2. • In 1973, Herbert Boyer and Stanley Cohen
successfully created the first recombinant
DNA molecule. Their research work
proved that it is possible to change the
genotype of organisms artificially. In 1977,
Walter Gilbert and Frederick Sanger
worked out methods to determine the
sequence of bases in DNA. All of these
progresses make it possible to isolate and
identify a gene, and induce the gene in
host cells to express functional protein.
3. • The genetic endowment of organisms can
now be precisely changed in designed
ways. The development of recombinant
DNA techniques has revolutionized
biology and is having an increasing impact
on clinical medicine. It offers a rational
approach to understanding the molecular
basis of a number of diseases, for
example, new insights are emerging into
the regulation of gene expression in
cancer.
4. • Clinically useful proteins are now
produced by recombinant DNA
techniques. This powerful technique is
also applied in diagnosis or therapy. And it
paves the way to the modern fields of
genomics and proteomics. The new
opportunities opened by recombinant DNA
technology promise to have broad effects.
6. • A clone is a large population of identical
molecules, cells or organism that is
descended from a single individual through
asexual reproduction and genetically
identical to a single common ancestor. A
DNA clone is a large number of identical
DNA molecules produced from a single
DNA molecule through an asexual process.
Molecular clone in the genetics sense
refers to DNA clone.
• 1.1 DNA clone
7. 1.1.1 DNA Cloning
• Cloning is the process of asexually
creating identical copies of an original.
DNA cloning(molecular cloning) refers to
the production of large number of identical
DNA molecules and usually involves the
use of bacterial cells as host for the DNA.
8. • DNA cloning involves the preparation of a
specific gene or DNA segment from a
larger chromosome, insertion of it to a
carrier DNA, and then introduction of the
modified DNA to host cells, selection of
the transformants containing the target
genes. The result is selective amplification
of a particular gene or DNA segment. So
DNA cloning is also called gene cloning or
DNA recombination.
9. • The methods used to accomplish these
and related tasks are collectively referred
to as recombinant DNA technology or,
more informally, genetic engineering.
Genetic engineering, proteomic
engineering, enzymatic engineering, and
cellular engineering constitute
biotechnology.
10. 1.2 Different enzymes are used in
DNA recombination
• The discovery of two types of enzymes
permitted the now common technique of
DNA cloning. One type of enzymes, called
restriction enzymes, cuts the DNA from
any organism at specific sequences of a
few nucleotides, generating a reproducible
set of fragments.
11. • The other type of enzymes , called DNA
ligases, can ligate DNA restriction
fragments, producing recombinant DNA.
In different situations, some other
enzymes may also be needed for DNA
recombination (Table 6-1).
12. Enzyme( s) Function
restriction endonucleases recognize specific nucleotide sequences and cleaves
the DNA within or near to the recognition sequences
DNA ligase covalently attaches a free 5'-phosphate group to a 3'-
hydroxyl group
DNA polymerase I synthesizes DNA
klenow DNA polymerase proteolytic fragment of DNA polymerase that lacks
the 5'-3' exonuclease activity
reverse transcriptase CRT) synthesizeds DNA with RNA as template
alkaline phosphatase removes phosphates from 5'ends of DNA molecules
terminal transferase catalyzes the attachment of any dNMP to the 3'end
of DNA
Table 6-1 Common enzymes used in recombinant DNA technology
13. 1.2.1 Restriction enzymes
• Restriction enzymes, also called restriction
endonucleases, recognize, bind to
specific sequences in double-stranded
DNA, and cleave the DNA. They are
usually isolated from bacteria. The role of
these enzymes in bacteria is to "restrict"
the invasion of foreign DNA by cutting it
into pieces.
14. • Hence, these enzymes are known as
restriction enzymes. The cell's own DNA is
not degraded, because the sites
recognized by its own restriction enzymes
are methylated. Many restriction enzymes
have been purified and characterized.
15. • The names of restriction enzymes consist
of a three-italic-letter abbreviation for the
host organism. For example, restriction
enzyme EcoR is from Escherichia coli.Ⅰ
The first three letters in the name of the
enzyme consist of the first letter of the
genus (E) and the first two letters of the
species (co), which are followed by a
strain designation (R) and a roman
numeral ( )to indicate the order ofⅠ
discovery.
16. • There are three types of restriction enzymes,
designated , ,andIII. Types and containⅠ Ⅱ Ⅰ Ⅲ
the activities of both the endonuclease and
methylase. Type restriction enzymes cleaveⅠ
DNA at random sites. Type restrictionⅢ
enzymes cleave the DNA about 25 bp from
the recognition sequence. Both types of
enzymes require ATP for energy supply.
17. • Type restriction enzymes, require noⅡ
ATP, and usually cleave the DNA within
the recognition sequence itself. So typeⅡ
restriction enzymes have extraordinary
utility in DNA recombination.
18. • Many type restriction enzymesⅡ
recognize specific sequences of 4 to 6
base pairs and cleave a phosphodiester
bond in each strand in this region. One
unique feature of restriction enzymes is
that the nucleotide sequences they
recognize are palindromic, or inverted
repeats. It cuts one strand of the DNA
double helix at one point and the second
strand at a different, complementary point.
19. • For example, the sequence recognized by
a restriction enzyme EcoR is GAATTC.Ⅰ
In each strand, the enzyme cleaves the
GA phosphodiester bond on the 5' side of
the symmetric axis. The arrow indicates
the cleavage site.
20. If the cleavage site is not at the center, the
restriction enzyme ( e.g., EcoR ) will generateⅠ
cohesive ends(sticky ends), which can base-
pair with other DNA fragments cleaved by the
same restriction enzyme. If the cleavage site
is at the center, the restriction enzyme (e.g.,
Hpa ) will generate blunt endsⅠ
21. • Any two pieces of DNA containing the
same sequences within their sticky ends
can anneal together and be covalently
ligated together in the presence of DNA
ligase. Any two blunt-ended fragments of
DNA can be ligated together irrespective of
the sequences at the ends of the duplexes.
22. Table 6-2 Commonly used restriction enzymes
The specificities of several of these enzymes
are shown in Table 6-2.
23. 1.3 The interested gene
• The purpose of recombinant DNA
technology is to clone a gene or DNA
fragment of interest or to obtain the
product of the gene-protein. The
interested gene in DNA recombination is
the gene or a segment of a gene to be
studied, or to be cloned, or to be
expressed in recipient cells. The
interested gene to be prepared could be
either cDNA or genomic DNA.
24. • cDNA is the single strand DNA
complementary to an mRNA, or double-
stranded DNA with one strand
complementary to an mRNA.
• Genomic DNA refers to any DNA fragment
coming from the genome of a cell or an
organism.
• In the process of DNA cloning, recombinant
DNA is constructed by ligating a vector with a
cDNA or genomic DNA, which is what we are
interested in. Any interested gene can be
cloned once it is introduced into a suitable
vector for transforming a bacterial host.
25. 1.4 Vectors
• Molecular cloning involves the amplification
of a given DNA molecule by replication in a
host cell. However, the DNA fragment to be
amplified does not have an origin of
replication, it must be inserted into a carrier
which can be replicated in bacterial cells
and passed to subsequent generations of
the bacteria.
26. The ideal cloning vectors display the
following properties:
• (1) they are not integrated into the host
genome so that they are easily isolated
and purified;
• (2) it is easy for them to enter into host
cells because of their small size;
27. • (3)they contain an origin of replication that
allows them to be replicated independently
in host cells;
• (4) they contain one or more selectable
markers that allow the easy identification
of the host cells harboring them;
• ( 5)they have single restriction sites at
which foreign DNA can be inserted.
28. • Such a carrier, termed “vector”, is usually
a DNA molecule which has a suitable
origin of replication. If a vector is used for
amplification of a DNA fragment, it is
called "cloning vector". If a vector is used
for the expression of a given gene, it is
called "expression vector". The vectors
used for cloning are derived from naturally
occurring bacterial plasmid and
bacteriophage.
29. • Plasmids are autonomous
extrachromosomal replicons found
commonly in prokaryotes. They are circular
duplex DNA molecules occurring naturally in
some bacteria and ranging in size from 1 to
300kb.
1.4.1 Plasmids
30. • They carry genes for antibiotics-
resistance, and can be replicated
independently in host cells. Any DNA
fragment can be inserted into a plasmid at
one or two given restriction sites, and
replicated along with the replication of the
plasmid.
31. • Many plasmids have been ingeniously
modified to enhance the delivery of
recombinant DNA molecules into bacteria
and to facilitate the selection of bacteria
harboring these vectors. Most plasmid
vectors in current use have a multiple
cloning site, a cluster of unique restriction
sites which provide a convenient position
for the introduction of donor DNA prepared
by using a variety of enzymes.
32. • One of the most useful plasmids for
cloning is pBR322 (Figure 6-1), which
contains genes for resistance to
tetracycline and ampicillin. Different
restriction enzymes can cleave this
plasmid at a variety of unique sites, at
which DNA fragments can be inserted.
34. • Transformation becomes less successful
as plasmid size increases, and it is difficult
to clone DNA segments longer than about
15kb when plasmids are used as the
vector.
35. • Another group of plasmids in common use
is pUC series(Figure 6-2), which contain a
multiple cloning sites recognized by
several different restriction enzymes, an
ampicillin-resistance gene (ampr
)for
selective amplification, and a replication
origin ( ori ) for replication in the host cell.
37. • Bacteriophage λvectors are used for DNA
library construction because they have a
greater capacity than plasmids. The
middle segment of a phage DNA is not
essential for productive infection and can
be replaced with foreign DNA, thus λ
phages designed for cloning have been
constructed.
38. • M13 phage is another very useful vector
for cloning DNA. It is especially useful for
sequencing the inserted DNA. That is can
be easily sequenced.
39. 2. Basic Principles Of DNA
Recombination
The basic process of DNA recombination
includes several major steps:
• (1) preparation of the DNA fragment of the
interested gene;
• (2)selection and preparation of a suitable
vector;
40. • (3) ligation of DNA fragment with vector;
• (4) transformation of host cells with
recombinant DNA;
• (5)screening the host cells harboring the
recombinant DNA;
• (6) identification of recombinant DNA.
Figure 6-3 illustrates the process of DNA
cloning using plasmid as vector.
41. Figure 6-3 The process of DNA cloning using plasmid as vector
42. 2.1 Preparation of the interested gene
• The fragments of the interested gene for
DNA recombination include genomic DNA,
cDNA, PCR product and chemically
synthesized DNA fragment. Different
strategies should be chosen for the
preparation of DNA fragments of the
interested gene according to different
purposes.
43. • If the sequence of the DNA fragment to be
cloned is known, at least the flanking
parts, the DNA fragment can be prepared
using the polymerase chain reaction
(PCR). The amplified DNA fragment can
be cloned directly.
44. 2.2 Selection and preparation of vector
• The selection of vector is dependent on
the purpose of the construction of
recombinant DNA. For the cloning of DNA
fragment, a cloning vector should be
chosen. For the expression of a gene in
the given host cell, a suitable expression
vector should be chosen according to the
host cell. Many expression vectors have
constructed for the expression of gene in
different cells such as mammalian cells ,
bacterial cells, yeast and so on.
45. 2.3 Ligation of DNA molecules
• After digestion with restriction enzyme(s) ,
different DNA fragments(including
linearized vector) can be ligated by DNA
ligase which fills in the nicks by catalyzing
the formation of the phosphodiester bonds.
Composite DNA molecules comprising
covalently linked segments from two or
more sources are called recombinant
DNAs.
46. • The ligation of DNA fragment with sticky
ends is facilitated by the annealing of
complementary sticky ends. The fragments
digested by the same restriction enzymes
generally will link together through base-
pairing so that it is very easy for DNA ligase
to fill the nicks.
47. • DNA fragments with same sticky
ends(compatible ends) are ligated in this
way, even if they are digested by different
restriction enzymes. For example, a
fragment generated by Mbo (▼ GATC)Ⅰ
will link to a fragment generated by BamH
(G▼ GATCC).Ⅰ
49. • The simplest strategy for cloning is thus to
cut the donor and vector DNA with the
same restriction enzyme and join them
with DNA ligase. However, this allows the
vector to reclose without an insert. There
are one procedures which prevent vector
self-ligation:
50. • the vector and donor can be prepared by
digestion with same pair of restriction
enzymes so that both vector and donor
themself will have two incompatible ends,
but compatible ends are existent between
vector and donor. A further advantage of
the second strategy is that the orientation
of the insert can be predicted (directional
cloning).
51. • Sticky-end ligation is technically easy, but
sticky-end sites may not be available. To
circumvent these problems, the strategy of
ligation with blunt ends is used. This
technique has the advantage of joining
together any pair of DNA fragments
without suitable restriction sites, albeit less
efficiently and nondirectionally.
52. • Blunt ends can be generated by filling or
trimming overhanging termini. The
disadvantages are that there is no control
over the orientation of insertion or the
number of molecules annealed together ,
and there is no easy way to retrieve the
insert.
53. 2.4 Introduction of recombinant DNA into
host cells
• The recombinant DNA molecules are then
introduced into host cells, which amplify
the DNA molecules in the course of many
generations of cell division. Plasmids can
be introduced into bacterial cells by a
process called transformation. Bacterial
cells are first treated with calcium chloride(
CaCI2 )to make the cell wall more
permeable to DNA. These cells are said to
be competent, and some of them can take
up DNA-Ca2+
complex.
54. 2.5 Screening and identification
• Neither DNA manipulation nor gene
transfer procedures are 100 efficient, so it﹪
is desirable to identify the recombinant
population. A method is needed to select
those that do. This process is termed
screening or selection. There are direct
and indirect selection systems employed
depending on the vector.
55. • The gene or genotype can be detected
directly based on some mark genes and
target gene. The usual strategy is to use a
plasmid that includes a gene that the host
cell requires for growth under specific
conditions, such as a gene that confers
resistance to an antibiotic.
56. • Only cells transformed by the plasmid can
grow in the presence of antibiotic. Cloning
vector often contains genes that confer
resistance to different antibiotics(tetr
, ampr
or kanr
), allowing the identification of cells
that contain the intact plasmid or a
recombinant version of the plasmid
because those bacteria with the antibiotic
resistance gene will survive, whereas
those without it will die.
antibiotics: a chemical substance derivable from a mold or bacterium
that kills microorganisms and cures infections
57. • Ideally, cloning sites are placed within a
selectable or visible marker gene to
facilitate recombinant selection. The
insertion of DNA into a functional region of
the vector will interrupt an essential
function of the vector. This concept
provides a selection technique of
insertional inactivation.
58. • For example, the common plasmid vector
pBR322 has both tetr
and ampr
genes.
Insertion of DNA at the Hind ,Ⅲ Sal , orⅠ
BamH restriction site inactivates theⅠ tetr
gene. Cells containing pBR322 with a
DNA insert at one of these restriction
sites are resistant to ampicillin but
sensitive to tetracycline. Cells that failed to
take up the vector are sensitive to both
antibiotics, whereas cells containing
pBR322 without a DNA insert are resistant
to both. And so they can be readily
selected.
59. • Specific DNA sequences are detectable
by marker rescue. Insertional inactivation
can therefore be used to distinguish
clones of plasmid that contain an insert. A
similar strategy is blue-white selection.
Plasmids carry a nonfunctional, truncated
allele of the LacZ gene, which encodes a
N-terminal fragment of the β-
galactosidase protein termed the α
peptide.
60. • This can be complemented by an allele
encoding the remainder of the
polypeptide, which is found in specially
modified host strains such as E.coli
JM101. Functionalβ-galactosidase
converts the colorless substrate X-gal into
a blue precipitate. The lacZ gene contains
multiple restriction site. Therefore
recombinant cells form white colonies and
non-recombinants form blue colonies on
the appropriate detection media.
62. • Specific DNA sequences are detectable
by PCR. If the sequence of the cloned
DNA is known, the primers can be
designed. Then the PCR product will
ensure the foreign DNA in the
transformants.
63. • Specific DNA sequences are detectable by
hybridization. Cells containing particular
DNA sequences can be identified by DNA
hybridization. There are many variations of
the basic method, most making use of a
labeled probe, complementary to the DNA
being sought. In one classic approach to
detect a particular DNA sequence within a
DNA library, nitrocellulose membrane is
pressed onto an agar plate containing
many individual bacterial colonies from the
library.
64. • The membrane is treated with alkali to
disrupt the cells and denature the DNA
which remains bound to the region of the
membrane around the colony from which it
comes. The radioactive DNA probe
anneals only to its complementary DNA.
After any unannealed probe DNA is
washed away, the hybridized DNA can be
detected by autoradiography (Figure 6-5).
66. Figure 6-5 Use of hybridization to identify a clone with a particular DNA segment
67. • Specific DNA sequences are identified by
DNA sequencing. The ultimate
characterization of a cloned DNA is to
determine its nucleotide sequence.
Transformants can be identified by
sequencing until a bacterial colony
containing a plasmid with the foreign gene
is found.
68. Drug Resistance Gene Transferred by Plasmid
Plasmid gets out and
into the host cell
Resistant Strain
New Resistance Strain
Non-resistant Strain
Plasmid
Enzyme
Hydrolyzing
Antibiotics
Drug Resistant Gene
mRNA
Juang RH (2004) BCbasics