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Tools of Biotechnology

Genetic engineering involves manipulating DNA through techniques like gene splicing and cloning. It has two main steps - synthesizing recombinant DNA by combining DNA fragments, and cloning the DNA by inserting it into a host organism. Restriction enzymes cut DNA at specific recognition sequences and are used to fragment DNA for recombination. Vectors are used to carry passenger DNA into host cells, and must be able to replicate autonomously within the host and allow for selection of transformed cells.

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324 views10 pages

Tools of Biotechnology

Genetic engineering involves manipulating DNA through techniques like gene splicing and cloning. It has two main steps - synthesizing recombinant DNA by combining DNA fragments, and cloning the DNA by inserting it into a host organism. Restriction enzymes cut DNA at specific recognition sequences and are used to fragment DNA for recombination. Vectors are used to carry passenger DNA into host cells, and must be able to replicate autonomously within the host and allow for selection of transformed cells.

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iamforu1
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Tools of

Biotechnology
What is Genetic
engineering?
• Most fundamental mechanics
of biotechnology.
• It involves gene splicing,
recombinant DNA cloning &
tissue culture technology.
Technique involves 2
steps
• Step 1:Involves synthesis of
recombinant DNA
• Step 2:is the cloning OR in other
words Genetic engineering is a
technique which involves the
process of manipulating DNA to
form new genes OR inserting
altered genes in different o’s
acquires the genetic abilities or
characters of donor…….
RECOMBINANT DNA:-

• A DNA formed by union of


fragments of genetic material
derived from two different o’s.The
two o’s may belong to the same
species or to different species
such a DNA is also called HYBRID
DNA…………..
BASIC TECHNIQUE OF G.E/DNA
RECOMBINANT TECHNOLOGY

• It involves various types:


1)Gene isolation
2)Selection of vector
3)Cloning of desired gene
4)Specific gene transfer
5)Expression of desired gene
RESTRICTION ENZYMES
• Also called as ENDONUCLEASES
• They produce internal cuts called
CLEAVAGES in DNA molecules.
• All bacteria produce atleast 1 type of
restriction enzyme.These are
chemically proteins & they serve
purpose of cutting up foreign DNA
such as that of invading viruses.They
are called as MOLECULAR SCISSORS.
• Now they are helping the recombinant
researchers to enable them to cut the
DNA.
• Restriction enzymes cut the DNA at very
specific places along its length.
• Example-The restriction enzyme ECOR-1
produced by the intestinal bacterium
E.coli recognizes the following sequence
(fig) AATT
3 TYPES OF R-ENDONUCLEASES
• Type-1 R-endonuclease are complex & have
recognition sequences of 15 base pair. They cleave
the DNA about 100 bp away from 5’ end

• Type 2 R-endonucleases are remarkably stable and


induces cleavage within their reorganization
sequences or very close to them. More than 350
different type of R-endonucleases Type 2 are
present. Only type 2 R-endonucleases are used for
restriction mapping and gene cloning.

• Type 3 Restriction endonucleases are intermediate


between type 1 and 2 enzymes. Eg: Eco K, EcoB,
Eco P1,Eco P15…….
LIGASES
Fragments of DNA from different o’s or
even from different species may be
joined together at their sticky ends
thereby producing recombinant DNA.
This is made possible by the use of
enzyme called LIGASE….
VECTORS

“DNA molecules which are


used to carry the passenger
DNA (to be cloned) are
called……
• SIZE: Should be less than
10 kilobytes
PROPERTIES OF GOOD VECTOR
• Should be able to replicate autonomously.
• Should be easy to isolate & purify.
• Should be easily introduced into the host cell.
• Should have suitable marker genes that allow easy detection &
selection of transformed host cell.
• When objective is gene transfer it should have ability to
integrate either itself or DNA insert it carries into the genome
of the host cell.
• Should contain unique target sites for as many restriction
enzymes as possible into which DNA insert can be integrated
without disrupting an essential function……….

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