Chapter 11 :- Biotechnology : Principles and Processes
Biotechnology :- Technical manipulation of different organisms to produce desirable products.
The DNA segments cut by restriction enzymes are palindromic i.e. the nucleotide sequence of
these DNA pieces read the same both backwards and forward.
e.g.madam
Blunt or flush ends are produced by many restriction enzymes which cleave both strands of DNA
at exactly the same nucleotide position, in the centre of recognition site.
5 CCCGGG 3
3 GGGCCC 5
It cuts both DNA strands producing blunt ends.
Sticky or cohesive ends are produced when restriction enzymes donot cut DNA at the same nucleotide
position but cut the recognition sequence unequally.
Electrophoresis :- It is a method of separation of charged molecules applying in an electric field. This
technique was pioneered by A.Tiselius in 1937. When the charged molecules are placed in an electric field
they migrate depending on their net charges, size, shape and applied current. This technique of
electrophoresis can be used for separation of DNA,RNA and proteins.
In gel electrophoresis, the gel acts as a molecular sieve to separate molecules. The
different types of gel electrophoresis used are :-
a) Agarose gel electrophoresis
b) Pulse Field Gel Electrophoresis (PFGE)
c) Polyacrylamide Gel Electrophoresis (PAGE)
d) Sodium Dodecyl sulphate polyacrylamide Gel Electrophoresis (SDS-PAGE)
,Agarose gel method is most widely used for separationof DNA. Agarose is a linear polymer of D-galactose
and 3,6- anhydro-L-galactose extracted from algal seaweeds. At neutral pH the negatively charged DNA
migrate toward the anode after the application of electric field across the gel.
In the agarose gel electrophoresis method, the DNA fragments separate according to their size
through sieving effect provided by the agarose gel.
The separated DNA fragments can be seen only after staining the DNA with ethidium bromide
followed by exposure to UV radiation. These DNA fragments are stained as bright orange coloured bands.
The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is
known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by
joining them with cloning vectors.
Polymerase Chain Reaction
It is a technique for exponential amplification of DNA sequence through in vitro DNA
synthesis. The technique was developed by Kary Mullis in 1985 and he shared Nobel Prize in chemistry with
Michael Smith in 1993 for the discovery of PCR.
, The tools of PCR are as follows :-
(i) DNA template :- Any desired DNA or target DNA can serve as DNA template for amplification.
(ii) Primers :- A pair of oligonucleotides of about 180 – 230 nucleotides with similar G+C contents act as
primers. The primers are designed to anneal(hybridize) on opposite strands of target sequence so that
they are extended toward each other by addition of nucleotides at their 3 ends.
(iii) Enzymes :- The most commonly used enzyme is thermostable Taq polymerase bacterium called
Thermus aquaticus. It survives at 950C for 1-2 minutes.
Procedure of PCR :- The mechanism of PCR has the following steps :-
(a) Denaturation
(b) Annealing
(c) Polymerization (extension)
The entire process is depicted in three steps and two complete cycles produce eight strands.
Similarly, the third cycle will produce 16 strands. It is estimated that 30 cycles will produce about one
billion copies.
Fig :- Diagrammatic representation of working of PCR
Biotechnology :- Technical manipulation of different organisms to produce desirable products.
The DNA segments cut by restriction enzymes are palindromic i.e. the nucleotide sequence of
these DNA pieces read the same both backwards and forward.
e.g.madam
Blunt or flush ends are produced by many restriction enzymes which cleave both strands of DNA
at exactly the same nucleotide position, in the centre of recognition site.
5 CCCGGG 3
3 GGGCCC 5
It cuts both DNA strands producing blunt ends.
Sticky or cohesive ends are produced when restriction enzymes donot cut DNA at the same nucleotide
position but cut the recognition sequence unequally.
Electrophoresis :- It is a method of separation of charged molecules applying in an electric field. This
technique was pioneered by A.Tiselius in 1937. When the charged molecules are placed in an electric field
they migrate depending on their net charges, size, shape and applied current. This technique of
electrophoresis can be used for separation of DNA,RNA and proteins.
In gel electrophoresis, the gel acts as a molecular sieve to separate molecules. The
different types of gel electrophoresis used are :-
a) Agarose gel electrophoresis
b) Pulse Field Gel Electrophoresis (PFGE)
c) Polyacrylamide Gel Electrophoresis (PAGE)
d) Sodium Dodecyl sulphate polyacrylamide Gel Electrophoresis (SDS-PAGE)
,Agarose gel method is most widely used for separationof DNA. Agarose is a linear polymer of D-galactose
and 3,6- anhydro-L-galactose extracted from algal seaweeds. At neutral pH the negatively charged DNA
migrate toward the anode after the application of electric field across the gel.
In the agarose gel electrophoresis method, the DNA fragments separate according to their size
through sieving effect provided by the agarose gel.
The separated DNA fragments can be seen only after staining the DNA with ethidium bromide
followed by exposure to UV radiation. These DNA fragments are stained as bright orange coloured bands.
The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is
known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by
joining them with cloning vectors.
Polymerase Chain Reaction
It is a technique for exponential amplification of DNA sequence through in vitro DNA
synthesis. The technique was developed by Kary Mullis in 1985 and he shared Nobel Prize in chemistry with
Michael Smith in 1993 for the discovery of PCR.
, The tools of PCR are as follows :-
(i) DNA template :- Any desired DNA or target DNA can serve as DNA template for amplification.
(ii) Primers :- A pair of oligonucleotides of about 180 – 230 nucleotides with similar G+C contents act as
primers. The primers are designed to anneal(hybridize) on opposite strands of target sequence so that
they are extended toward each other by addition of nucleotides at their 3 ends.
(iii) Enzymes :- The most commonly used enzyme is thermostable Taq polymerase bacterium called
Thermus aquaticus. It survives at 950C for 1-2 minutes.
Procedure of PCR :- The mechanism of PCR has the following steps :-
(a) Denaturation
(b) Annealing
(c) Polymerization (extension)
The entire process is depicted in three steps and two complete cycles produce eight strands.
Similarly, the third cycle will produce 16 strands. It is estimated that 30 cycles will produce about one
billion copies.
Fig :- Diagrammatic representation of working of PCR
