICTION ENZYMES

an enzyme is used to cleave the plasmid and creates a linear plasmid with
sticky end
the enzyme used to cleave the human chromosomal DNA which contain
the gene of interest into many fragment with sticky end.

CREATING RECOMBINANT PLASMIDS

the sticky end will made two DNA join by base pairing, result in circular
plasmid contain new DNA.
DNA ligase is used to close the gap
A plasmid carry foreign gene is known as a recombinant plasmid.
a. If the cell reanneals itself without insert the foreign DNA, the lac
Z gene will remain uninterrupted and produce B-galactose, then
the X-Gal will turn the cell blue

b. If the cell working (insert foreign DNA), lac Z will not produce
functional group and unable to generate active B-galactose and
the X-Gal will turn the cell colourless
INSERT THE RECOMBINANT PLASMID INTO A HOST CELL THROUGH
TRANSFORMATION

The recombinant plasmid are introduced to bacterial cell

The plasmid are added to flask containing E.coli
Calcium ion in form calcium chloride, are added to the flask followed by
a brief heat shock.
This will effect of making holes appear in the cell wall membranes.

SCREENING OF CLONES AND IDENTIFY CLONES CARRYING GENE OF INTEREST

To identify bacteria have been transformed, we will put the bacteria in a

petri disk [late containing X-Gal and amplicin.
Those bacteria which contain the plasmid with amp are able to grow and
multiply to form a bacteria colony in medium containing ampilicin.
Bacteria that did not have plasmid with amp will be killed by ampicillin.
Bacteria with functional B-galactosidase enzyme will form blue colonies
when they grown in medium with the presence of X-Gal and colourless
colonies if they do not
Bacteria which form colourless colonies are the one containing the
recombinant plasmid and can be isolated for futher cloning.

POLYMERASE CHAIN REACTION

Polymerase chain is another way to copy DNA without vector and host cell
I.
II.

III.

The starting materials contains a sample of DNA, known as template DNA

The double mixture stranded DNA is mixed with excess primer. This
mixture is then heated up at 98C to bring denaturation of the double
stranded DNA into separate single.
Cool the solution about 60C. the primer will bind to the DNA as the
temperature lowered. The strand will pair with the base of complimentary
primer, leaving the rest of the fragment single stranded. This stage called
annealing

IV.

After that the Taq polymerase is added, the polymerase will copied the rest
of the fragment as it were replicating DNA. This known as primer
extension.