resurrection enzyme , dna library, gene cloning

2. Delvadiya Indrajay R.
Roll No. :
Reg.no. : 2010116027
M.Sc. (Agri) student
Dept. of Genetics and Plant Breeding
JAU , Junagadh

3. Restriction Enzymes
Gene cloning
Gene Library
Genomic Library
C- DNA Library

4. DNA RESTRICTION
AND
RESTRICTION ENZYME

5. – The process of cutting DNA at or near specific
recognition nucleotide sequences (known
as restriction sites), using restriction enzyme is
known as DNA restriction.
– The enzyme used for DNA Restriction is known
as Restriction Enzyme.

6. – Restriction enzymes are a type of endonucleases, i.e., enzymes that
produces cuts within DNA molecules.
– As a rule, they are recognising highly specific, short base sequences
for binding to DNA molecules. They may, however produces the cut
either within the recognition site or at some distance from this site.
– It is found naturally in a wide variety of prokaryotes.
– Restriction enzymes are also known as molecular scissors

7. Discovery of Restriction Enzymes:
– Arbor and Dussoix in 1962 discovered that certain bacteria
contain Endonucleases which have the ability to cleave
DNA.
In 1970, Hamilton O. Smith, Thomas Kelly and Kent
Wilcox isolated and characterized the first type II restriction
enzyme, HindI, from the bacterium Haemophilus influenzae.
Werner Arbor, Hamilton Smith and Daniel Nathans
shared the 1978 Nobel Prize for Medicine and Physiology for
their discovery of Restriction Enzymes.

8. Biological Role in Bacteria :
– Most bacteria use Restriction Enzymes as a defence against
bacteriophages.
– Restriction enzymes prevent the replication of the phage by cleaving
its DNA at specific sites.
– The host DNA is protected by Methylases which add methyl groups to
adenine or cytosine bases within the recognition site thereby modifying
the site and protecting the DNA.

9. Mechanism of Action:
– Restriction Endonuclease scan the length of the DNA, binds
to the DNA molecule when it recognizes a specific sequence
and makes one cut in each of the sugar phosphate backbones
of the double helix – by hydrolyzing the phoshphodiester
bond. Specifically, the bond between the 3’ O atom and the P
atom is broken.

10. Restriction Site:
– Restriction Endonuclease is an enzyme that cuts double-
stranded or single stranded DNA at specific recognition
nucleotide sequences (mostly palindromic or symmetry)
known as restriction sites.
 Palindromic Sequence:
– The mirror-like palindrome in which the same forward
and backwards are on a single strand of DNA strand.

11. – The inverted repeat palindrome is also a sequence
that reads the same forward and backwards, but the
forward and backward sequences are found in
complementary DNA strands.
• Inverted repeat palindromes are more common and
have greater biological importance than mirror-like
palindromes.

12. Types of Restriction Enzyme
Type I Restriction Enzymes:
– These restriction enzymes recognize the recognition site, but
cleave the DNA somewhere between 400 base pairs (bp) to 10,000
bp or 10 kbp right or left. The cleavage site is not specific.
– These enzymes are made up of three peptides with multiple
functions. These enzymes require Mg++, ATP and S adenosyl
methionine for cleavage or for enzymatic hydrolysis of DNA.
These enzymes are studied for general interest rather than as useful
tools for genetic engineering.
– E.g., EcoK I, EcoA I, CfrA I

13. Type II Restriction Enzymes
– Restriction enzymes of this type recognize the restriction site and
cleave the DNA within the recognition site or sequence.
– Type II Restriction enzyme are widely used for gene manipulation
studies since they have specific cleavage sites.
– These enzymes require Mg++ as cofactor for cleavage activity and
can generate 5 -PO4 or 3 -OH. Enzymes of this type are highly
important because of their specificity.

14. Blunt End Cutters:
– Blunt end cutters Type II restriction enzymes of this class cut
the DNA strands at same points on both the strands of DNA
within the recognition sequence. The DNA strands generated
are completely base paired. Such fragments are called as blunt
ended or flush ended fragments.
– E.g., AluI – (Arthrobacter luteus)

15. Type III Restriction Enzymes
– Type III Restriction enzymes of this type recognize the recognition
site, but cut the DNA 1 kbp away from the restriction site.
– These enzymes are made up of two peptides or subunits. These
enzymes require ATP, Mg++ and S-adenosyl methionine for action.
– E.g., EcoP I, Hind III, EcoP15 I

16. Property Type I Type II Type III
Structure Enzyme complex of 500-600 k
dal composed of three separate
subunits
Normally homodimers of 20-70
k dal
Heterodimers with subunits
of 70 and 100 k dal
Composition Multienzyme complex with R
(endonuclease), M (methylase)
and S (specificity) subunits
Separate enzymes;
endonuclease is a homodimer,
methylase a monomer
subunit provides specificity
on its own; functions as
methylase; as heterodimer
with R subunit; functions as
methylase- endonuclease
Cofactors Mg2+, ATP,
Sadenosylmethionine (SAM)
(needed for cleavage as well as
methylation)
Mg2+, SAM (for methylation
only)
Mg2+, ATP (for cleavage),
SAM (needed for
methylation: stimulate
cleavage)
Recognition sites Asymmetric, bipartite, may be
degenerate; 1315 base pairs
containing interruption of 6 to 8
base pairs
symmetric, may be bipartite,
may be degenerate; 4 to 8 base
pairs normally 180° rotational
symmetry
Asymmetric, uninterrupted,
5-6 nucleotide long with no
rotational symmetry
Cleavage Non-specific, variable distance
(100-1000 nucleotides) from
recognition site
Precise cleavage within
recognition site at defined
distance
Precise cleavage at a fixed
distance; 25-27 nucleotides
from recognition site
Example EcoK I, EcoA I, CfrA I Eco RI, BamHI, HindI, Hind III EcoP I, HindII, EcoP15
The properties of three types of restriction endonucleases are given below.

17. Isoschizomers and Neoschizomers
– Restriction enzymes that have the same recognition sequence as
well as the same cleavage site are Isoschizomers.
– Restriction enzymes that have the same recognition sequence
but cleave the DNA at a different site within that sequence are
Neoschizomers. Eg:SmaI and XmaI
5’ C C C G G G 3’ 5’ C C C G G G 3’
3’ G G G C C C 5’ 3’ G G G C C C 5’
Xma I Sma I

18. – Star activity : Under extreme conditions such as elevated pH
or low ionic strength, RE are capable of cleaving sequences
which are similar but not identical to their recognition
sequence.
– Linkers: These are short, chemically synthesized, self
complementary, double stranded oligo nucleotides, which
contain within there one or more restriction endonuclease sites.
Linkers are joined with blunt ended DNA fragments, cleavage
of the linker with the appropriate restriction enzyme creates
suitable cohesive protruding ends.
– Adaptors: Adoptors are short, chemically synthesized DNA
double strands which can be used to link the ends of two DNA
molecules that have different sequences at their ends.

20. Why Bacteria does not cleave their own DNA by restriction enzyme?
• Most bacteria use Restriction Enzymes as a defence against bacteriophages.
• Restriction enzymes prevent the replication of the phage by cleaving its DNA at
specific sites.
•The host DNA is protected by Methylases which add methyl groups to adenine or
cytosine bases within the recognition site thereby modifying the site and protecting the
DNA.
(a) Unmethylated DNA cleaved by RE. (b) Methylated DNA cannot cleaved by RE.

21. Applications of DNA
Restriction
– They are used in gene cloning
– Restriction enzymes are most widely used in recombinant DNA
technology.
– Detection of RFLPs
– DNA Mapping
– Genotype a DNA sample by SNP

22. Molecular Cloning
or
gene cloning
or
Genetic Engineering
or
Recombinant DNA
Technology

23. – To clone means to make identical copies. DNA cloning involves
separating a specific gene or DNA segment from a larger chromosome,
attaching it to a small carrier DNA. The resultant hybrid DNA is
called recombinant DNA, which is transferred to a proper host
(bacteria, virus or yeast) and replicated to make multiple copy of
the selected gene.
– When cloned under an appropriate expression vector, a gene can be
expressed (I.e. transcribed and translated), at desired level to produce
recombinant proteins.
– Most methods for cloning pieces of DNA in the laboratory share
general features, such as the use of bacteria and their plasmids.
Plasmids are small circular DNA molecules that replicate separately
from the bacterial chromosome

24. – To work directly with specific genes, scientists prepare
well-defined segments of DNA in identical copies, a
process called DNA cloning.
– This technology has made it possible to isolate, clone
and produce DNA for all the genes in appropriate
quantity so that they can be sequenced and
characterized. Similarly, some of the genes which are
expressed at very low level, can be cloned and desired
amount of recombinant proteins can be produced.

25. – Five steps of cloning:
1. Cutting the DNA to be cloned from the chromosomal using sequence specific
Restriction Endonuclease.
2. Selecting a cloning vector (a small molecule capable of self-replicating inside
host cells), and cutting the cloning vector with the same restriction
endonuclease producing the cohessive ends.
3. Incubating the vector and subject DNA togather to aneal and then joining
them using DNA ligase. The resultant DNA is called recombinant DNA.
4. Transferring the reconbinant DNA to an appropriate host such as bacteria,
virus or yeast which will provide necessory biomachinary for DNA replication.
5. Identifying the host cells that contain the recombinant DNA.

26. STEP -1 : Isolation of DNA (Gene of
Interest) Fragments to be Cloned
– Before we carry out the operation of gene cloning we need two basic
things in their purified state – the gene of our interest (GI) and the
vector. A GI is a fragment of gene whose product (a protein, enzyme
or a hormone) interests us. For example, gene encoding for the
hormone insulin.
– Similarly, the vector is a carrier molecule which can carry our GI into
a host, replicate there along with the GI making its multiple copies.
In this state the GI can also be expressed in the host cell producing
the product of the gene which is needed by us.

27. STEP-2 : Insertion of Isolated DNA into the a
Suitable Vector to Form the Recombinant DNA
– Once the ingredients are ready we can start the operation. Our next step
will be to cut both the vectors as well as the GI by using a special type
of enzyme, called restriction endonuclease. A restriction endonuclease
is an enzyme that cuts double-stranded or single-stranded DNA at
specific recognition nucleotide sequences known as restriction sites
towards the inner region (hence endonuclease).
– They are also regarded as molecular scissors as they cut open the DNA
strands. After this cutting step we move to pasting. Here the GI is taken
and pasted to the cut vector. This procedure also needs an enzyme,
called DNA ligase. They are also considered as molecular glue.

28. – The resulting DNA molecule is a hybrid of two DNA molecules – our GI and
the vector. In the terminology of genetics this intermixing of different DNA
strands is called recombination (which naturally takes place in the prophase 1
of meiosis 1). Hence, this new hybrid DNA molecule is also called a
recombinant DNA molecule and this technology is called recombinant DNA
technology (RDT).
A good vector must have the following properties:
1. It should be able to replicate autonomously. When the objective of cloning
is to obtain a large number of copies of the DNA insert, the vector replicon
must be under relaxed control so that it can generate multiple copies of itself
in a single host cell.
2. It should be easy to isolate and purify.
3. It should be easily introduced into the host cells, i.e. transformation of the
host with the vector should be easy.

29. 4. The vector should have suitable marker genes that allow easy
detection and/or selection of the transformed host cells.
5. When the objective is gene transfer, it should have the ability to
integrate either itself or the DNA insert it carries into the genome of the
host cell.
6. The cells transformed with the vector molecules containing the DNA
insert (recombinant or chimaeric vector) should be identifiable or
selectable from those transformed by the vector molecules only.
7. A vector should contain unique target sites for as may restriction
enzymes as possible into which the DNA insert can be integrated
without disrupting an essential function.
8. When the expression of the DNA insert is desired, the vector should
contain atleast suitable control elements, e.g. promoter, operator and

30. Figure 20.2a
Bacterium
Bacterial
chromosome
Plasmid
2
1 Gene inserted into
plasmid
Cell containing
gene of interest
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
(“foreign” DNA)
Recombinant
bacterium

31. STEP-3: Introduction of the Recombinant DNA into a
Suitable Organism known as Host:
– When our recombinant DNA molecule is ready we need to introduce it into a
living system known as host.
– This is done either for one or both of the following reasons:
– (a) To replicate the recombinant DNA molecule in order to get the multiple copies
of our GI.
– (b) To let our GI get express and produce the protein which is needed by us.
– Introduction of the recombinant DNA into the host cell is done by various ways
and strictly depends upon the size of the DNA molecule and the nature of GI.
Some of the methods followed to carry out this step includes electroporation,
micro-injection, lipofection, etc.
– When we carry out this process some of the host cells will take up the re-
combinant DNA and some will not. The host cells which have taken up the
recombinant DNA are called transformed cells and the process is called
transformation.

32. STEP-4: Selection of the Transformed Host Cells
and Identification of the Clone Containing the Gene
of Interest:
– The transformation process generates a mixed population of
transformed and non-trans- formed host cells. As we are
interested only in transformed host cells it becomes necessary
to filter them out. This is exactly what is done in the selection
process. There are many existing selection strategies some of
which include taking the help of reporter genes, colony
hybridization technique, etc.

33. STEP-5: Multiplication/Expression of the
Introduced Gene in the Host:
– Once we have purified our transformed host cells by the screening
process; it is now our job to provide them optimum parameters to grow
and multiply. In this step the transformed host cells are introduced into
fresh culture media which provide them rich nourishment followed by
an incubation in the oven at right temperature.
– At this stage the host cells divide and re-divide along with the
replication of the recombinant DNA carried by them. Now at this point
we have two choices.
– When the aim of the cloning process is to generate a gene library, then
our target will be obtaining numerous copies of GI. So with this plan in
our mind we will simply go for the replication of the recombinant
DNA and not beyond that.

34. – If the aim of the cloning experiment is to obtain the product of
GI, then we will go for a step ahead where we will provide
favourable conditions to the host cells in which the GI sitting in
the vector can express our product of interest (PI).

35. STEP-6: Isolation of the Multiplied Gene
Copies/Protein Expressed by the Introduced Gene:
– In this step we isolate our multiplied GI which is present attached
with the vector or the protein encoded by it. This can be rightly com-
pared with the process of harvesting where we collect the crop from
the field. There are many processes of isolation, the selection of
which varies from case to case.
– STEP-7: Purification of the Isolated Gene Copy/Protein:
– After the harvesting of the isolated gene copy or the protein it is
now our job to purify them.

36. Figure 20.2b
Host cell grown in
culture to form a clone
of cells containing the
“cloned” gene of interest
Gene of
interest
Protein expressed from
gene of interest
Protein harvestedCopies of gene
Basic research
and various
applications
3
4
Basic
research
on protein
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth
hormone treats
stunted growth

37. Figure 20.2
Bacterium
Bacterial
chromosome
Plasmid
2
1
3
4
Gene inserted into
plasmid
Cell containing gene
of interest
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
(“foreign” DNA)
Recombinant
bacterium
Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Gene of
interest
Protein expressed from
gene of interest
Protein harvestedCopies of gene
Basic research
and various
applications
Basic
research
on protein
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth
hormone treats
stunted growth

41. GENOME -
Introduction
– What is a GENOME ????
– Genome: “ The Book Of Life”
– The word “ genome” was coined in about 1930.
– The total DNA present in the nucleus of each cell of an organism
– is its genome.
– It comes from the terms gene and chromosome
– It corresponds to all the organism’s bases: A, T, C, G.
–

42. GENE LIBRARY

43. WHAT IS GENE LIBRARY????
– A gene library is a collection of different DNA sequences from
an organism, which has been cloned into a vector for ease of
purification, storage and analysis.
– There are two types of gene library that can be made depending
upon the source of the DNA used.
1.Genomic library.
2.cDNA library.

44. Two types of GENE libraries
– The Genomic library contains DNA fragments
representing the entire genome of an organism.
– The cDNA library contains only complementary DNA
molecules synthesized from mRNA molecules in a cell.

45. Genomic library
– Genomic library are made from total nuclear DNA of an organism or
species.
– DNA is cut into clonable pieces as randomly possible using restriction
endonuclease.
– Genomic libraries contain whole genomic fragments including gene
exons and introns, gene promoters, intragenic DNA, origins of
replication, etc

46. Construction of genomic
libraries
– There are following main steps in gene cloning:
1. Isolation of genomic DNA and vector.
2. Cleavage of Genomic DNA and vector by
Restriction Endonucleases.
3. Ligation of fragmented DNA with the vector.
4. Transformation of r-DNA in the bacterial cell.

47. Step 1 :- extraction of genomic
DNA
– Isolation of genomic DNA includes four steps:
 Culture of bacteria is grown and then harvested.
 Cell are broken to remove their contents.
 Cell extract is treated to remove all component except DNA.
 Concentrating DNA from resulting DNA solution.

49. Step- 2 :- Cut with the restriction
endonuclease enzyme

50. Step 3 :- Ligate the gene with the vector
which cleaves by same enzyme

51. Step 4:- Transfer (transform) into bacteria
Cells which are able to undergo
this treatment are termed as
component cells.
CaCl2 cause DNA to precipitate
on the outside walls of bacterial
cells.

52. Step 5 :- Amplification
– E. coli cells are
grown in an agar
medium containing
amplicillin or
tetracyclin at 37°C.

53. CONSTRUCTION OF GENOMIC LIBRARY

55. Screening of genomic library
– Once the genomic library has been created, it is screened
to identify the genes of interest. One of the most common
library screening technique is called colony
hybridization.
– In the process of library construction, phage vectors are
used then the process of identification of genes of interest
involved is the Plaques hybridization

56. COLONY HYBRIDIZATION
– Colony hybridization is the screening
of a library with a labeled probe(
radioactive substance )to identify a
specific sequence of DNA, RNA,
Enzyme, protein, or antibody.

57. Plaque hybridization
– The plaques are screed by a
technique based on the
hybridization of oligonucleotide
probe to target DNA.
– In this case, DNA is transferred
directly from the petri dish to the
filter, which is then incubated with
labeled probes.

58. Application of genomic library
 Genomic library construction is the first steps in any DNA sequencing
projects.
 Genomic library helps in identification of the novel pharmaceutically
important genes.
 Genomic library helps in identification of new genes which were silent in
the host.
 It helps us in understanding the complexity of genomes.

59. Serving as a source of genomic sequence for generation of
transgenic animals through genetic engineering.
Study of the function regulatory sequences invitro.
Study of genetic mutations in cancer tissues.
Creates cDNA libraries to determine what genes are being
expressed at a particular time.

60. What is cDNA???
– cDNA a means the complementary DNA or copy DNA.
– According to the central dogma of the molecular biology DNA is
transcribed into mRNA.
– mRNA gets translated to produce protein.
– Therefore , the flow the biological information is the from DNA to
RNA to protein.

61. cDNA Is Reverse Transcribed from mRNA
mature mRNA
poly A tail
5’ 3’
TTTTReverse
transcription
CCC
3’ 5’
3’
5’ 3’GGG
DNA polymerase
RNA hydrolysis
5’
3’ 5’

62. What is cDNA library???
– cDNA library is a population of bacterial transformants or phage
lysates in which each mRNA isolated insertion in a plasmid or a
phase vector.
– The frequency of a specific cDNA in such a library would ordinarily
depend on the frequency of the concerned mRNA in the tissue or
organism.

63. Intron and Exon in Eukaryotic Cells
mRNA
DNA
5’ 3’
cap
poly A
tail
exon exonexon
intron intron
mature mRNA
Processing
Transcription
Splicing
promotor
3’ 5’
Take place in nucleus
start codon stop codon
To cytoplasm
Intron deleted

64. CONSTRUCTION OF cDNA
LIBRARY
 Isolation of mRNA.
 Preparation of cDNA.
 Cloning of cDNAs.

65. Isolation of mRNA
– Total mRNA is extracted from a suitable organism or tissue.
Several procedures listed below:
– Chromatography on poly –U sepharose or oligo- Tcellulose.
– Density gradient centrifugation.
– Precipitated polysomes and purified. E.g. maize :zein(seed
storage protein)
– mRNA preparations from specific tissue. E.g.seed storages
protein genes in developing seeds, globin gene in erthrocytes ,
insulin gene in cells of pancreas, etc

66. Preparation of cDNA
– When eukaryotic mRNA is used as a template, a poly-T
oligonuceotide (oligodeoxynuclotide) is used as the primers
since these mRNAs have a poly a poly-A tail at their 3’ ends.
– Special tricks are required to utilize primers for other RNAs.
E.g. prokaryotic RNA , rRNA, RNA virus genome etc.
– In such cases poly-A tail is added to 3’ end to make it analogus
to eukaryotic mRNA and reaction catalyzed by enzyme poly-A
polymerase.
– Appropriate oligonucleotide primers is annealed with mRNA.

67. – Reverse transcriptase extend the 3’ end of the primers using mRNA
molecules as a templete.
– Produces RNA –DNA hybrid molecules.
– The DNA strand of this hybrid is the DNA a copy of the mRNA.
– The RNA strand is digested by either Rnase or alkaline hydrolysis this
frees the single stranded cDNA.
– This cDNA end serves as its own primer and provides the free 3’OH
required for synthesis of complementary strand.
– Synthesis of complementary strand is either by reverse transcriptase or
by E.coli DNA polymerase.

68. cDNA construction

69. Cloning of cDNAs
– cDNA are cloned by phage insertion vectors.
– 105- 106 cDNA clones are sufficient for the isolation
– It is advisable to enrich the mRNA preparation before library
construction.
– Size fractionation coupled with testing the various fractions for the
presence of desired mRNAs , .e.g. by injecting mRNAs into xenopus
oocytes and assaying for the protein product of the concerned mRNA.

70. cDNA Library
mRNA
Reverse transcription
Genes in expression
Complete gene
Smaller
Library
Vector: Plasmid
cDNA

72. Problems in cDNA preparation
 Incomplete copying of the mRNA by reverse transcriptase(5’ end)
mRNA missing from cDNA.
 Incomplete copying of the cDNA single strand so that the 3’end of
mRNA will be missing from cDNA single strand so that the 3’end of
mRNA will be missing cDNA.
 The nuclease used for cleaving the hairpin loop may also nibble away
the end of the duplex.

73. Applications in cDNA libraries
– cDNA libraries are commonly used when reproducing
eukaryotic genomes, as the amount of information is reduced to
remove the large numbers of non-coding regions from the
library.
– cDNA libraries are used to express eukaryotic genes in
prokaryotes. Prokaryotes do not have introns in their DNA and
therefore do not possess any enzymes that can cut it out during
transcription process. cDNA do not have introns and therefore
can be expressed in prokaryotic cells.

74. – cDNA libraries are most useful in reverse genetics where the
additional genomic information is of less use.
– Also, it is useful for subsequently isolating the gene that codes for
that mRNA.
– Discovery of novel genes.
– Cloning of full length cDNA molecules for in vitro study of gene
function.
– Study of the repertoire if mRNAs expressed in different cells or
tissues.
– Study of alternative splicing in different cells or tissues .

75. Sr.
no
Genomic library cDNA library
1. It includes all possible fragments of
DNA from a given cell or organism.
cDNA library carries only expressed gene
sequences.
2. It is larger It is smaller
3. It represents the entire genome of
an organism having both coding and
non coding regions.
It represents only the expressed part of
the genome and contain only coding
sequences called ESTs(Expressed sequence
tags)
4. Expression of genes taken from
genomic library is difficult in
prokaryotic systems like bacteria
due to absence of splicing
mechanism.
cDNA has only coding or exon sequences
therefore can be directly expressed in
prokaryotic system.
5. Vector used genomic library include
plasmid, cosmid, lambda phage, BAC
and YAC in order to accommodate
large fragments.
Vector used cDNA library includes plasmid,
phagemids, lambda phages etc to
accommodate small fragment as cDNA has
no introns.
6. Uses of genomic library Uses of cDNA library