1. II. Restriction EndonucleasesII. Restriction Endonucleases
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. • Restriction Enzymes
– Bacterial enzymes that cut DNA molecules
only at restriction sites.
– Categorized into two groups based on type
of cut
• Cuts with sticky ends
• Cuts with blunt ends

3. A. Origin and functionA. Origin and function
• Bacterial origin = enzymes that cleave foreign
DNA
• Named after the organism from which they
were derived
– EcoRI from Escherichia coli
– BamHI from Bacillus amyloliquefaciens
• Protect bacteria from bacteriophage infection
– Restricts viral replication
• Bacterium protects it’s own DNA by
methylating those specific sequence.

4. B. AvailabilityB. Availability
• More than 800 types of REs are known;
and more than 400 of them are
available commercially.

5. C. ClassesC. Classes
• Type I
– Cuts the DNA on both strands but at a non-
specific location at varying distances from
the particular sequence that is recognized
by the restriction enzyme
– Therefore random/imprecise cuts
– Not very useful for rDNA applications

6. • Type II
– Cuts both strands of DNA within the
particular sequence recognized by the
restriction enzyme
– Used widely for molecular biology
procedures
– DNA sequence = symmetrical

7. • Reads the same in the 5’ 3’ direction on
both strands = Palindromic Sequence
• Some enzymes generate “blunt ends” (cut in
middle)
• Others generate “sticky ends” (staggered
cuts)
– H-bonding possible with complementary tails
– DNA ligase covalently links the two fragments
together by forming phosphodiester bonds of the
phosphate-sugar backbones

10. Figure 8.2Figure 8.2 Actions of restriction enzymes-overviewActions of restriction enzymes-overview

12. DNA Ligase in Action!DNA Ligase in Action!

13. III. Vectors for GeneIII. Vectors for Gene
CloningCloning

14. •Vectors:
– Nucleic acid molecules that deliver a gene
into a cell
– Useful properties:
• Small enough to manipulate in a lab
• Survive inside cells
• Contain recognizable genetic marker
• Ensure genetic expression of gene
• Autonomous replication.

15. A. Requirements of a vector toA. Requirements of a vector to
serve as a carrier moleculeserve as a carrier molecule
• The choice of a vector depends on the
design of the experimental system and how
the cloned gene will be screened or utilized
subsequently
• Most vectors contain a prokaryotic origin of
replication allowing maintenance in bacterial
cells.

16. • Some vectors contain an additional
eukaryotic origin of replication allowing
autonomous, episomal replication in
eukaryotic cells.
• Multiple unique cloning sites are often
included for versatility and easier library
construction.
CONTD…

17. B. Main types of vectorsB. Main types of vectors
i. Bacterial Plasmids
ii. Bacteriophages
iii. Cosmids
iv. Others:
 Bacterial artificial chromosome (BAC),
 Yeast artificial chromosome (YAC), Human
artificial chromosome (HAC), yeast 2 micron
plasmid, retrovirus, baculovirus vector……

18. C. Choice of vectorC. Choice of vector
• Depends on nature of protocol or
experiment
• Type of host cell to accommodate rDNA
– Prokaryotic
– Eukaryotic

19. D. Plasmid vectorD. Plasmid vector
• Covalently closed, circular, double stranded DNA
molecules that occur naturally and replicate
extrachromosomally in bacteria
• Many confer drug resistance to bacterial strains
• Origin of replication present (ORI)

20. • Examples
– pBR322
• One of the original plasmids used
• Two selectable markers (Amp and Tet resistance)
• Several unique restriction sites scattered throughout
plasmid (some lie within antibiotic resistance genes =
means of screening for inserts)

21. EE. Common steps. Common steps involved in isolating ainvolved in isolating a
particular DNA fragment from a complexparticular DNA fragment from a complex
mixture of DNA fragments or moleculesmixture of DNA fragments or molecules
1. DNA molecules are digested with enzymes
called restriction endonucleases which reduces
the size of the fragments  Renders them more
manageable for cloning purposes
2. These products of digestion are inserted into a
DNA molecule called a vector  Enables
desired fragment to be replicated in cell culture
to very high levels in a given cell (copy #)

22. 3. Introduction of recombinant DNA
molecule into an appropriate host cell
– Transformation or transfection
– Each cell receiving rDNA = CLONE
– May have thousands of copies of rDNA
molecules/cell after DNA replication
– As host cell divides, rDNA partitioned
into daughter cells
CONTD…

23. • 4. Population of cells of a given clone is
expanded, and therefore so is the rDNA.
– Amplification
– DNA can be extracted, purified and used for
molecular analyses
• Investigate organization of genes
• Structure/function
• Activation
• Processing
CONTD…

24. • Gene product encoded by that rDNA can be
characterized or modified through mutational
experiments
• Antibiotic resistance genes and/or other selectable
markers enable identification of cells that have
acquired the vector construct.
• Some vectors contain inducible or tissue-specific
promoters permitting controlled expression of
introduced genes in transfected cells or transgenic
animals.
CONTD…

25. • Modern vectors contain multi-functional
elements designed to permit a combination
of cloning, DNA sequencing, in vitro
mutagenesis and transcription and episomal
replication.

26. – pUC18
• Derivative of pBR322
• Advantages over pBR322:
– Smaller – so can accommodate larger DNA
fragments during cloning (5-10kbp)
– Higher copy # per cell (500 per cell = 5-10x more
than pBR322)
– Multiple cloning sites clustered in same location =
“Polylinker”

27. • Vectors able to survive under antibiotic
selection are amplified in bacterial hosts by
autonomous replication
• Plasmid DNA containing the gene of
interest is purified from large scale cultures

28. • Subsequent steps in the experimental design
are undertaken:
– Sub-cloning
– Mutagenesis
– Sequencing
– Transfection of eukaryotic cell lines (calcium
phosphate precipitation, lipofection, electroporation,
dextran sulfate, microinjection,…..)
– Fragment isolation for transgenic mice production
(microinjection)
– PCR

29. E. Lambda vectorE. Lambda vector
(Bacteriophages)(Bacteriophages)
• Bacteriophage lambda (λ) infects E. coli
• Lambda phage is another choice vector. In view of
the disadvantage in case of plasmids (the plasmids
can not carry large fragments of DNA) there is a
need of other cloning vectors capable of carrying
larger fragments.
• λ- phages were found to be capable of carrying
much larger DNA inserts, as large as 40 kb.
• Double-stranded, linear DNA vector – suitable for
library construction
• Can accommodate large segments of foreign DNA

30. F. Cosmid vectorsF. Cosmid vectors
 An artificially constructed circular DNA
molecule of 5000 – 7000 base pairs.
It combines useful features of both, the
plasmid and the lambda-phase, because of the
presence of the following elements:
i. An origin of replication.
ii.Packaging signals of λ-phage, called cos-sites.

31. Contd…Contd…
iii. In addition, the cosmid contains an
antibiotic resistant gene and several
restriction sites where foreign DNA can be
inserted.
Note: Cosmid permits insertion of large
fragments of DNA, up to 35 – 50kb long.

32. G. Shuttle vectorsG. Shuttle vectors
• Hybrid molecules designed for use in
multiple cell types
• Multiple ORIs allow replication in both
prokaryotic and eukaryotic host cells
allowing transfer between different cell
types
– Examples:
• E. coli  yeast cells
• E. coli  human cell lines
• Selectable markers and cloning sites

33. H. Bacterial artificialH. Bacterial artificial
chromosomes (BACs)chromosomes (BACs)
• Based on F factor of bacteria (imp. In conjugation)
• Can accommodate 1 Mb of DNA (= 1000kbp)
• F factor components for replication and copy #
control are present
• Selectable markers and cloning sites available
• Other useful features:
• Direct RNA synthesis
• RNA probes for hybridization experiments
• RNA for in vitro translation

34. I. Yeast artificial chromosomesI. Yeast artificial chromosomes
(YACs)(YACs)
• Hybrid molecule containing components of yeast,
protozoa and bacterial plasmids
– Yeast:
• ORI = ARS (autonomously replicating sequence)
• Selectable markers on each arm (TRP1 and URA3)
• Yeast centromere
– Protozoa= Tetrahymena
• Telomere sequences (yeast telomeres may also be used)
– Bacterial plasmid
• Polylinker
• Can accommodate >1Mb (1000kbp = 106
bp)

35. J. Human artificialJ. Human artificial
chromosomes (HACs)chromosomes (HACs)
• Developed in 1997 – synthetic, self-replicating
• ~1/10 size of normal chromosome
• Micro-chromosome that passes to cells during mitosis
• Contains:
– ORI
– Centromere
– Telomere
– Protective cap of repeating DNA sequences at ends
of chromosome (protects from shortening during
mitosis)
– Histones provided by host cell

36. Figure 8.3Figure 8.3 Producing a recombinant vectorProducing a recombinant vector
Antibiotic
resistance
gene
Restriction
site
mRNA for human
growth hormone (HGH)
Reverse
transcription
Plasmid (vector)
cDNA for HGH
Restriction
enzyme
Restriction
enzyme
Sticky ends
Gene for human
growth hormone
Ligase
Recombinant plasmid
Introduce recombinant
plasmid into bacteria.
Recombinant
plasmid
Bacterial
chromosome
Inoculate bacteria
on media containing
antibiotic.
Bacteria containing
the plasmid with
HGH gene survive
because they also
have resistance gene.