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CLONING
METHODOLOGIES
By
M.NAGAPRADHEESH(2K21/MSBT/18)
I M.Sc., BIO TECHNOLOGY
r DNA Technology
STEPS INVOLVED IN MOLECULAR CLONING
* A traditional molecular cloning experiment (or) research can be divided into
Nine steps:
1.Selection of the host organisms
2.Selection of Cloning vector
3.Preparation of the Vector
4.Preparation for the Insert
5.Generation of the Recombinant DNA (rDNA)
6.Introduction of the rDNA into the Host cell or Organism
7.Selection of the clones of organism containing the vectors
8.Screening of clones with the desired rDNA molecules
9.Expression and Isolation of the recombinant DNA.
NOTE:
* Before performing a cloning experiment (or) research it is always
recommended to perform an in silico simulation of the procedure using dedicated
software or websites(Eg.,NCBI,EMBL etc..) for DNA or gene sequence manipulation.
*This software or websites is also useful to align DNA Sequences and create
publication quality genomic or plasmid maps.
PRINCIPLES AND STEPS INVOLVED IN GENOMIC OR
CDNA CLONING:
☆ Complementary DNA (cDNA) cloning is termed for the gene cloning(cloning of DNA
fragments) obtained from cDNA.
☆ The principle of cDNA cloning is that it involves the copying of mRNA transcripts into
DNA, which are then inserted into bacterial plasmids and then placed into bacteria
by Transformation
☆ At this stage, it should be clear that mRNA used for cDNA preparation is a processed
transcript and not the original one transcribed from DNA.
☆ In-order to clone a DNA sequence that codes for a required gene product, the gene
should be removed from the organism and cloned it in the vector molecule.
☆ A gene library is a random collection of cloned fragments in an appropriate vector that
particularly consists of all the genetic information about that species.
☆ There are two methods for the formation of gene libraries. They are:
● Complementary DNA (cDNA)
● Genomic DNA libraries
STEPS OF CDNA CLONING
1.Isolation of mRNA
2.Synthesis of first strand of cDNA
3.Synthesis of second strand of cDNA
4.Cloning of cDNA
5.Introduction to host cells
6.Clone selection
1.ISOLATION OF MRNA:
• A crude extract of the tissue with the gene of interest is prepared.
• The extract must be free from proteins, polysaccharides and all other contaminants.
• The technique of oligo-deoxythymine (oligo-dT) cellulose chromatography is used for the
further purification of many eukaryotic mRNAs from the total or polysomal fraction.
• mRNAs consist of poly A (adenosine residues) tail at their 3′ end.
• Under favourable conditions, this tail will bind to a string of thymidine residues
immobilized on cellulose and then poly (A)+ fraction can be eluted.
• Two or three passages of the poly (A)+ fraction through such a column produces a
fraction highly enriched for mRNA.
• This fraction includes different mRNA sequences, however certain techniques can be
employed for extracting a particular mRNA species.
• After the preparation of the fraction, it is essential to confirm if the extracted mRNA
consists of the sequence of interest.
• It is performed by translation of mRNA in vitro and identification of suitable polypeptides
in the products obtained.
2.SYNTHESIS OF FIRST STRAND OF CDNA:
• Reverse transcriptase is a RNA dependent DNA polymerase which is used to
copy the mRNA fraction into the first strand of DNA.
• This enzyme, like all other DNA polymerases, can only add residues at the
3′-OH group of an existing primer, which is base paired with the template.
• The most commonly used primer is oligo-dT for cloning of cDNAs.
• Oligo-dT primer is 12-18 nucleotides in length, that binds to the poly (A)
tract at the 3′ end of mRNA molecules.
• The RNA strand of the resulting RNA-DNA hybrid is destroyed prior to
second strand synthesis through alkaline hydrolysis.
3.SYNTHESIS OF SECONDARY STAND OF CDNA:
The second strand of cDNA can be synthesized by two techniques. They are:
i. Self-priming cDNA:
• In Self-priming, the mRNA hybrid obtained is denaturated for the synthesis of second strand on the single strand of cDNA by the klenow
fragment of DNA polymerase I.
• The transitory hairpin structure at the 3′ end of single-stranded DNA can be used to prime the synthesis of second strand of cDNA by the
klenow fragment of Escherichia coli DNA polymerase I.
• Single-strand specific S1 nuclease digests the hairpin loop and any single-stranded overhung at the other end.
• The ultimate product is a population of double-stranded, blunt-ended DNA molecules complementray to the original mRNA fraction.
ii. Replacement synthesis:
• In this method, the cDNA:mRNA hybrid works as a template for a nick translation reaction.
• In the mRNA strand of the hybrid, RNase H produces nicks and gaps, creating a series of RNA primers.
• These RNA primers are used by E. coli DNA polymerase I during the synthesis of second strand of cDNA.
• The advantages of this technique are:
– very efficient
– can be performed directly using the products of the first strand reaction
– eliminates the need to use nuclease S1 to cleave the single-stranded hairpin loop in the double stranded cDNA.
4.CLONING OF CDNA:
• The most frequently used technique for cloning cDNAs involves the
addition of complementary homopolymeric tracts to double stranded
cDNA and to the plasmid vector.
• To the cDNA, strings of cytosine residues are added using the enzyme
terminal transferase to form oligo-dC tails on the 3′ ends.
• Likewise, a plasmid is cut open at a unique restriction endonuclease site
and tailed with oligo-dG.
• Now, the vector and the double stranded cDNA are joined by hydrogen
bonding between the complementary homopolymers.
• It results in the formation of open circular hybrid molecules capable of
transforming E. coli.
5.INTRODUCTION INTO HOST CELL:
• For the transforming of bacteria, the recombinant plasmids are used, usually the E. coli K-12 strain.
• The uptake of plasmid molecules from the surrounding medium is performed by E. coli cells treated with
calcium chloride.
• Any gaps in the recombinant plasmid will be repaired by the host cells.
• The transformed bacteria can be isolated from non-transformed ones on the basis of antibiotic
resistance.
• Majority of cloning plasmids contain two antibiotic resistance genes, one of which is destroyed during
cloning.
• For instance, in the case of pBR322, cloning into unique PstI site destroys ampicillin resistance but
leaves tetracycline resistance intact.
• Bacteria transformed with a recombinant plasmid will be sensitive to ampicillin but resistant to
tetracycline.
6.CLONE SELECTION:
• The antibiotic resistance selection already performed has recognized which clones carry a recombinant
plasmid, however there will be thousands of various inserts.
• The cloning process generally commences with a whole population of mRNA sequences.
• Selection of clones carrying the sequence of interest is the tough job.
• If the gene is expressed, then the simplest selection is to screen for the presence of the protein.
• It can be screened either by bacterial phenotype it produces or by the protein detection methods usually
based on immunological or enzymological techniques.
• If the protein is not expressed, then other methods such as nucleic acid hybridization are used.
• Identification of the gene is discussed after the genomic DNA cloning.
OTHER TECHNIQUES INVOLVED IN CLONING OR
FOREIGN DNA TRANSFER :
☆TRANSDUCTION (in Bacterial cells)
☆ELECTROPORATION (in both Plant and Bacterial cells)
☆TRANSFECTION (in both Plant and Animal cells)
☆ULTRASONICATION (in Plant cells)
☆BIOLISTICS OR PARTICLE BOMBARDMENT (in Plant cells)
☆RETROVIRAL METHOD (in Animal cells)
☆MICROINJECTION (in Animal cells)
☆EMBROYONIC STEM CELL METHOD (in Animal cells)
EXPRESSION CLONING AND PROTEIN-PROTEIN
INTEREACTIVE CLONING TECHNIQUES:
• Expression cloning is a technique in DNA cloning that uses expression vectors to generate a
library of clones, with each clone expressing one protein. This expression library is then screened
for the property of interest and clones of interest are recovered for further analysis. An example,
would be using an expression library to isolate genes that could confer antibiotic resistance.
• The following are techniques involves in protein-protein interaction:
1.Yeast Two Hybrid System
2.Phage Display System.
• These above two techniques is also helpul in maximising gene expressions.
CDNA AND GENOMIC DNA LIBRARY CONSTRUCTION:
• A copy of DNA generated from messenger RNA (mRNA) with the help of enzyme reverse transcriptase is
termed as cDNA.
• A set of cDNA fragments, each of which has been cloned into a separate vector molecule, which constitute
a some portion of transcriptome of the organism and stored as a library is known as a cDNA library.
STEPS INVOLVED IN CDNA LIBRARY CONSTRUCTION:
• To construct cDNA libraries, DNA copies from mRNA sequences of organism are produced and
then they are cloned.
• The term cDNA is given as all the DNA in the library are complementary to the mRNAs and are
produced by reverse transcription of mRNAs.
• Most eukaryotic DNA consists of repeated sequences that are not transcribed into mRNA, and in
a cDNA library the sequences are not represented.
• It should be remembered that prokaryotes and lower eukaryotes do not contain introns, and
cDNA preparation for these species is usually needless.
• Therefore, cDNA libraries are only created from higher eukaryotes.
• For the construction of cDNA library, both the bacterial and bacteriophage DNA can be used as
vectors.
SIMILARITIES BETWEEN CDNA AND GENOMIC DNA
LJBRARY:
• cDNA Library (Complementary DNA library)
• cDNA – It is a DNA copy of an mRNA molecule generated by reverse transcriptase, a DNA polymerase that can use
either RNA or DNA as their template
• These are prepared using mRNA as templates, their starting material
• They are representative of only those genes of the genome that are expressed given specific conditions
• cDNA does not have introns, and hence can be expressed in prokaryotic cells
Genomic DNA library
• Genomic DNA is the chromosomal DNA of an entity representative of the collection of its genomic content. They are
different from that of complementary DNA, mitochondrial DNA or bacterial plasmid DNA. These are directly
prepared from the genomic DNA, and represent the complete genome of an entity
• For their construction, ligases and restriction endonucleases are vital
• It can also represent the DNA of both eukaryotic and prokaryotic entities and carries introns
• As it carries introns, they are incapable of expression in the prokaryotes. Moreover, prokaryotes lack the machinery
for processing introns
ADVANTAGES & DISADVANTAGES OF CDNA &
GENOMIC LIBRARY:
Advantages of cDNA library:
• There are two major benefits of a cDNA library.
• First, it is enriched with fragments from genes that have been actively transcribed.
• Second, introns do not disrupt the cloned sequences; if the goal is to create a eukaryotic protein in bacteria,
introns will pose a problem, since most bacteria have no means of eliminating the introns.
Disadvantages of cDNA library:
• A cDNA library has the drawback that it only includes sequences that are present in mature mRNA.
• There are no introns and any other sequences that are modified during transcription; sequences that are
not transcribed into RNA, such as promoters and enhancers, are also not present in a library of cDNA.
• It is also important to remember that only certain gene sequences expressed in the tissue from which the
RNA has been isolated constitute the cDNA library.
• In addition, in a cDNA library, the frequency of a specific DNA sequence depends on the abundance of the
corresponding mRNA in the given tissue.
• In contrast, in a genomic DNA library, almost all genes are present at the same frequency
REFRENCES:
WEBSITES AND HTML:
#www.microbenotes.com/(cDNA CLONING)
#www.onlinebiology.com/(cDNA CLONING)
#www.byjus.in/(CLONING)
BOOK:
#Name:TEXTBOOK OF BIOTECHNOLOGY(4th EDITION)
#Authour Name:H.K.DAS
#Publication Name:WILEY INDIA
CLONING METHODOLOGIES(M.Nagapradheesh).pptx

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CLONING METHODOLOGIES(M.Nagapradheesh).pptx

  • 2. STEPS INVOLVED IN MOLECULAR CLONING * A traditional molecular cloning experiment (or) research can be divided into Nine steps: 1.Selection of the host organisms 2.Selection of Cloning vector 3.Preparation of the Vector 4.Preparation for the Insert 5.Generation of the Recombinant DNA (rDNA) 6.Introduction of the rDNA into the Host cell or Organism 7.Selection of the clones of organism containing the vectors 8.Screening of clones with the desired rDNA molecules 9.Expression and Isolation of the recombinant DNA. NOTE: * Before performing a cloning experiment (or) research it is always recommended to perform an in silico simulation of the procedure using dedicated software or websites(Eg.,NCBI,EMBL etc..) for DNA or gene sequence manipulation. *This software or websites is also useful to align DNA Sequences and create publication quality genomic or plasmid maps.
  • 3. PRINCIPLES AND STEPS INVOLVED IN GENOMIC OR CDNA CLONING: ☆ Complementary DNA (cDNA) cloning is termed for the gene cloning(cloning of DNA fragments) obtained from cDNA. ☆ The principle of cDNA cloning is that it involves the copying of mRNA transcripts into DNA, which are then inserted into bacterial plasmids and then placed into bacteria by Transformation ☆ At this stage, it should be clear that mRNA used for cDNA preparation is a processed transcript and not the original one transcribed from DNA. ☆ In-order to clone a DNA sequence that codes for a required gene product, the gene should be removed from the organism and cloned it in the vector molecule. ☆ A gene library is a random collection of cloned fragments in an appropriate vector that particularly consists of all the genetic information about that species. ☆ There are two methods for the formation of gene libraries. They are: ● Complementary DNA (cDNA) ● Genomic DNA libraries
  • 4. STEPS OF CDNA CLONING 1.Isolation of mRNA 2.Synthesis of first strand of cDNA 3.Synthesis of second strand of cDNA 4.Cloning of cDNA 5.Introduction to host cells 6.Clone selection
  • 5. 1.ISOLATION OF MRNA: • A crude extract of the tissue with the gene of interest is prepared. • The extract must be free from proteins, polysaccharides and all other contaminants. • The technique of oligo-deoxythymine (oligo-dT) cellulose chromatography is used for the further purification of many eukaryotic mRNAs from the total or polysomal fraction. • mRNAs consist of poly A (adenosine residues) tail at their 3′ end. • Under favourable conditions, this tail will bind to a string of thymidine residues immobilized on cellulose and then poly (A)+ fraction can be eluted. • Two or three passages of the poly (A)+ fraction through such a column produces a fraction highly enriched for mRNA. • This fraction includes different mRNA sequences, however certain techniques can be employed for extracting a particular mRNA species. • After the preparation of the fraction, it is essential to confirm if the extracted mRNA consists of the sequence of interest. • It is performed by translation of mRNA in vitro and identification of suitable polypeptides in the products obtained.
  • 6. 2.SYNTHESIS OF FIRST STRAND OF CDNA: • Reverse transcriptase is a RNA dependent DNA polymerase which is used to copy the mRNA fraction into the first strand of DNA. • This enzyme, like all other DNA polymerases, can only add residues at the 3′-OH group of an existing primer, which is base paired with the template. • The most commonly used primer is oligo-dT for cloning of cDNAs. • Oligo-dT primer is 12-18 nucleotides in length, that binds to the poly (A) tract at the 3′ end of mRNA molecules. • The RNA strand of the resulting RNA-DNA hybrid is destroyed prior to second strand synthesis through alkaline hydrolysis.
  • 7. 3.SYNTHESIS OF SECONDARY STAND OF CDNA: The second strand of cDNA can be synthesized by two techniques. They are: i. Self-priming cDNA: • In Self-priming, the mRNA hybrid obtained is denaturated for the synthesis of second strand on the single strand of cDNA by the klenow fragment of DNA polymerase I. • The transitory hairpin structure at the 3′ end of single-stranded DNA can be used to prime the synthesis of second strand of cDNA by the klenow fragment of Escherichia coli DNA polymerase I. • Single-strand specific S1 nuclease digests the hairpin loop and any single-stranded overhung at the other end. • The ultimate product is a population of double-stranded, blunt-ended DNA molecules complementray to the original mRNA fraction. ii. Replacement synthesis: • In this method, the cDNA:mRNA hybrid works as a template for a nick translation reaction. • In the mRNA strand of the hybrid, RNase H produces nicks and gaps, creating a series of RNA primers. • These RNA primers are used by E. coli DNA polymerase I during the synthesis of second strand of cDNA. • The advantages of this technique are: – very efficient – can be performed directly using the products of the first strand reaction – eliminates the need to use nuclease S1 to cleave the single-stranded hairpin loop in the double stranded cDNA.
  • 8. 4.CLONING OF CDNA: • The most frequently used technique for cloning cDNAs involves the addition of complementary homopolymeric tracts to double stranded cDNA and to the plasmid vector. • To the cDNA, strings of cytosine residues are added using the enzyme terminal transferase to form oligo-dC tails on the 3′ ends. • Likewise, a plasmid is cut open at a unique restriction endonuclease site and tailed with oligo-dG. • Now, the vector and the double stranded cDNA are joined by hydrogen bonding between the complementary homopolymers. • It results in the formation of open circular hybrid molecules capable of transforming E. coli.
  • 9. 5.INTRODUCTION INTO HOST CELL: • For the transforming of bacteria, the recombinant plasmids are used, usually the E. coli K-12 strain. • The uptake of plasmid molecules from the surrounding medium is performed by E. coli cells treated with calcium chloride. • Any gaps in the recombinant plasmid will be repaired by the host cells. • The transformed bacteria can be isolated from non-transformed ones on the basis of antibiotic resistance. • Majority of cloning plasmids contain two antibiotic resistance genes, one of which is destroyed during cloning. • For instance, in the case of pBR322, cloning into unique PstI site destroys ampicillin resistance but leaves tetracycline resistance intact. • Bacteria transformed with a recombinant plasmid will be sensitive to ampicillin but resistant to tetracycline.
  • 10. 6.CLONE SELECTION: • The antibiotic resistance selection already performed has recognized which clones carry a recombinant plasmid, however there will be thousands of various inserts. • The cloning process generally commences with a whole population of mRNA sequences. • Selection of clones carrying the sequence of interest is the tough job. • If the gene is expressed, then the simplest selection is to screen for the presence of the protein. • It can be screened either by bacterial phenotype it produces or by the protein detection methods usually based on immunological or enzymological techniques. • If the protein is not expressed, then other methods such as nucleic acid hybridization are used. • Identification of the gene is discussed after the genomic DNA cloning.
  • 11. OTHER TECHNIQUES INVOLVED IN CLONING OR FOREIGN DNA TRANSFER : ☆TRANSDUCTION (in Bacterial cells) ☆ELECTROPORATION (in both Plant and Bacterial cells) ☆TRANSFECTION (in both Plant and Animal cells) ☆ULTRASONICATION (in Plant cells) ☆BIOLISTICS OR PARTICLE BOMBARDMENT (in Plant cells) ☆RETROVIRAL METHOD (in Animal cells) ☆MICROINJECTION (in Animal cells) ☆EMBROYONIC STEM CELL METHOD (in Animal cells)
  • 12. EXPRESSION CLONING AND PROTEIN-PROTEIN INTEREACTIVE CLONING TECHNIQUES: • Expression cloning is a technique in DNA cloning that uses expression vectors to generate a library of clones, with each clone expressing one protein. This expression library is then screened for the property of interest and clones of interest are recovered for further analysis. An example, would be using an expression library to isolate genes that could confer antibiotic resistance. • The following are techniques involves in protein-protein interaction: 1.Yeast Two Hybrid System 2.Phage Display System. • These above two techniques is also helpul in maximising gene expressions.
  • 13. CDNA AND GENOMIC DNA LIBRARY CONSTRUCTION: • A copy of DNA generated from messenger RNA (mRNA) with the help of enzyme reverse transcriptase is termed as cDNA. • A set of cDNA fragments, each of which has been cloned into a separate vector molecule, which constitute a some portion of transcriptome of the organism and stored as a library is known as a cDNA library. STEPS INVOLVED IN CDNA LIBRARY CONSTRUCTION: • To construct cDNA libraries, DNA copies from mRNA sequences of organism are produced and then they are cloned. • The term cDNA is given as all the DNA in the library are complementary to the mRNAs and are produced by reverse transcription of mRNAs. • Most eukaryotic DNA consists of repeated sequences that are not transcribed into mRNA, and in a cDNA library the sequences are not represented. • It should be remembered that prokaryotes and lower eukaryotes do not contain introns, and cDNA preparation for these species is usually needless. • Therefore, cDNA libraries are only created from higher eukaryotes. • For the construction of cDNA library, both the bacterial and bacteriophage DNA can be used as vectors.
  • 14. SIMILARITIES BETWEEN CDNA AND GENOMIC DNA LJBRARY: • cDNA Library (Complementary DNA library) • cDNA – It is a DNA copy of an mRNA molecule generated by reverse transcriptase, a DNA polymerase that can use either RNA or DNA as their template • These are prepared using mRNA as templates, their starting material • They are representative of only those genes of the genome that are expressed given specific conditions • cDNA does not have introns, and hence can be expressed in prokaryotic cells Genomic DNA library • Genomic DNA is the chromosomal DNA of an entity representative of the collection of its genomic content. They are different from that of complementary DNA, mitochondrial DNA or bacterial plasmid DNA. These are directly prepared from the genomic DNA, and represent the complete genome of an entity • For their construction, ligases and restriction endonucleases are vital • It can also represent the DNA of both eukaryotic and prokaryotic entities and carries introns • As it carries introns, they are incapable of expression in the prokaryotes. Moreover, prokaryotes lack the machinery for processing introns
  • 15. ADVANTAGES & DISADVANTAGES OF CDNA & GENOMIC LIBRARY: Advantages of cDNA library: • There are two major benefits of a cDNA library. • First, it is enriched with fragments from genes that have been actively transcribed. • Second, introns do not disrupt the cloned sequences; if the goal is to create a eukaryotic protein in bacteria, introns will pose a problem, since most bacteria have no means of eliminating the introns. Disadvantages of cDNA library: • A cDNA library has the drawback that it only includes sequences that are present in mature mRNA. • There are no introns and any other sequences that are modified during transcription; sequences that are not transcribed into RNA, such as promoters and enhancers, are also not present in a library of cDNA. • It is also important to remember that only certain gene sequences expressed in the tissue from which the RNA has been isolated constitute the cDNA library. • In addition, in a cDNA library, the frequency of a specific DNA sequence depends on the abundance of the corresponding mRNA in the given tissue. • In contrast, in a genomic DNA library, almost all genes are present at the same frequency
  • 16. REFRENCES: WEBSITES AND HTML: #www.microbenotes.com/(cDNA CLONING) #www.onlinebiology.com/(cDNA CLONING) #www.byjus.in/(CLONING) BOOK: #Name:TEXTBOOK OF BIOTECHNOLOGY(4th EDITION) #Authour Name:H.K.DAS #Publication Name:WILEY INDIA