1. M.Sc. Biotech./Biochem./Microbio. (Sem – II)
Genetic Engineering
Enzymes for Gene Cloning
By – Dr. Ravi Kant
Assistant Professor (Biotechnology)
Email – ravi.kant@nirmauni.ac.in
2. How do we generate recombinant plasmid / DNA ?
• Vector and target DNA to be cloned must be cut at specific locations and joined.
• Cutting and joining are some of the basic DNA manipulation techniques.
• Cutting DNA at specific locations: Restriction endonucleases.
• Joining DNA fragments in a controlled manner: DNA ligases.
4. Nucleases
• Nucleases, enzymes that cut or degrade nucleic acids (DNA/RNA) i.e. cleave
phosphodiester bonds between two nucleotides.
• Nucleases: two types exonucleases and endonucleases.
• Exonucleases: remove one nucleotide at a time from ends.
• Endonucleases: cuts (cleaves phosphodiester bond) within the DNA molecule.
5. Exonucleases
• remove one nucleotide at a time from ends.
• Examples:
Bal31, an exonuclease from
Alteromonas espejiana degrade dsDNA.
Exonuclease III: an exonuclease from
E.coli, degrades just one strand of dsDNA.
6. Exonucleases – more examples
• S1 endonuclease from Aspergillus oryzae,
degrades ssDNA including single stranded
nicks.
• DNAse I, from cow pancreas, degrades
both single and double stranded DNA
molecules
8. Restriction endonucleases
• In 1950s it was noted that certain bacterial trains are immune to bacteriophage
infection, in other words certain strains of bacteria restricts bacteriophage
infection.
• Later it was found that it was because of certain enzymes that bacteria encodes
that degrades bacteriophage DNA before it could replicate.
• Bacteria protects its own DNA from its restriction endonucleases by keeping it
methylated.
9. Restriction endonucleases
• Restriction endonuclease, the nuclease found in several bacterial species
(perhaps all) that protects them from bacteriophages.
• Restriction endonucleases recognises specific sequences and cleaves certain
phosphodiester bond.
• Was an important discovery with which a new technology discipline,
recombinant DNA technology (genetic engineering) started.
• W. Arber, H. Smith, and D. Nathans were awarded Nobel prize their discovery of
restriction endonucleases in 1978.
10. Restriction endonucleases - types
• 5 Types.
• Type I: cleave at sites remote from a recognition site; require both ATP and S-
adenosyl-L-methionine to function; multifunctional protein with both
restriction digestion and methylase activities.
• Type II: cleave within or at short specific distances from a recognition site;
most require magnesium.
• Type III: cleave at sites a short distance from a recognition site; require ATP (but
do not hydrolyse it); S-adenosyl-L-methionine stimulates the reaction but is not
required; exist as part of a complex with a modification methylase.
• Type IV: cleave only modified DNA, e.g. methylated, hydroxymethylated and
glucosyl-hydroxymethylated DNA
• Type V: enzymes utilize guide RNAs (gRNAs).
11. Type II Restriction endonucleases
• cleaves within or at short specific distances from a recognition site; requires
magnesium.
• are homodimers that recognize specific
4-8 bp long palindromic nucleotide
sequences.
• palindromic sequence, a sequence that
has 2 fold symmetry.
• either generates blunt ends (cutting both strands at the same position) or sticky
ends when cuts at staggered positions).
13. Type II Restriction endonucleases –
sticky and blunt ends
• blunt ends (cutting both strands at the
same position) or sticky ends when
cuts at staggered positions).
• e.g. AluI cleavage generates blunt ends.
• e.g. EcoRI cleavage generates sticky
end.
• BamHI, BglII, Sau3A generates the
same sticky ends.
• Let’s assume that all four nucleotides are randomly distributed in a 5000bp long
DNA fragment (GC content 50%) then how many fragments should a restriction
enzyme generate that recognises a 6bp long palindromic sequence??
14. • isoschizomers: restriction endonucleases that recognise same sequence and
cleave at the same site. e.g. SacI and SstI
• neoschizomers: restriction endonucleases that recognise same sequence but
cleave at different sites location. e.g. SmaI and XmaI
• Number of recognition sequences for give restriction endonuclease may be
calculated mathematically. e.g a tetranucleotide sequence (GATC) should occur
after every 256 bp (44), a pentanucleotide sequence should occur after every
1024 bp (45), and so on. assuming all four nucleotides are equally abundant and
randomly distributed.
15. How do we perform restriction digestion in the lab and do we analyze
• Restriction digestion.
• Analysis.
16. How do we generate recombinant plasmid / DNA ?
• Vector and target DNA to be cloned must be cut at specific locations and joined.
• Cutting and joining are some of the basic DNA manipulation techniques.
• Cutting DNA at specific locations: Restriction endonucleases.
• Joining DNA fragments in a controlled manner: DNA ligases.
17. Ligases
• Ligation: joining together vector and target DNA fragment.
• Ligases: enzymes that join DNA fragments.
• T4 DNA ligase: most common ligase in use. Purified from E.coli infected with T4
bacteriophage.
• Most living cells have ligases in them and their physiological function is to seal
the discontinuities (a missing phosphodiester bond) that may arise during DNA
replication, recombination, etc.
18. Ligation
• Blunt end ligation:
• Sticky end ligation:
• Whether blunt end or sticky end ligation would be more effective??
19. Ligation
• Blunt end ligation:
• Sticky end ligation:
• Whether blunt end or sticky end ligation would be more effective?? Sticky end.
20. Ligation (molecular mechanism)
• The complementary nucleotide sequence (sticky ends) from 2 different
molecules forms hydrogen bonds.
• T4 DNA ligase catalyzes the formation of a phosphodiester bond between the
3'OH at one end of a strand of DNA and the 5'-phosphate group of another.
• T4 DNA ligase uses ATP as the energy source for ligation.
21. Ligation (molecular mechanism)
• T4 DNA ligase first reacts with ATP, forming a ligase-
AMP intermediate with the AMP linked to the ε-amino
group of lysine in the active site of the ligase via a
phosphoamide bond.
• Adenylyl group is then transferred to the phosphate
group at the 5' end of a DNA chain, forming a DNA-
adenylate complex.
• Finally, a phosphodiester bond between the two DNA
ends is formed via the nucleophilic attack of the 3'-
hydroxyl at the end of a DNA strand on the activated
5′-phosphoryl group of another.
22. Ligases
• Taq DNA ligase: catalyze a phosphodiester
bond between two adjacent oligonucleotides
which are hybridized to a complementary
DNA strand.
• is active at a relatively high temperature (45-
65oC). Requires NAD as a cofactor.
• Taq RNA ligase: catalyze the formation of a
phosphodiester bond between RNA/RNA
oligonucleotides, RNA/DNA oligonucleotides,
or DNA/DNA oligonucleotides. Requires ATP
as a cofactor.
• may be used to generate DNA – RNA hybrids.
23. Putting sticky ends to blunt ends
• Digest vector and target DNA with the same restriction endonuclease.
• Linkers: short dsDNA of known sequence containing recognition site for a RE
e.g. BamHI linker, are synthesized in-vitro.
• Target DNA with blunt ends is incubated with a high concentration of linkers
and DNA ligase.
• Here, ligation would be more
efficient because large amount of
linker would be present in the
reaction mixture.
• Several linkers would attach to blunt
ended target DNA.
• RE digestion would yield target DNA
with sticky ends. as the energy source for ligation.
24. Putting sticky ends to blunt ends
• Problem with Linkers: internal restriction site.
• Adapters: are linkers with sticky ends. So there is no
need to digest, no problem even if there is internal
restriction site.
• Problem: adapter could self ligate.
• Blunt side of the adapter is normal (has 5 phosphate and 3’ OH) but sticky end lacks 5’
phosphate. Thus adapter can ligate to target DNA from blunt side but could not self ligate
because phosphate group is essential for the formation of phosphodiester bond.
• Once adapter is attached phosphate group may be added using polynucleotide kinases.
25. Putting sticky ends to blunt ends
• Problem with Linkers: internal restriction site.
• Adapters: are linkers with sticky ends. So there is no
need to digest, no problem even if there is internal
restriction site.
• Problem: adapter could self ligate.
• Blunt side of the adapter is normal (has 5 phosphate and 3’ OH) but sticky end lacks 5’
phosphate. Thus adapter can ligate to target DNA from blunt side but could not self ligate
because phosphate group is essential for the formation of phosphodiester bond.
• Once adapter is attached phosphate group may be added using polynucleotide kinases.
26. Putting sticky ends to blunt ends
• Homopolymer tailing: adding multiple nucleotides to 3’OH termini
of dsDNA employing terminal deoxynucleotidyl transferase.
• For sticky end ligation homopolymer tails are synthesized on
target and vector DNA which is complementary to each other.
• Usually poly dG and poly dC are used…why?
• poly dGs or poly dCs synthesized are never of the same size.
after base-pairing polymerase like Klenow fragment is used fillin
the gaps and ligase to seal the nicks (form phosphodiester bond).
• Practically, treatment with the Klenow fragment is not required,
base-pairing involving poly C and poly G is strong and recombinant
plasmid with some gaps may be introduced into the host and
there host DNA polymerase will fill in the gaps and ligase would
seal the nicks.
27. Enzymes for gene cloning - polymerases
• DNA polymerases: enzymes that synthesize a new strand of DNA complementary to existing
DNA or RNA template.
• DNA polymerases require short double-stranded regions (primers) for initiation.
• Different types of polymerases that are in use.
• DNA polymerase I: isolated from E.coli, attaches to short single stranded region (a nick), and
synthesizes completely new strand. Since it has both 5’ to 3’ polymerase activity and 5’ to 3
exonuclease activity.
28. Enzymes for gene cloning -
Klenow fragment
• In DNA polymerase I, polymerase and
exonuclease activities have been
assigned to different domains.
• Exonuclease activity is confined to first
323 amino acids.
• Klenow fragment, DNA polymerase I
with first 323 amino acids, without
exonuclease activity has only
polymerase activity.
• Used to fill in the gaps.
29. Enzymes for gene cloning - Taq DNA polymerase
• Taq DNA polymerase: DNA polymerase isolated from Thermus aquaticus, a bacteria found in
hot springs.
• Is a thermostable DNA polymerase, that works even at 94 degree centigrade, a feature that
makes it suitable for PCR.
Enzymes for gene cloning - reverse transcriptase
• Reverse transcriptase: is RNA dependent DNA polymerase, that synthesizes a
complementary DNA strand for a given RNA template.
• Special application in complementary DNA synthesis and cloning.
30. Enzymes for gene cloning - DNA modifying
enzymes
• Alkaline phosphatase: isolated from
E.coli, calf intestine, removes a
phosphate group from 5’ end.
• Polynucleotide kinase: from E.coli
infected with T4 bacteriophage, adds
phosphate to 5’ OH.
• Terminal deoxynucleotidyl transferase:
isolated from calf thymus, multiple
nucleotides to single or double-stranded
DNA, used for homopolymer tailing.
31. Enzymes for gene cloning - DNA modifying enzymes
• From the restriction-modification system, we know that certain bacterial strains are immune
to infection by bacteriophages because they have endonucleases that cuts bacteriophage
DNA before I could multiply.
• Bacterial strain protects its own DNA from its endonucleases by methylating.
• Methylases: recognition sequences for methylases are the same as that of the respective
endonuclease. e.g. EcoRI methylase recognizes GAATTC and EcoRI restriction endonuclease
also recognizes GAATTC.
• Methylases transfers a methyl group from S-adenosyl methionine to recognition sequence
and protect them from endonucleases.
• Methylases like SssI has broad specificity recognizes –CG- and methylates C, thus protecting
from a wide variety of restriction endonucleases.
• Some restriction endonucleases like DpnI cuts at their recognition sequence only if it is
methylated.
• While some restriction endonucleases cuts both methylated and non-methylated
recognitions sequences e.g. BamHI.
32. Enzymes for gene cloning - DNA modifying enzymes
• dam and dcm Methylation: The methylase encoded by the dam gene (dam methylase)
transfers a methyl group from SAM to the N6 position of the adenine base in the sequence
5' … GATC … 3’.
• The methylase encoded by the dcm gene (dcm methylase) methylates the internal cytosine
base, at the C5 position, in the sequences 5' … CCAGG … 3' and 5' … CCTGG … 3’.
• Why are we talking about dam and dcm methylation system???
33. Enzymes for gene cloning - DNA modifying enzymes
• dam and dcm Methylation: The methylase encoded by the dam gene (dam methylase)
transfers a methyl group from SAM to the N6 position of the adenine base in the sequence
5' … GATC … 3’.
• The methylase encoded by the dcm gene (dcm methylase) methylates the internal cytosine
base, at the C5 position, in the sequences 5' … CCAGG … 3' and 5' … CCTGG … 3’.
• Why are we talking about dam and dcm methylation system???
• Almost all strains of E. coli commonly used in cloning have a dam+dcm+ genotype.
• So while choosing a restriction enzyme, one should also be aware of its sensitivity
methylation else dam-dcm- E.coli strain would be required.
34. Restriction ligation using DNA topoisomerase
• DNA topoisomerases: enzymes that removes positive and negative
supercoiling.
• Works by creating double-strand breaks, has both nuclease and ligase
activity.
• Uses a special vector that has a unique CCCTT sequence.
Topoisomerase from vaccinia virus recognizes and cleaves at CCCTT to
linearize it.
• After cutting topoisomerase may remain covalently attached to blunt
ends, reaction may be stored at that point vector may be stored until
needed.
35. Restriction ligation using DNA topoisomerase contd..
• Cleavage by topoisomerases generates 3’P and 5’OH termini. Target molecule generated by
digestion would have 5’P and 3’OH termini.
• for cloning target DNA is dephosphorylated
using alkaline phosphatase.
• When mixed with a vector having
topoisomerase attached, its ligase activity will
form a phosphodiester bond.
• Resulting recombinant molecule with nicks
when introduced into the host, host enzymes
will repair it.
• group from 5’ end.
