NCERT Textbook: Gene Cloning | Biotechnology for Class 12 - NEET PDF Download

 Page 1


Gene cloning is a customary procedure to use a gene for its 
product in the biotechnology industry and various other 
purposes. Traditionally, it engages the transfer of a DNA 
fragment containing the gene of interest to a host cell by a 
vector so that many copies of the gene will be available for 
its characterisation and future application. Technological 
breakthrough in the ??eld of genetic engineering have made 
it possible to analyse DNA, isolate a speci??c gene from a 
genome, enzymatically inserting it into an autonomously 
replicating vector (e.g. plasmid) to generate rDNA molecule 
and ultimately introducing into host (e.g., bacteria) to 
produce a virtually unlimited number of copies (clones) of 
it. This chapter will expose students to all the procedures 
involved in gene cloning.
3.1 Ident If Icat Ion of c and Idate Gene Over the past decades, rDNA technology has been utilised 
to produce crops that are resistant to pests, diseases, 
herbicides and pathogens. This is possible by manipulating 
the speci??c gene of interest of one organism followed by 
its transfer into the genome of another organism, which 
upon expression results in the desired product or activity. 
Gene Cloning
3.1 Identification of 
Candidate Gene
3.2 Isolation of Nucleic 
Acids
3.3 Enzymes used for 
Recombinant DNA 
Technology
3.4 Modes of DNA 
Transfer 
3.5 Screening and 
Selection
3.6  Blotting 
Techniques
3.7 Polymerase Chain 
Reaction (PCR)
3.8 DNA Libraries 
3
Chapter 
Chapter 3_Gene Cloning.indd   27 23-01-2025   11:21:08
Reprint 2025-26
Page 2


Gene cloning is a customary procedure to use a gene for its 
product in the biotechnology industry and various other 
purposes. Traditionally, it engages the transfer of a DNA 
fragment containing the gene of interest to a host cell by a 
vector so that many copies of the gene will be available for 
its characterisation and future application. Technological 
breakthrough in the ??eld of genetic engineering have made 
it possible to analyse DNA, isolate a speci??c gene from a 
genome, enzymatically inserting it into an autonomously 
replicating vector (e.g. plasmid) to generate rDNA molecule 
and ultimately introducing into host (e.g., bacteria) to 
produce a virtually unlimited number of copies (clones) of 
it. This chapter will expose students to all the procedures 
involved in gene cloning.
3.1 Ident If Icat Ion of c and Idate Gene Over the past decades, rDNA technology has been utilised 
to produce crops that are resistant to pests, diseases, 
herbicides and pathogens. This is possible by manipulating 
the speci??c gene of interest of one organism followed by 
its transfer into the genome of another organism, which 
upon expression results in the desired product or activity. 
Gene Cloning
3.1 Identification of 
Candidate Gene
3.2 Isolation of Nucleic 
Acids
3.3 Enzymes used for 
Recombinant DNA 
Technology
3.4 Modes of DNA 
Transfer 
3.5 Screening and 
Selection
3.6  Blotting 
Techniques
3.7 Polymerase Chain 
Reaction (PCR)
3.8 DNA Libraries 
3
Chapter 
Chapter 3_Gene Cloning.indd   27 23-01-2025   11:21:08
Reprint 2025-26
Biotechnology XII
28
The ??rst and most formidable problem in this process is to 
identify the candidate gene in the genome of an organism.
Identi??cation of a gene to be cloned depends upon 
its signi??cance with regard to its role in biomedical, 
economical and evolutionary ??elds. This information 
on a gene comes from its biochemical and physiological 
studies. For example, the cause of diseases (diabetes 
in human beings due to de??ciency of insulin) or defect 
in the metabolic pathways (iron de??ciency leading to 
chlorosis in plants) or resistance to environment (salinity 
tolerance in plants) or resistance to infection (both in 
plants and animals) or economically important genes 
(milk protein, blood clotting factors, etc.) are candidate 
genes for the improvement of human health and needs. 
Once a gene of interest is identi??ed, it is explored in 
new sources and the same is cloned as mentioned in the 
subsequent sections.  
Searching a gene of interest is not an easy task. This 
will be clear from the following example. As you know, a 
haploid human genome contains approximately 3.2 billion 
bp. Therefore, searching a gene of interest having a size of 
3000 to 3500 bp, which is one-millionth of the genome; 
is perhaps more dif??cult than looking for a needle in 
the haystack.
There are a few methods developed to achieve this task. 
One of these methods is to deduce the DNA sequence of the 
gene coding for a speci??c polypeptide chain based on its 
amino acid sequence. Another way to synthesize candidate 
gene is to isolate the mRNA of the desired gene from 
speci??c tissue, then synthesizing single stranded cDNA by 
using reverse transcriptase enzyme and converting that in 
to double stranded cDNA as candidate gene, which can be 
cloned (as discussed in the subsequent section).
3.2 Isolat Ion of n ucle Ic a c Ids The ??rst and foremost requirement for any molecular 
biology experiment is isolation of nucleic acids from 
organisms. Extraction of nucleic acids is encountered by 
two big challenges. First one is related to their availability 
in cells as DNA and RNA, both of which are present in very 
Chapter 3_Gene Cloning.indd   28 23-01-2025   11:21:08
Reprint 2025-26
Page 3


Gene cloning is a customary procedure to use a gene for its 
product in the biotechnology industry and various other 
purposes. Traditionally, it engages the transfer of a DNA 
fragment containing the gene of interest to a host cell by a 
vector so that many copies of the gene will be available for 
its characterisation and future application. Technological 
breakthrough in the ??eld of genetic engineering have made 
it possible to analyse DNA, isolate a speci??c gene from a 
genome, enzymatically inserting it into an autonomously 
replicating vector (e.g. plasmid) to generate rDNA molecule 
and ultimately introducing into host (e.g., bacteria) to 
produce a virtually unlimited number of copies (clones) of 
it. This chapter will expose students to all the procedures 
involved in gene cloning.
3.1 Ident If Icat Ion of c and Idate Gene Over the past decades, rDNA technology has been utilised 
to produce crops that are resistant to pests, diseases, 
herbicides and pathogens. This is possible by manipulating 
the speci??c gene of interest of one organism followed by 
its transfer into the genome of another organism, which 
upon expression results in the desired product or activity. 
Gene Cloning
3.1 Identification of 
Candidate Gene
3.2 Isolation of Nucleic 
Acids
3.3 Enzymes used for 
Recombinant DNA 
Technology
3.4 Modes of DNA 
Transfer 
3.5 Screening and 
Selection
3.6  Blotting 
Techniques
3.7 Polymerase Chain 
Reaction (PCR)
3.8 DNA Libraries 
3
Chapter 
Chapter 3_Gene Cloning.indd   27 23-01-2025   11:21:08
Reprint 2025-26
Biotechnology XII
28
The ??rst and most formidable problem in this process is to 
identify the candidate gene in the genome of an organism.
Identi??cation of a gene to be cloned depends upon 
its signi??cance with regard to its role in biomedical, 
economical and evolutionary ??elds. This information 
on a gene comes from its biochemical and physiological 
studies. For example, the cause of diseases (diabetes 
in human beings due to de??ciency of insulin) or defect 
in the metabolic pathways (iron de??ciency leading to 
chlorosis in plants) or resistance to environment (salinity 
tolerance in plants) or resistance to infection (both in 
plants and animals) or economically important genes 
(milk protein, blood clotting factors, etc.) are candidate 
genes for the improvement of human health and needs. 
Once a gene of interest is identi??ed, it is explored in 
new sources and the same is cloned as mentioned in the 
subsequent sections.  
Searching a gene of interest is not an easy task. This 
will be clear from the following example. As you know, a 
haploid human genome contains approximately 3.2 billion 
bp. Therefore, searching a gene of interest having a size of 
3000 to 3500 bp, which is one-millionth of the genome; 
is perhaps more dif??cult than looking for a needle in 
the haystack.
There are a few methods developed to achieve this task. 
One of these methods is to deduce the DNA sequence of the 
gene coding for a speci??c polypeptide chain based on its 
amino acid sequence. Another way to synthesize candidate 
gene is to isolate the mRNA of the desired gene from 
speci??c tissue, then synthesizing single stranded cDNA by 
using reverse transcriptase enzyme and converting that in 
to double stranded cDNA as candidate gene, which can be 
cloned (as discussed in the subsequent section).
3.2 Isolat Ion of n ucle Ic a c Ids The ??rst and foremost requirement for any molecular 
biology experiment is isolation of nucleic acids from 
organisms. Extraction of nucleic acids is encountered by 
two big challenges. First one is related to their availability 
in cells as DNA and RNA, both of which are present in very 
Chapter 3_Gene Cloning.indd   28 23-01-2025   11:21:08
Reprint 2025-26
Gene Cloning 29
small amounts in cells in comparison to other biological 
macromolecules, such as proteins, carbohydrates and 
lipids. Second, the enormous length of nucleic acids, 
particularly makes it susceptible to cleavage when exposed 
to harsh physical stress. In addition, the chemical bonds 
by which different components of nucleic acids are joined 
with each other and various groups present in them make 
nucleic acid vulnerable to chemical agents.
Four important steps are involved during the extraction 
of nucleic acids. The ??rst step involves the effective rupture 
of cell membrane or walls to release the nucleic acids and 
other cellular molecules. The second step involves the 
protection of nucleic acids from their respective degrading 
Fig. 3.1: Steps involved in the isolation of DNA
Ground up cells 
transferred
CTAB, SDS 
buffer, EDTA
Interface  
(Lysed proteins)
Aqueous phase 
(Nucleic acids)
Nucleic acid 
transferred
Nucleic acid 
pellet
RNase 
added
Organic Phase  
(Broken down proteins, lipids, 
and other cell debris)
Chloroform Chloroform
Phenol
Phenol Isoamyl 
alcohol
Cell lysate
Isoamyl 
alcohol
Aqueous phase is 
pipatted to another tube
Aqueous 
phase 
(DNA)
DNA
Chilled ethanol 
Centrifuged
Centrifuged
Chilled ethanol 
Centrifuged
The cells and tissues are broken 
down in a lyses buffer
Chapter 3_Gene Cloning.indd   29 23-01-2025   11:21:08
Reprint 2025-26
Page 4


Gene cloning is a customary procedure to use a gene for its 
product in the biotechnology industry and various other 
purposes. Traditionally, it engages the transfer of a DNA 
fragment containing the gene of interest to a host cell by a 
vector so that many copies of the gene will be available for 
its characterisation and future application. Technological 
breakthrough in the ??eld of genetic engineering have made 
it possible to analyse DNA, isolate a speci??c gene from a 
genome, enzymatically inserting it into an autonomously 
replicating vector (e.g. plasmid) to generate rDNA molecule 
and ultimately introducing into host (e.g., bacteria) to 
produce a virtually unlimited number of copies (clones) of 
it. This chapter will expose students to all the procedures 
involved in gene cloning.
3.1 Ident If Icat Ion of c and Idate Gene Over the past decades, rDNA technology has been utilised 
to produce crops that are resistant to pests, diseases, 
herbicides and pathogens. This is possible by manipulating 
the speci??c gene of interest of one organism followed by 
its transfer into the genome of another organism, which 
upon expression results in the desired product or activity. 
Gene Cloning
3.1 Identification of 
Candidate Gene
3.2 Isolation of Nucleic 
Acids
3.3 Enzymes used for 
Recombinant DNA 
Technology
3.4 Modes of DNA 
Transfer 
3.5 Screening and 
Selection
3.6  Blotting 
Techniques
3.7 Polymerase Chain 
Reaction (PCR)
3.8 DNA Libraries 
3
Chapter 
Chapter 3_Gene Cloning.indd   27 23-01-2025   11:21:08
Reprint 2025-26
Biotechnology XII
28
The ??rst and most formidable problem in this process is to 
identify the candidate gene in the genome of an organism.
Identi??cation of a gene to be cloned depends upon 
its signi??cance with regard to its role in biomedical, 
economical and evolutionary ??elds. This information 
on a gene comes from its biochemical and physiological 
studies. For example, the cause of diseases (diabetes 
in human beings due to de??ciency of insulin) or defect 
in the metabolic pathways (iron de??ciency leading to 
chlorosis in plants) or resistance to environment (salinity 
tolerance in plants) or resistance to infection (both in 
plants and animals) or economically important genes 
(milk protein, blood clotting factors, etc.) are candidate 
genes for the improvement of human health and needs. 
Once a gene of interest is identi??ed, it is explored in 
new sources and the same is cloned as mentioned in the 
subsequent sections.  
Searching a gene of interest is not an easy task. This 
will be clear from the following example. As you know, a 
haploid human genome contains approximately 3.2 billion 
bp. Therefore, searching a gene of interest having a size of 
3000 to 3500 bp, which is one-millionth of the genome; 
is perhaps more dif??cult than looking for a needle in 
the haystack.
There are a few methods developed to achieve this task. 
One of these methods is to deduce the DNA sequence of the 
gene coding for a speci??c polypeptide chain based on its 
amino acid sequence. Another way to synthesize candidate 
gene is to isolate the mRNA of the desired gene from 
speci??c tissue, then synthesizing single stranded cDNA by 
using reverse transcriptase enzyme and converting that in 
to double stranded cDNA as candidate gene, which can be 
cloned (as discussed in the subsequent section).
3.2 Isolat Ion of n ucle Ic a c Ids The ??rst and foremost requirement for any molecular 
biology experiment is isolation of nucleic acids from 
organisms. Extraction of nucleic acids is encountered by 
two big challenges. First one is related to their availability 
in cells as DNA and RNA, both of which are present in very 
Chapter 3_Gene Cloning.indd   28 23-01-2025   11:21:08
Reprint 2025-26
Gene Cloning 29
small amounts in cells in comparison to other biological 
macromolecules, such as proteins, carbohydrates and 
lipids. Second, the enormous length of nucleic acids, 
particularly makes it susceptible to cleavage when exposed 
to harsh physical stress. In addition, the chemical bonds 
by which different components of nucleic acids are joined 
with each other and various groups present in them make 
nucleic acid vulnerable to chemical agents.
Four important steps are involved during the extraction 
of nucleic acids. The ??rst step involves the effective rupture 
of cell membrane or walls to release the nucleic acids and 
other cellular molecules. The second step involves the 
protection of nucleic acids from their respective degrading 
Fig. 3.1: Steps involved in the isolation of DNA
Ground up cells 
transferred
CTAB, SDS 
buffer, EDTA
Interface  
(Lysed proteins)
Aqueous phase 
(Nucleic acids)
Nucleic acid 
transferred
Nucleic acid 
pellet
RNase 
added
Organic Phase  
(Broken down proteins, lipids, 
and other cell debris)
Chloroform Chloroform
Phenol
Phenol Isoamyl 
alcohol
Cell lysate
Isoamyl 
alcohol
Aqueous phase is 
pipatted to another tube
Aqueous 
phase 
(DNA)
DNA
Chilled ethanol 
Centrifuged
Centrifuged
Chilled ethanol 
Centrifuged
The cells and tissues are broken 
down in a lyses buffer
Chapter 3_Gene Cloning.indd   29 23-01-2025   11:21:08
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Biotechnology XII
30
enzymes, which are released in the isolation medium 
with other proteins. In the third step, the nucleic acids 
are separated from other molecules. In the fourth and the 
last step, the isolated nucleic acids are precipitated and 
concentrated by adding ethanol or isopropanol.
Although, chemical and physical properties of nucleic 
acids are similar in all organisms, the outer boundary of 
cells differs from one organism to another. Therefore, in 
order to disrupt the cell boundaries for releasing nucleic 
acids into extraction medium, different strategies are 
adopted. Animal cells have plasma membrane that can 
be easily disrupted. On the contrary, plant cells and 
bacteria are protected by tough layers (e.g., cell wall), 
which need different approaches for their lysis. These 
include homogenisation, grinding, sonication or enzymatic 
treatment. Such mechanical or enzymatic treatment 
ruptures plasma membrane or cell wall so that nucleic 
acids get released from cells and exposes them to nuclease 
enzymes (deoxyribonuclease and ribonuclease), which are 
also released simultaneously.
Isolat Ion of dna As bacterial cells have little structure beyond the cell wall 
and cell membrane, isolating DNA from them is much 
easier. An enzyme called lysozyme digest the peptidoglycan, 
the main component of bacterial cell wall. Detergents 
like sodium dodecyl sulphate (SDS) is used to lyse the 
cell membranes by disrupting the lipid bilayer. Plant 
and animal cells are ground to release the intracellular 
components. Plant cells are mechanically ruptured in a 
blender to break open the tough cell walls. For isolation 
of DNA from plant cells, cetyl trimethyl ammonium 
bromide (CTAB) is used as detergent (a cationic detergent).  
Plant cells have high concentration of polysaccharide 
and polyphenols in comparison to animal cells and pose 
problems during isolation of DNA. The solubility of DNA 
and polysaccharides to CTAB depends on ionic strength of 
the solution. At low ionic strength, DNA is soluble in CTAB 
solution while polysaccharides are insoluble; whereas 
at high ionic strength, polysaccharides are soluble and 
DNA is insoluble. In addition, being a detergent, it also 
Chapter 3_Gene Cloning.indd   30 23-01-2025   11:21:08
Reprint 2025-26
Page 5


Gene cloning is a customary procedure to use a gene for its 
product in the biotechnology industry and various other 
purposes. Traditionally, it engages the transfer of a DNA 
fragment containing the gene of interest to a host cell by a 
vector so that many copies of the gene will be available for 
its characterisation and future application. Technological 
breakthrough in the ??eld of genetic engineering have made 
it possible to analyse DNA, isolate a speci??c gene from a 
genome, enzymatically inserting it into an autonomously 
replicating vector (e.g. plasmid) to generate rDNA molecule 
and ultimately introducing into host (e.g., bacteria) to 
produce a virtually unlimited number of copies (clones) of 
it. This chapter will expose students to all the procedures 
involved in gene cloning.
3.1 Ident If Icat Ion of c and Idate Gene Over the past decades, rDNA technology has been utilised 
to produce crops that are resistant to pests, diseases, 
herbicides and pathogens. This is possible by manipulating 
the speci??c gene of interest of one organism followed by 
its transfer into the genome of another organism, which 
upon expression results in the desired product or activity. 
Gene Cloning
3.1 Identification of 
Candidate Gene
3.2 Isolation of Nucleic 
Acids
3.3 Enzymes used for 
Recombinant DNA 
Technology
3.4 Modes of DNA 
Transfer 
3.5 Screening and 
Selection
3.6  Blotting 
Techniques
3.7 Polymerase Chain 
Reaction (PCR)
3.8 DNA Libraries 
3
Chapter 
Chapter 3_Gene Cloning.indd   27 23-01-2025   11:21:08
Reprint 2025-26
Biotechnology XII
28
The ??rst and most formidable problem in this process is to 
identify the candidate gene in the genome of an organism.
Identi??cation of a gene to be cloned depends upon 
its signi??cance with regard to its role in biomedical, 
economical and evolutionary ??elds. This information 
on a gene comes from its biochemical and physiological 
studies. For example, the cause of diseases (diabetes 
in human beings due to de??ciency of insulin) or defect 
in the metabolic pathways (iron de??ciency leading to 
chlorosis in plants) or resistance to environment (salinity 
tolerance in plants) or resistance to infection (both in 
plants and animals) or economically important genes 
(milk protein, blood clotting factors, etc.) are candidate 
genes for the improvement of human health and needs. 
Once a gene of interest is identi??ed, it is explored in 
new sources and the same is cloned as mentioned in the 
subsequent sections.  
Searching a gene of interest is not an easy task. This 
will be clear from the following example. As you know, a 
haploid human genome contains approximately 3.2 billion 
bp. Therefore, searching a gene of interest having a size of 
3000 to 3500 bp, which is one-millionth of the genome; 
is perhaps more dif??cult than looking for a needle in 
the haystack.
There are a few methods developed to achieve this task. 
One of these methods is to deduce the DNA sequence of the 
gene coding for a speci??c polypeptide chain based on its 
amino acid sequence. Another way to synthesize candidate 
gene is to isolate the mRNA of the desired gene from 
speci??c tissue, then synthesizing single stranded cDNA by 
using reverse transcriptase enzyme and converting that in 
to double stranded cDNA as candidate gene, which can be 
cloned (as discussed in the subsequent section).
3.2 Isolat Ion of n ucle Ic a c Ids The ??rst and foremost requirement for any molecular 
biology experiment is isolation of nucleic acids from 
organisms. Extraction of nucleic acids is encountered by 
two big challenges. First one is related to their availability 
in cells as DNA and RNA, both of which are present in very 
Chapter 3_Gene Cloning.indd   28 23-01-2025   11:21:08
Reprint 2025-26
Gene Cloning 29
small amounts in cells in comparison to other biological 
macromolecules, such as proteins, carbohydrates and 
lipids. Second, the enormous length of nucleic acids, 
particularly makes it susceptible to cleavage when exposed 
to harsh physical stress. In addition, the chemical bonds 
by which different components of nucleic acids are joined 
with each other and various groups present in them make 
nucleic acid vulnerable to chemical agents.
Four important steps are involved during the extraction 
of nucleic acids. The ??rst step involves the effective rupture 
of cell membrane or walls to release the nucleic acids and 
other cellular molecules. The second step involves the 
protection of nucleic acids from their respective degrading 
Fig. 3.1: Steps involved in the isolation of DNA
Ground up cells 
transferred
CTAB, SDS 
buffer, EDTA
Interface  
(Lysed proteins)
Aqueous phase 
(Nucleic acids)
Nucleic acid 
transferred
Nucleic acid 
pellet
RNase 
added
Organic Phase  
(Broken down proteins, lipids, 
and other cell debris)
Chloroform Chloroform
Phenol
Phenol Isoamyl 
alcohol
Cell lysate
Isoamyl 
alcohol
Aqueous phase is 
pipatted to another tube
Aqueous 
phase 
(DNA)
DNA
Chilled ethanol 
Centrifuged
Centrifuged
Chilled ethanol 
Centrifuged
The cells and tissues are broken 
down in a lyses buffer
Chapter 3_Gene Cloning.indd   29 23-01-2025   11:21:08
Reprint 2025-26
Biotechnology XII
30
enzymes, which are released in the isolation medium 
with other proteins. In the third step, the nucleic acids 
are separated from other molecules. In the fourth and the 
last step, the isolated nucleic acids are precipitated and 
concentrated by adding ethanol or isopropanol.
Although, chemical and physical properties of nucleic 
acids are similar in all organisms, the outer boundary of 
cells differs from one organism to another. Therefore, in 
order to disrupt the cell boundaries for releasing nucleic 
acids into extraction medium, different strategies are 
adopted. Animal cells have plasma membrane that can 
be easily disrupted. On the contrary, plant cells and 
bacteria are protected by tough layers (e.g., cell wall), 
which need different approaches for their lysis. These 
include homogenisation, grinding, sonication or enzymatic 
treatment. Such mechanical or enzymatic treatment 
ruptures plasma membrane or cell wall so that nucleic 
acids get released from cells and exposes them to nuclease 
enzymes (deoxyribonuclease and ribonuclease), which are 
also released simultaneously.
Isolat Ion of dna As bacterial cells have little structure beyond the cell wall 
and cell membrane, isolating DNA from them is much 
easier. An enzyme called lysozyme digest the peptidoglycan, 
the main component of bacterial cell wall. Detergents 
like sodium dodecyl sulphate (SDS) is used to lyse the 
cell membranes by disrupting the lipid bilayer. Plant 
and animal cells are ground to release the intracellular 
components. Plant cells are mechanically ruptured in a 
blender to break open the tough cell walls. For isolation 
of DNA from plant cells, cetyl trimethyl ammonium 
bromide (CTAB) is used as detergent (a cationic detergent).  
Plant cells have high concentration of polysaccharide 
and polyphenols in comparison to animal cells and pose 
problems during isolation of DNA. The solubility of DNA 
and polysaccharides to CTAB depends on ionic strength of 
the solution. At low ionic strength, DNA is soluble in CTAB 
solution while polysaccharides are insoluble; whereas 
at high ionic strength, polysaccharides are soluble and 
DNA is insoluble. In addition, being a detergent, it also 
Chapter 3_Gene Cloning.indd   30 23-01-2025   11:21:08
Reprint 2025-26
Gene Cloning 31
lyses cell wall. Both the molecules are separated based 
on their differential af??nity to CTAB. Addition of polyvinyl 
pyrrolidone (PVP) to CTAB extraction medium neutralises 
phenols. Soluble DNA present in supernatant is extracted 
with chloroform-isoamyl solution. DNA present in aqueous 
phase is precipitated using ethanol or isopropanol. In case 
of animal cells, the cell membrane is disrupted by detergent 
to release the  intracellular components.
The cells and tissues from which nucleic acids are to be 
extracted are broken down in a medium either mechanically 
or enzymatically. The media is usually a buffer having 
mild alkaline pH with minimum ionic strength (0.05 M) 
containing chelating agent ethylene diamine tetraacetic 
acid (EDTA). The mild alkaline pH facilitates the reduction 
of electrostatic interaction between DNA and basic proteins 
(histones) released during cell disruption. Chelating of 
divalent cations particularly Mn
2+
 and Mg
2+
 prevents  the 
action of nucleases. Further, inhibition of their activities 
is achieved due to alkaline pH of the buffer. In addition, 
chelating of divalent cations prevents the formation of their 
respective salts with phosphate groups of nucleic acids.
The next step is to separate nucleic acids from its 
bound proteins. This is achieved by decreasing interaction 
between proteins and nucleic acids so that nucleic acids 
are free of proteins, by exposing to detergents, like SDS, an 
anionic detergent. Exposure to SDS makes all the protein 
molecules anionic. Consequently, basic proteins that 
are positively charged and bound to negatively charged 
nucleic acids become negatively charged and dissociate 
from the nucleic acids. In addition, SDS also prevents  the 
activities of nucleases thereby giving additional protection 
to nucleic acids from nucleases. Then sodium chloride is 
added to the medium at high concentration. Increased salt 
concentration diminishes the ionic interaction between 
DNA and cations thus ensuring complete dissociation 
of DNA and protein complexes. Deproteinisation of the 
medium is achieved by exposing it to chloroform and 
isoamyl alcohol. These solvents are non-polar in nature 
when it is added to the medium that is polar in nature and 
subjected to centrifugation, it gives three distinct layers. 
Since, the density of organic solvent mixture is higher than 
water, it forms a lower layer (which contains denatured 
Chapter 3_Gene Cloning.indd   31 23-01-2025   11:21:08
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