SlideShare une entreprise Scribd logo
1  sur  71
Télécharger pour lire hors ligne
Chapter 6
Genetic Recombination and
Genetic Engineering
• 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.
• 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.
• 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.
1. Concepts Involved In DNA
Recombination
• 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
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.
• 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.
• 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.
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.
• 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).
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
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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
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
• 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.
Table 6-2 Commonly used restriction enzymes
The specificities of several of these enzymes
are shown in Table 6-2.
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.
• 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.
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.
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;
• (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.
• 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.
• 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
• 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.
• 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.
• 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.
Figure 6-1 Plasmid pBR322
• 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.
• 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.
Figure 6-2 Plasmid pUC18
• 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.
• 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.
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;
• (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.
Figure 6-3 The process of DNA cloning using plasmid as vector
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.
• 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.
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.
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.
• 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.
• 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
Genetic recombination and genetic engineering
• 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:
• 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).
• 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.
• 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.
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.
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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
Genetic recombination and genetic engineering
• 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.
• 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.
• 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).
Genetic recombination and genetic engineering
Figure 6-5 Use of hybridization to identify a clone with a particular DNA segment
• 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.
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
Target Genes Carried by Plasmid
1 plasmid
1 cellRecombinant
Plasmid
Transformation
Target Gene
Recombination
Restriction
Enzyme
Restriction
Enzyme
ChromosomalDNA
Target Genes
DNA Recombination
Transformation
Host Cells
Juang RH (2004) BCbasics
Amplification and Screening of Target Gene
1
1 cell line, 1 colony
X100
X1,000
Plasmid
DuplicationBacteria
Duplication
Plating
Pick the colony
containing target gene
=100,000 Juang RH (2004) BCbasics
www.themegallery.com
Your company slogan in here
LOGO

Contenu connexe

Tendances

Tendances (20)

Cloning vector
Cloning vectorCloning vector
Cloning vector
 
PHAGEMID.pdf
PHAGEMID.pdfPHAGEMID.pdf
PHAGEMID.pdf
 
Molecular markers
Molecular markersMolecular markers
Molecular markers
 
Vector construction
Vector constructionVector construction
Vector construction
 
Tetrad analysis by rk
Tetrad analysis by rkTetrad analysis by rk
Tetrad analysis by rk
 
Transposons in drosophila - P element
Transposons in drosophila - P elementTransposons in drosophila - P element
Transposons in drosophila - P element
 
Genomic and c dna library
Genomic and c dna libraryGenomic and c dna library
Genomic and c dna library
 
Prokaryotic genome organization
Prokaryotic genome organizationProkaryotic genome organization
Prokaryotic genome organization
 
Cosmids pagemids
Cosmids pagemidsCosmids pagemids
Cosmids pagemids
 
Recombination
RecombinationRecombination
Recombination
 
Restriction Mapping
Restriction MappingRestriction Mapping
Restriction Mapping
 
C dna
C dnaC dna
C dna
 
Mammalian artificial chromosome
Mammalian artificial chromosomeMammalian artificial chromosome
Mammalian artificial chromosome
 
cDNA Library Construction
cDNA Library ConstructioncDNA Library Construction
cDNA Library Construction
 
Molecular markers
Molecular markersMolecular markers
Molecular markers
 
Agrobacterium mediated gene transfer in plants.
Agrobacterium mediated gene transfer in plants.Agrobacterium mediated gene transfer in plants.
Agrobacterium mediated gene transfer in plants.
 
Construction of gene library
Construction of gene libraryConstruction of gene library
Construction of gene library
 
Gene Silencing by Histone Modification
Gene Silencing by Histone ModificationGene Silencing by Histone Modification
Gene Silencing by Histone Modification
 
Genome mapping
Genome mappingGenome mapping
Genome mapping
 
Nucleases
NucleasesNucleases
Nucleases
 

En vedette

En vedette (20)

Genetic recombination
Genetic recombinationGenetic recombination
Genetic recombination
 
Genetic recombination mechanism
Genetic recombination mechanismGenetic recombination mechanism
Genetic recombination mechanism
 
Bacterial recombination (1)
Bacterial recombination (1)Bacterial recombination (1)
Bacterial recombination (1)
 
Recombination
RecombinationRecombination
Recombination
 
Dna recombination mechanisms new
Dna recombination mechanisms newDna recombination mechanisms new
Dna recombination mechanisms new
 
Genetic engineering project
Genetic engineering projectGenetic engineering project
Genetic engineering project
 
Homologous Recombination (HR)
Homologous Recombination (HR)Homologous Recombination (HR)
Homologous Recombination (HR)
 
Cloning
CloningCloning
Cloning
 
Genetic engineering and recombinant DNA technology
Genetic engineering and recombinant DNA  technologyGenetic engineering and recombinant DNA  technology
Genetic engineering and recombinant DNA technology
 
Enzymes used in Genetic Engineering
Enzymes used in Genetic EngineeringEnzymes used in Genetic Engineering
Enzymes used in Genetic Engineering
 
Genetic engineering
Genetic engineeringGenetic engineering
Genetic engineering
 
Gene cloning
Gene cloningGene cloning
Gene cloning
 
Session ii g2 overview chemical modeling mmc
Session ii g2 overview chemical modeling mmcSession ii g2 overview chemical modeling mmc
Session ii g2 overview chemical modeling mmc
 
6872125 physics-of-semiconductor-devices (1)
6872125 physics-of-semiconductor-devices (1)6872125 physics-of-semiconductor-devices (1)
6872125 physics-of-semiconductor-devices (1)
 
Semiconductor
SemiconductorSemiconductor
Semiconductor
 
Silicon in Semiconductor devices
Silicon in Semiconductor devices Silicon in Semiconductor devices
Silicon in Semiconductor devices
 
The process of genetic engineering in detail
The process of genetic engineering in detailThe process of genetic engineering in detail
The process of genetic engineering in detail
 
GMO Food and I-522 Labeling
GMO Food and I-522 LabelingGMO Food and I-522 Labeling
GMO Food and I-522 Labeling
 
Genetic engineering
Genetic engineeringGenetic engineering
Genetic engineering
 
Restriction enzymes
Restriction enzymesRestriction enzymes
Restriction enzymes
 

Similaire à Genetic recombination and genetic engineering

Recombinant DNA Technology- Part 1.pdf
Recombinant DNA Technology- Part 1.pdfRecombinant DNA Technology- Part 1.pdf
Recombinant DNA Technology- Part 1.pdfNamrata Chhabra
 
Recombinant DNA technology
Recombinant DNA technologyRecombinant DNA technology
Recombinant DNA technologyArfaShazad
 
Recombinant dna technology 2017 cloning and pcr
Recombinant dna technology 2017 cloning and pcrRecombinant dna technology 2017 cloning and pcr
Recombinant dna technology 2017 cloning and pcrLama K Banna
 
Restriction Digestion and its Applications
Restriction Digestion and its ApplicationsRestriction Digestion and its Applications
Restriction Digestion and its ApplicationsASHIKH SEETHY
 
Enzymes involved in rDNA technology.pptx
Enzymes involved in rDNA technology.pptxEnzymes involved in rDNA technology.pptx
Enzymes involved in rDNA technology.pptxPoonam Patil
 
16. A. RECOMBINANT DNA.pptx
16. A. RECOMBINANT DNA.pptx16. A. RECOMBINANT DNA.pptx
16. A. RECOMBINANT DNA.pptxKiranChoudhari6
 
DNA CLONING
DNA CLONINGDNA CLONING
DNA CLONINGastro60
 
Dna cloning
Dna cloningDna cloning
Dna cloningastro60
 
Dna cloning
Dna cloningDna cloning
Dna cloningastro60
 
Recombinant dna technology
Recombinant dna technology Recombinant dna technology
Recombinant dna technology utsav parmar
 
Gene cloning lecture notes 5 for 2010
Gene cloning lecture notes 5 for 2010Gene cloning lecture notes 5 for 2010
Gene cloning lecture notes 5 for 2010Shadrach Meynscer
 
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCRB.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCRRai University
 
Lecture 1 molecular tech. rdt 11 50-2020
Lecture 1 molecular tech. rdt 11 50-2020 Lecture 1 molecular tech. rdt 11 50-2020
Lecture 1 molecular tech. rdt 11 50-2020 Dr Vishnu Kumar
 
Lecture 1 molecular tech. RDT By Dr Vishnu Kumar Professor, Biochemistry
Lecture 1 molecular tech. RDT  By Dr Vishnu Kumar Professor, BiochemistryLecture 1 molecular tech. RDT  By Dr Vishnu Kumar Professor, Biochemistry
Lecture 1 molecular tech. RDT By Dr Vishnu Kumar Professor, BiochemistryDr Vishnu Kumar
 
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCRB.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCRRai University
 

Similaire à Genetic recombination and genetic engineering (20)

Recombinant DNA Technology- Part 1.pdf
Recombinant DNA Technology- Part 1.pdfRecombinant DNA Technology- Part 1.pdf
Recombinant DNA Technology- Part 1.pdf
 
Recombinant DNA technology
Recombinant DNA technologyRecombinant DNA technology
Recombinant DNA technology
 
Recombinant dna technology 2017 cloning and pcr
Recombinant dna technology 2017 cloning and pcrRecombinant dna technology 2017 cloning and pcr
Recombinant dna technology 2017 cloning and pcr
 
Recombinant DNA Technology
Recombinant DNA TechnologyRecombinant DNA Technology
Recombinant DNA Technology
 
Restriction Digestion and its Applications
Restriction Digestion and its ApplicationsRestriction Digestion and its Applications
Restriction Digestion and its Applications
 
gene cloning principles an technique
gene cloning principles an techniquegene cloning principles an technique
gene cloning principles an technique
 
Enzymes involved in rDNA technology.pptx
Enzymes involved in rDNA technology.pptxEnzymes involved in rDNA technology.pptx
Enzymes involved in rDNA technology.pptx
 
16. A. RECOMBINANT DNA.pptx
16. A. RECOMBINANT DNA.pptx16. A. RECOMBINANT DNA.pptx
16. A. RECOMBINANT DNA.pptx
 
DNA CLONING
DNA CLONINGDNA CLONING
DNA CLONING
 
Dna cloning
Dna cloningDna cloning
Dna cloning
 
Dna cloning
Dna cloningDna cloning
Dna cloning
 
Recombinant dna technology
Recombinant dna technology Recombinant dna technology
Recombinant dna technology
 
Dna cloning
Dna cloningDna cloning
Dna cloning
 
Gene cloning lecture notes 5 for 2010
Gene cloning lecture notes 5 for 2010Gene cloning lecture notes 5 for 2010
Gene cloning lecture notes 5 for 2010
 
Recombinant dna
Recombinant dnaRecombinant dna
Recombinant dna
 
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCRB.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT & PCR
 
Lecture 1 molecular tech. rdt 11 50-2020
Lecture 1 molecular tech. rdt 11 50-2020 Lecture 1 molecular tech. rdt 11 50-2020
Lecture 1 molecular tech. rdt 11 50-2020
 
Lecture 1 molecular tech. RDT By Dr Vishnu Kumar Professor, Biochemistry
Lecture 1 molecular tech. RDT  By Dr Vishnu Kumar Professor, BiochemistryLecture 1 molecular tech. RDT  By Dr Vishnu Kumar Professor, Biochemistry
Lecture 1 molecular tech. RDT By Dr Vishnu Kumar Professor, Biochemistry
 
5 Recombinant DNA Technology.ppt
5  Recombinant DNA Technology.ppt5  Recombinant DNA Technology.ppt
5 Recombinant DNA Technology.ppt
 
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCRB.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCR
B.Tech Biotechnology II Elements of Biotechnology Unit 3 RDT and PCR
 

Plus de shobejee

Pmdc forensic
Pmdc forensicPmdc forensic
Pmdc forensicshobejee
 
Cs101lec01 100130102405-phpapp02
Cs101lec01 100130102405-phpapp02Cs101lec01 100130102405-phpapp02
Cs101lec01 100130102405-phpapp02shobejee
 
Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02
Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02
Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02shobejee
 
Molecular hybridization of nucleic acids
Molecular hybridization of nucleic acidsMolecular hybridization of nucleic acids
Molecular hybridization of nucleic acidsshobejee
 
Chapter1 cell structure of bacteria
Chapter1 cell structure of bacteriaChapter1 cell structure of bacteria
Chapter1 cell structure of bacteriashobejee
 
145 527-1-pb
145 527-1-pb145 527-1-pb
145 527-1-pbshobejee
 

Plus de shobejee (7)

Pmdc forensic
Pmdc forensicPmdc forensic
Pmdc forensic
 
Cs101lec01 100130102405-phpapp02
Cs101lec01 100130102405-phpapp02Cs101lec01 100130102405-phpapp02
Cs101lec01 100130102405-phpapp02
 
Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02
Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02
Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02
 
Molecular hybridization of nucleic acids
Molecular hybridization of nucleic acidsMolecular hybridization of nucleic acids
Molecular hybridization of nucleic acids
 
Chapter1 cell structure of bacteria
Chapter1 cell structure of bacteriaChapter1 cell structure of bacteria
Chapter1 cell structure of bacteria
 
Marketing
MarketingMarketing
Marketing
 
145 527-1-pb
145 527-1-pb145 527-1-pb
145 527-1-pb
 

Dernier

Machine Learning Model Validation (Aijun Zhang 2024).pdf
Machine Learning Model Validation (Aijun Zhang 2024).pdfMachine Learning Model Validation (Aijun Zhang 2024).pdf
Machine Learning Model Validation (Aijun Zhang 2024).pdfAijun Zhang
 
9 Steps For Building Winning Founding Team
9 Steps For Building Winning Founding Team9 Steps For Building Winning Founding Team
9 Steps For Building Winning Founding TeamAdam Moalla
 
Designing A Time bound resource download URL
Designing A Time bound resource download URLDesigning A Time bound resource download URL
Designing A Time bound resource download URLRuncy Oommen
 
Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...
Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...
Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...Will Schroeder
 
UiPath Solutions Management Preview - Northern CA Chapter - March 22.pdf
UiPath Solutions Management Preview - Northern CA Chapter - March 22.pdfUiPath Solutions Management Preview - Northern CA Chapter - March 22.pdf
UiPath Solutions Management Preview - Northern CA Chapter - March 22.pdfDianaGray10
 
20230202 - Introduction to tis-py
20230202 - Introduction to tis-py20230202 - Introduction to tis-py
20230202 - Introduction to tis-pyJamie (Taka) Wang
 
UiPath Studio Web workshop series - Day 8
UiPath Studio Web workshop series - Day 8UiPath Studio Web workshop series - Day 8
UiPath Studio Web workshop series - Day 8DianaGray10
 
OpenShift Commons Paris - Choose Your Own Observability Adventure
OpenShift Commons Paris - Choose Your Own Observability AdventureOpenShift Commons Paris - Choose Your Own Observability Adventure
OpenShift Commons Paris - Choose Your Own Observability AdventureEric D. Schabell
 
Empowering Africa's Next Generation: The AI Leadership Blueprint
Empowering Africa's Next Generation: The AI Leadership BlueprintEmpowering Africa's Next Generation: The AI Leadership Blueprint
Empowering Africa's Next Generation: The AI Leadership BlueprintMahmoud Rabie
 
KubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCost
KubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCostKubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCost
KubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCostMatt Ray
 
Nanopower In Semiconductor Industry.pdf
Nanopower  In Semiconductor Industry.pdfNanopower  In Semiconductor Industry.pdf
Nanopower In Semiconductor Industry.pdfPedro Manuel
 
Linked Data in Production: Moving Beyond Ontologies
Linked Data in Production: Moving Beyond OntologiesLinked Data in Production: Moving Beyond Ontologies
Linked Data in Production: Moving Beyond OntologiesDavid Newbury
 
The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...
The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...
The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...Aggregage
 
UiPath Studio Web workshop series - Day 7
UiPath Studio Web workshop series - Day 7UiPath Studio Web workshop series - Day 7
UiPath Studio Web workshop series - Day 7DianaGray10
 
Videogame localization & technology_ how to enhance the power of translation.pdf
Videogame localization & technology_ how to enhance the power of translation.pdfVideogame localization & technology_ how to enhance the power of translation.pdf
Videogame localization & technology_ how to enhance the power of translation.pdfinfogdgmi
 
Meet the new FSP 3000 M-Flex800™
Meet the new FSP 3000 M-Flex800™Meet the new FSP 3000 M-Flex800™
Meet the new FSP 3000 M-Flex800™Adtran
 
Salesforce Miami User Group Event - 1st Quarter 2024
Salesforce Miami User Group Event - 1st Quarter 2024Salesforce Miami User Group Event - 1st Quarter 2024
Salesforce Miami User Group Event - 1st Quarter 2024SkyPlanner
 
Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...
Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...
Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...DianaGray10
 

Dernier (20)

Machine Learning Model Validation (Aijun Zhang 2024).pdf
Machine Learning Model Validation (Aijun Zhang 2024).pdfMachine Learning Model Validation (Aijun Zhang 2024).pdf
Machine Learning Model Validation (Aijun Zhang 2024).pdf
 
9 Steps For Building Winning Founding Team
9 Steps For Building Winning Founding Team9 Steps For Building Winning Founding Team
9 Steps For Building Winning Founding Team
 
Designing A Time bound resource download URL
Designing A Time bound resource download URLDesigning A Time bound resource download URL
Designing A Time bound resource download URL
 
201610817 - edge part1
201610817 - edge part1201610817 - edge part1
201610817 - edge part1
 
Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...
Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...
Apres-Cyber - The Data Dilemma: Bridging Offensive Operations and Machine Lea...
 
UiPath Solutions Management Preview - Northern CA Chapter - March 22.pdf
UiPath Solutions Management Preview - Northern CA Chapter - March 22.pdfUiPath Solutions Management Preview - Northern CA Chapter - March 22.pdf
UiPath Solutions Management Preview - Northern CA Chapter - March 22.pdf
 
20230202 - Introduction to tis-py
20230202 - Introduction to tis-py20230202 - Introduction to tis-py
20230202 - Introduction to tis-py
 
UiPath Studio Web workshop series - Day 8
UiPath Studio Web workshop series - Day 8UiPath Studio Web workshop series - Day 8
UiPath Studio Web workshop series - Day 8
 
OpenShift Commons Paris - Choose Your Own Observability Adventure
OpenShift Commons Paris - Choose Your Own Observability AdventureOpenShift Commons Paris - Choose Your Own Observability Adventure
OpenShift Commons Paris - Choose Your Own Observability Adventure
 
Empowering Africa's Next Generation: The AI Leadership Blueprint
Empowering Africa's Next Generation: The AI Leadership BlueprintEmpowering Africa's Next Generation: The AI Leadership Blueprint
Empowering Africa's Next Generation: The AI Leadership Blueprint
 
KubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCost
KubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCostKubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCost
KubeConEU24-Monitoring Kubernetes and Cloud Spend with OpenCost
 
Nanopower In Semiconductor Industry.pdf
Nanopower  In Semiconductor Industry.pdfNanopower  In Semiconductor Industry.pdf
Nanopower In Semiconductor Industry.pdf
 
Linked Data in Production: Moving Beyond Ontologies
Linked Data in Production: Moving Beyond OntologiesLinked Data in Production: Moving Beyond Ontologies
Linked Data in Production: Moving Beyond Ontologies
 
The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...
The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...
The Data Metaverse: Unpacking the Roles, Use Cases, and Tech Trends in Data a...
 
UiPath Studio Web workshop series - Day 7
UiPath Studio Web workshop series - Day 7UiPath Studio Web workshop series - Day 7
UiPath Studio Web workshop series - Day 7
 
Videogame localization & technology_ how to enhance the power of translation.pdf
Videogame localization & technology_ how to enhance the power of translation.pdfVideogame localization & technology_ how to enhance the power of translation.pdf
Videogame localization & technology_ how to enhance the power of translation.pdf
 
Meet the new FSP 3000 M-Flex800™
Meet the new FSP 3000 M-Flex800™Meet the new FSP 3000 M-Flex800™
Meet the new FSP 3000 M-Flex800™
 
Salesforce Miami User Group Event - 1st Quarter 2024
Salesforce Miami User Group Event - 1st Quarter 2024Salesforce Miami User Group Event - 1st Quarter 2024
Salesforce Miami User Group Event - 1st Quarter 2024
 
Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...
Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...
Connector Corner: Extending LLM automation use cases with UiPath GenAI connec...
 
20150722 - AGV
20150722 - AGV20150722 - AGV
20150722 - AGV
 

Genetic recombination and genetic engineering

  • 1. Chapter 6 Genetic Recombination and Genetic Engineering
  • 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.
  • 5. 1. Concepts Involved In DNA Recombination
  • 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
  • 69. Target Genes Carried by Plasmid 1 plasmid 1 cellRecombinant Plasmid Transformation Target Gene Recombination Restriction Enzyme Restriction Enzyme ChromosomalDNA Target Genes DNA Recombination Transformation Host Cells Juang RH (2004) BCbasics
  • 70. Amplification and Screening of Target Gene 1 1 cell line, 1 colony X100 X1,000 Plasmid DuplicationBacteria Duplication Plating Pick the colony containing target gene =100,000 Juang RH (2004) BCbasics

Notes de l'éditeur

  1. 質體 可以由一種宿主傳到另一種宿主菌,因此抗藥性也可以因此傳播開來,使得很多細菌很快對抗生素產生抗藥性。 遺傳工程也是利用質體的這種傳播功能,把一個被修改過的重組質體,放到宿主細菌中去表現,生產我們所要的物質。
  2. 質體 用限制脢切開後,可以插入各種外來的 DNA ,形成重組質體,後者可以轉形到宿主細胞中。 但是,一個細菌只能接受一種重組質體,而這個轉進去宿主的質體,可以在細菌中大量複製相同的質體。
  3. 質體 在細菌中會大量複製,大約每一隻細菌可以累積 5~200 個質體 ( 以 100 計算 ) ;若把這些轉形菌途佈在培養皿上,則由單一細菌所形成的菌落,將只含有一種轉形菌,也就是只含有一種重組質體。 每一個菌落若有 1000 隻細菌,而每隻細菌含有 100 個重組質體,則每個菌落就有 1×100×1000 個均質的純系重組質體,沒有雜夾其他的質體;因為一個細菌只能包容一種質體,而一個菌落只有一種細菌。 因此這種 分子群殖 (molecular cloning) 手法,有點像在『 種植分子 』,把一個目標基因,經過上述的 純化 與 放大 的過程,得到數目眾多的複製 DNA ,可以抽取出來應用。 最後所得到的群落,可以用探針進行雜合反應,挑選出含有目標基因的菌落 (N3-13) 。