This document discusses various methods for constructing DNA libraries, which involve breaking large DNA molecules into smaller fragments that can then be joined to vector DNA. DNA can be fragmented through restriction enzyme digestion, mechanical shearing, or enzymatic treatment. The fragments are then ligated into a vector using DNA ligase or other techniques. Recombinant vectors can be introduced into host cells and clones containing the inserted DNA fragment can be selected and stored.

1. Construction of DNA Libraries
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Lecture- 19

2. Introduction of DNA fragment into the vector
DNA molecule
• Isolation of DNA fragment containing the gene of interest
• Cleavage of vector DNA
• Joining of the DNA fragment containing the gene of interest to the
cleaved vector DNA molecule
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3. Breaking of large DNA molecules into small
fragments
• Digestion with restriction enzymes
• Cleavage by nonspecific endonucleases
• Mechanical shearing
• Automated shearing using high performance liquid
chromatography (HPLC)
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4. Digestion with restriction enzymes
• Restriction digestion using site-
specific restriction
endonucleases
• Routinely used for obtaining
DNA fragments in precise and
reproducible manner
• Simple, reliable, and generates
specific sized fragments
Examples of how restriction enzymes cleave DNA4

5. Cleavage by nonspecific endonucleases
• Nonspecific endonuclease-catalyzed
cleavage
• Gives nonuniform and random fragments
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6. Mechanical shearing
• By high speed stirring in a blender, controlled shearing can be
achieved
• High molecular weight DNA is sheared to fragments with a
mean size of ̴8 kbp by stirring at 1,500 rev/min for 30 min.
• Fragments of high molecular weight DNA can also be obtained
by passing through the orifice of a 28-gauge hypodermic
needle
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7. Automated shearing using HPLC
• Hydrodynamic shearing based on HPLC
• DNA is repeatedly passed through a small hole, using an HPLC
pump, until the sample is fragmented to products of certain
size
• The final size is determined by the flow rate of the sample and
size of the opening and these parameters are monitored by
the automated system
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8. Sonication
• Isolated DNA is subjected to
hydrodynamic shearing by
exposure to brief pulses of sound
waves
• Most sonicators shear DNA to a size
of 300-500 bp
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9. Nebulization
• Hydrodynamic shearing is
performed by collecting the
fine mist created by forcing
DNA in solution through a
small hole in the nebulizer
unit
• The speed of passage of DNA
solution through the hole,
the viscosity of the solution
and the temperature
regulate the size of the
fragments
Functioning of nebulizer
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10. Other methods of obtaining the gene of
interest
• If the gene of interest is small and its sequence is
known, it can be synthesized chemically for cloning in
expression vector
• If the gene sequences from same or related sources
are known, DNA fragments can be obtained from
PCR amplification gene specific primers and the
resulting PCR product may be cloned directly into an
expression vector
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11. • mRNA purified from total cell RNA may be reverse
transcribed to cDNA which when cloned into vector
form a cDNA library
• The only way to generate precise and defined
fragments is to cleave with restriction enzymes
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12. Joining of DNA fragments to vector DNA
molecule
• Ligation by E. coli DNA ligase
• Ligation by T4 DNA ligase from T4 phage-infected E.
coli
• Homopolymer tailing catalyzed by terminal
deoxynucleotidyl transferase (TdT)
• Short synthetic oligonucleotides such as linkers and
adaptors may be used to increase the versatility of
ligation reaction
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13. Ligation of DNA fragments using DNA ligase
• DNA ligase catalyzes the formation of a
phosphodiester bond between a 3’-OH and a 5’-
Phosphate group of DNA
• Both the DNA fragments and the vector should have
compatible ends or else compatibility between them
may be acquired by partial filling-in or trimming back
or with the addition of linkers and adapters
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14. Recombinant formation using
blunt end ligation
Recombinant formation using
sticky end ligation
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15. Factors affecting DNA ligation under in
vitro reaction conditions
• Temperature
• DNA concentration
• Length of insert DNA
• ATP concentration (in case of T4 DNA ligase)
• Inhibitory materials
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16. Joining using homopolymer tailing
• Terminal deoxynucleotidyl transferase is used
to add a series of nucleotide (oligo dA or oligo
dT) on to 3’-OH of dsDNA molecule
Cloning using homopolymer tailing method
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17. Increasing versatility and efficiency of
joining of DNA molecules
I. Trimming back and filling-in
II. Use of linkers
III.Use of adaptors
IV.Homopolymer tailing
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18. Trimming back and filling-in
• Trimming back and filling-in strategies are
employed for converting a sticky end into a
blunt end
Conversion of sticky end into blunt end by filling-in
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19. Creation of new restriction enzyme sites by filling-in
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20. Linkers
• These are chemically synthesized short pieces of DNA
molecules of known nucleotide sequence; blunt
ended, self complementary and contain an inherent
restriction enzyme site
Eco RI linker
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21. Cloning using linkers
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22. Adaptors
• Like linkers , adaptors are short synthetic
oligonuleotides that also increase the versatility of
ligation
• Two pairs of such oligonucleotides are designed to
anneal together in such a way as to create a short
dsDNA fragment with one sticky and one blunt end
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23. (a) Bam HI adaptor; (b) Bam HI-Eco RI adaptor
Use of Bam HI adaptor molecule in cloning
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24. Schematic representation of recombinant DNA technology 24

25. Introduction of recombinant DNA
molecules into host cells
• Hosts used:
E. coli
Bacillus subtilis, Saccharomyces cerevisiae, cultured
mammalian cells and cultured plant cells
• Several techniques are used for introduction of the
recombinant DNA molecule into host:
transformation
transfection
in vitro packaging 25

26. Selection of recombinants
Recombinants are either distinguished from non
recombinants by colour reaction or are the only host
capable of growth on that medium
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27. Storage of desired bacterial clones
• For long term storage, glycerin or dimethylsulfoxide
(DMSO) stocks are prepared
• These stocks are stored at -20o or -70o C
• Another method for long term storage of bacteria is
freeze drying or its low cost version, vaccum drying
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28. Subcloning
• The transfer of a cloned fragment of DNA from one
vector to another is termed as subcloning
• It is done for investigation of a short region of a large
cloned fragment in more detail or to transfer a gene
to a vector designed to express it in a particular
species or for transferring in M13 vectors
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29. Advantages of gene or cDNA cloning
• To get homogenous preparations of any desired DNA
• To get high yield of recombinant DNA
• To obtain a pure sample of a gene
• Isolation and manipulation of fragments of an
organisms genome
• Isolation of long genes and unknown genes
• Elucidation of gene function, promoter analysis and
identification of mutations
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30. Advantages….
• Information about cell type and developmental stage
specific genes, locations of splice sites and
alternatively spliced genes
• DNA or genome sequencing
• Site directed mutagenesis
• Large scale commercial production of proteins and
other molecules of biological importance
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