Biotechnology Principles and Processes L5

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Coordination and L1
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Biotechnology
Principles & Processes
Biotechnology
Separation and Isolation of DNA Fragments
➔ Fragments formed after cutting the molecule of DNA are separated according to
their size/length by technique called as Gel Electrophoresis
➔ Electrophoresis- It is a technique of separation of DNA/RNA or proteins molecule
according to their size, under the influence of electric field.
Agarose Gel Electrophoresis
➔ Fragments formed after cutting the molecule of DNA are separated according to
their size/length by technique called as Gel Electrophoresis
➔ Electrophoresis- It is a technique of separation of DNA/RNA or proteins molecule
according to their size, under the influence of electric field.
➔ Agarose powder is dissolved in buffer and heated.
➔ Choose an appropriate comb for forming the sample slots in the gel.
➔ After the solution cools, the agarose hardens.
➔ After mounting the gel in electrophoresis tank, add the buffer to cover the gel and
attack the tank to positive and negative electrodes.
➔ DNA molecules have negative charges, and so when placed in an electric field will
migrate towards the positive pole (anode).
➔ Polymerized agarose acts as a sieve with small holes between the tangled chains
of agarose.
➔ Hence, the smaller fragment size, the farther it moves.
➔ The process is repeated for Vector DNA also.
Visualisation of DNA Fragment
➔ To visualize the DNA, the agarose gel is immersed into a solution of ethidium
bromide.
➔ Bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to
UV light can be seen.
Isolation of DNA Fragment
➔ Elution- The separated bands of DNA are cut out from the agarose gel and
extracted from the gel piece.
➔ These purified DNA fragments are used in constructing recombinant DNA by
linking them with cloning vectors.
Isolation of DNA Fragment
Amplification of Gene of Interest
PCR (Polymerase Chain Reaction)
➔ Invented by Kary Mullis in 1985, PCR has revolutionised the field of Biotechnology
➔ It allows- Production and amplification of billion of copies of an original piece of
DNA in a tube within minutes.
➔ Also called as “Molecular Photocopying”.
➔ It has revolutionized the study of DNA to such extent that its creator, Kary B.Mullis,
was awarded the Nobel Prize for chemistry in 1993.
Amplification of Gene of Interest
PCR (Polymerase Chain Reaction)
➔ PCR is a three-step process that is carried out in repeated cycles.
➔ Principle of PCR- When double stranded DNA is heated to high temperature, the
two DNA strands separate giving rise to two single stranded DNA molecules.
➔ If these single stranded DNA molecules are copied by DNA Polymerase, this would
lead to duplication of original DNA.
Requirements for PCR
➔ A pair of primers (small chemically synthesized oligonucleotides that are
complementary to the regions of DNA) with exposed 3’ - OH groups that will bind
to the particular sequence of interest in the DNA template.
◆ Target DNA (few nanogram)
◆ Primers
◆ Taq polymerase
◆ Mg2+
◆ dNTPs (four deoxyribonucleoside triphosphates)
Requirements for PCR
Steps in PCR
Denaturation
Annealing
Extension
Denaturation
➔ The target DNA Is heated to high temperature (), which will break the hydrogen
bonds between two strands of DNA and will lead to separation of these two
strands.
➔ It will provide necessary single strand template.
Annealing
➔ The mixture is cooled down to 50 - 600C.
➔ Two oligonucleotide primers will anneal to each of the single stranded DNA
template since the primer sequence is complementary to 3’ end of DNA Template.
Extension
➔ The sample is next heated to 720C for sometime, during which time the DNA
polymerase adds nucleotides to the primer, synthesizing a new DNA strand using
only the template sequences that bind the primers.
Applications of PCR
➔ If the process of replication of DNA is repeated many times, the segment of DNA
can be amplified to approximately billion times., 1 billion copies are made at the
end of 30 PCR cycles.
➔ It is possible to generate ‘2n’ molecules after ‘n’ number of cycles.
Applications of PCR
➔ Detection of Pathogen
◆ It have been widely used in recent times to diagnose and detect various
pathogen like HIV
◆ DNA Fingerprinting
◆ Detection of specific mutations
◆ Prenatal diagnosis
◆ Palaeontology
◆ Diagnosis of plant pathogen
PCR
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