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Asst. Prof. Dr. Sawitree Chiampanichayakul
Dept. of Medical Technology
Fac. of Associated Medical Sciences
Chiang Mai University
Asst. Prof. Dr. Sawitree Chiampanichayakul
Dept. of Medical Technology
Fac. of Associated Medical Sciences
Chiang Mai University
DNA Cloning – the act of making many identical copies
of a particular piece of DNA (often a gene).
DNA Cloning
There are several steps involved in cloning a gene in a cell. The
specific steps in an individual procedure may vary, but most follow
these steps:
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Overall of DNA Overall of DNA CloningCloning
Introduction of recombinant DNA
into host cells
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make
recombinant DNA
Selection of host cells containing the recombinant
DNA
How is the DNA removed from the cells?How is the DNA removed from the cells?
How is the DNA cut into pieces?How is the DNA cut into pieces?
Purification of DNA from living cellsPurification of DNA from living cells
Total cell DNA (DNA of interest)Total cell DNA (DNA of interest)
Genomic DNA libraryGenomic DNA library
cDNA librarycDNA library
Vector DNAVector DNA
Plasmid DNAPlasmid DNA
Phage DNAPhage DNA
Genomic DNA Libraries
Genomic library is all DNA, introns, exons and non-coding
The genomic DNA is digested by a restriction endonuclease,
All fragments cloned at random into a plasmid vector.
Cultures of the bacteria, with each containing only a fraction of t
he genome,
Collectively contain all the genes and are called a library.
Construction of a human genomic DNA library
Construction of a human genomic DNA library
Genomic DNA Libraries
Foreign genomecut up withrestrictionenzyme
Recombinantplasmids Recombinant
phage DNA Phageclones
(b) Phage library(a) Plasmid library
or
Bacterialclones
cDNA library
cDNA made in vitro by reverse transcription of all the
mRNA produced by a particular cell.
Making cDNA from mRNA using reverse
transcriptase and DNA polymerase
Making cDNA from mRNA using reverse
transcriptase and DNA polymerase
• Plasmid DNA : Stringent plasmid
: Relaxed plasmid
Plasmid DNA separation
Plasmid DNA separation on the basis of size
Physical property: most plasmid DNA are much smaller than bacterial DNA
Treatment cells with EDTA and lysozyme in the presence of sucrose, the spheroplasts are formed
Cell lysis is induced by non-ionic detergent (Triton X-100)
After centrifugation, the cleared lysate consisting of plasmid DNA
Plasmid DNA separation
Plasmid DNA separation on the basis of conformation
Most plasmid DNA exist in the cell as supercoiled molecules
Two different methods are commonly used:
Alkaline denaturation
Ethidium bromide-Caesium chloride density gradient centrifugation
Alkaline denaturation
Linear DNA
Supercoiled plasmids
pH 12-12.5
Single-stranded linear DNA
pH 7
Tangled mass of linear DNA
Ethidium bromide-Caesium chloride density gradient centrifugation
1.7
1.651.6
1.81.75
Protein
DNA
RNA
Protein
Linear DNA
RNA
Supercolied DNA
CsCl CsCl+EtBr
How are the pieces of DNA put back together?How are the pieces of DNA put back together?
Restriction enzyme
Linn S. and Arber, W. (1960s) found RE in bacteria, where they are used as a defense against bacteriophage infection by cutting bacteriophage DNA inside of bacteria.
Bacterial DNA is protected from restriction enzymes by the addition of methyl groups to bacterial DNA to adenine or cytosine.
Also called “restriction endonucleases”, they cut DNA like scissors at specific sites called “restriction sites” or “restriction sequences”.
They cut across the sugar-phosphate backbone of DNA by breaking the covalent bond holding the sugar and phosphate together.
Sticky end
Sticky end
Restriction enzyme Type II
Restriction sequences are usually four to six base pairs in length, and are palindromic, which means that the sequence of
both DNA strands are the same when the top strand is read from left to right, and then when the bottom strand is read from
right to left. Can cut in to result in pieces of DNA with two possible ends:
Blunt ends: The enzyme cuts directly across the two strands of DNA.
Sticky ends: The enzyme cuts both strands in different places, leaving a short single-stranded piece of DNA
to hang over the end of the molecule. This piece can base pair with other single-stranded pieces of DNA to
form recombinant DNA
Separating Restriction Fragments and Visualizing DNA
Pieces of DNA are generated by restriction enzymes Pieces of DNA are generated by restriction enzymes can be separated and viewed.can be separated and viewed.
Restriction site
DNA 53 5
3G A A T T C
C T T A A G
Sticky end
Fragment from differentDNA molecule cut by thesame restriction enzyme
One possible combination
Recombinant DNA molecule
G
C T T A A
A A T T CG
A A T T C
C T T A AG
G
G GA A T T C A A T T C
C T T A A G C T T A A G
Restriction enzymes and recombinant DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
DNA fragment from another source is added. Base pairing of sticky ends produces various combinations.
2
DNA ligaseseals the strands.
3
DNA Cloning
There are several steps involved in cloning a gene in a cell. The
specific steps in an individual procedure may vary, but most follow
these steps:
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Cloning Vectors
A vector is used to amplify a single molecule of DNA into many copes.
A DNA fragment must be inserted into a cloning vector.
A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a
bacterial cell. Most vectors are genetically engineered plasmids or
phages.
Cloning Vectors
The DNA of interest is inserted into a cloning vector to transport it The DNA of interest is inserted into a cloning vector to transport it into the host cell. into the host cell.
The vector needs to have the following characteristics :The vector needs to have the following characteristics :
Have Have an origin of replicationan origin of replication so that the DNA can be replicated so that the DNA can be replicated within a host cell. within a host cell.
Be small enough to be isolated without being degraded during Be small enough to be isolated without being degraded during purification. purification.
Have several unique Have several unique restriction sitesrestriction sites for cloning a DNA fragment for cloning a DNA fragment (called a “multiple cloning site,” or “MCS”) so that the vector (called a “multiple cloning site,” or “MCS”) so that the vector
will be cut only once to open it. will be cut only once to open it. Have Have selectable markersselectable markers for determining whether the cloning for determining whether the cloning
vehicle has been transferred into cells and to indicate whether vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector. the foreign DNA has been inserted into the vector.
The DNA of interest is inserted into a cloning vector to transport it The DNA of interest is inserted into a cloning vector to transport it into the host cell. into the host cell.
The vector needs to have the following characteristics :The vector needs to have the following characteristics :
Have Have an origin of replicationan origin of replication so that the DNA can be replicated so that the DNA can be replicated within a host cell. within a host cell.
Be small enough to be isolated without being degraded during Be small enough to be isolated without being degraded during purification. purification.
Have several unique Have several unique restriction sitesrestriction sites for cloning a DNA fragment for cloning a DNA fragment (called a “multiple cloning site,” or “MCS”) so that the vector (called a “multiple cloning site,” or “MCS”) so that the vector
will be cut only once to open it. will be cut only once to open it. Have Have selectable markersselectable markers for determining whether the cloning for determining whether the cloning
vehicle has been transferred into cells and to indicate whether vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector. the foreign DNA has been inserted into the vector.
Cloning Vectors
A. Bacterial Vectors
Plasmids
Bacteriophage
Cosmids
B. Vectors for Other Organisms
Yeast Artificial Chromosomes (YACs)
Bacterial Artificial Chromosomes (BACs)
Plant Cloning Vectors
Mammalian Cell Vectors
Plasmid
Extrachromosomal DNA in a bacterial cell which can replicate independently.
Drug resistance plasmids are not essential for the cell's growth, but confer antibiotic resistance.
Plasmids used for molecular cloning have been artificially created by recombining fragments of various existing plasmids.
Plasmids contain multiple cloning sites with several restriction endonuclease sites.
Engineered as vectors that fit pieces of DNA of up to 10 kilobases in length
Bacterial Vectors
A bacterial plasmid used
as a cloning vector
Plasmid Cloning Vectors
Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells.
They can replicate independently of the host cell. The size of plasmids ranges from a few kb to near 100 kb
Can hold up to 10 kb fragments Plasmids have an origin of replication, antibiotic
resistance genes as markers, and several unique restriction sites.
After culture growth, the clone fragment can be recovered easily. The cells are lysed and the DNA is isolated and purified.
A DNA fragment can be kept indefinitely if mixed with glycerol in a –70oC freezer.
pBR322 :• The molecule is small, and can be isolated easily.
• This vector can accommodate DNA of up to 5 to 10 kb.• pBR322 has several unique restriction sites where the plasmid
can be opened for inserting a DNA fragment. • The genes encoding resistance to ampicillin (ampr) and
tetracycline (tetr) are used for plasmid and DNA insert selection. • Provides for the insertable selection of a selectable marker:
• If one of the antibiotic resistance genes is broken with the DNA of interest, then the bacteria receiving the plasmid will be sensitive to the antibiotic and die if treated with the antibiotic.
• A method that determines if a recombinant plasmid was created correctly and inserted correctly into the bacteria.
pUC19
Polylinker: restriction sites
Origin sequence Ampicillin
resistance gene
lacZ+
gene
Fig. 7.5
*Cut with same restriction enzyme
*DNA ligase
A virus that infects bacteria, and whose DNA can be engineered A virus that infects bacteria, and whose DNA can be engineered
into a cloning vector.into a cloning vector.
Kills bacteria by two pathways: Kills bacteria by two pathways:
Lytic pathway:Lytic pathway: Bacteriophage DNA is inserted into the Bacteriophage DNA is inserted into the
bacteria, and phage DNA and phage proteins are made, bacteria, and phage DNA and phage proteins are made,
assembled, and burst out of the bacteria, causing the bacteria assembled, and burst out of the bacteria, causing the bacteria
to burst (called “lysis”). to burst (called “lysis”).
Lysogenic pathway:Lysogenic pathway: The bacteriophage genome is The bacteriophage genome is
integrated into the bacterial DNA, replicating along with the integrated into the bacterial DNA, replicating along with the
bacterial cell genome. bacterial cell genome.
Bacteriophage cloning vector
Phage Cloning Vectors
Fragments up to 23 kb can be may be accommodated by a phage vector
Lambda phage and M13 phage are most common phage Segments of the Lambda DNA is removed and a stuffer
fragment is put in. The stuffer fragment keeps the vector at a correct size and
carries marker genes that are removed when foreign DNA is inserted into the vector.
Example: Charon 4A Lambda
Cosmid Cloning Vectors
Cosmids combine essential elements of a plasmid and Lambda systems; a small plasmid that
contains the cos (cohesive termini) sites from phage DNA, a plasmid origin of replication, and
genes for antibiotic resistance. Packaged into a bacteriophage protein coat, but
the plasmid replicates like a plasmid instead of phage DNA once it is in the bacteria.
Fragments from 30 to 46 kb can be accommodated by a cosmid vector.
Recombinant cosmids are packaged into lambda caspids
Recombinant cosmid is injected into the bacterial cell where the rcosmid arranges into a circle and replicates as a plasmid. It can be maintained and recovered just as plasmids.
Shown above is a 50,000 base-pair long DNA molecule bound with six EcoRI molecules, and imaged using the atomic force microscope. This image clearly indicates the six EcoRI "sites" and allows an accurate restriction enzyme map of the cosmid to be generated.
http://homer.ornl.gov/cbps/afmimaging.htm
Yeast Artificial Chromosomes(YACs) YACs can hold up to 500 kbs. YACs are designed to replicate as
plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.
YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers.
YAC plasmidYeast chromosome DNA is inserted to a unique restriction
site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.
Mammalian Cell Vectors
Mammalian cells are used because bacteria are not able to produce complex
eukaryotic proteins that are modified by processes such as glycosylation, or if
the mRNA needs to be processed after transcription.
There are several mammalian cell vectors:
Simian virus 40 (SV40) - a small DNA tumor virus, could only hold a
small piece of DNA and caused only transient (temporary) expression of
the inserted DNA.
Retrovirus- a single-stranded RNA virus that contains a gene for the
enzyme reverse transcriptase to create double-stranded DNA from RNA
template, so that the DNA can integrate into the host cell’s genome. It
needs to infect actively dividing cells.
Adenovirus- a double-stranded DNA virus that can infect many types of
host cells with high efficiency, with a low chance for causing disease. It
does not have to infect actively dividing cells.
DNA Cloning
There are several steps involved in cloning a gene in a cell. The
specific steps in an individual procedure may vary, but most follow
these steps:
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Creating Recombinant DNA
A plasmid vector is digested with EcoRI at a single site to produ
ce two sticky ends.
A sample of human DNA is also digested with EcoRI to produc
e pieces with the same sticky ends.
The two samples are mixed and allowed to hybridize, some mol
ecules will form with pieces of human DNA inserted into the plas
mid vector at the EcoRI site.
DNA ligase is used to covalently link the fragments.
Chemical Reaction of DNA Ligase
Phosphodiester bond (covalent bond)
ATP, NAD+
DNA Cloning
There are several steps involved in cloning a gene in a cell. The
specific steps in an individual procedure may vary, but most follow
these steps:
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
TransformationTransformation:: cell made competent to take up DNAcell made competent to take up DNA
TransTransducductiontion: : when the cloning vector used has aspects of a when the cloning vector used has aspects of a
virus, the host cell can be infected (transfected) to insert the virus, the host cell can be infected (transfected) to insert the
recombinant moleculerecombinant molecule
ElectroporationElectroporation: : the cell is placed in an electric field such the cell is placed in an electric field such
that small that small pores are temporarily opened in the membrane. pores are temporarily opened in the membrane.
Added DNA can enter through these pores.Added DNA can enter through these pores.
Microinjection of DNAMicroinjection of DNA in the cell nucleus, such as an animal in the cell nucleus, such as an animal
egg, is needed to introduce DNA into an entire animal.egg, is needed to introduce DNA into an entire animal. The The
DNA integrates into the animal chromosomes, the egg is DNA integrates into the animal chromosomes, the egg is
implanted, and the animal is born with the desired traits. implanted, and the animal is born with the desired traits.
Inserting vectors into host cellsInserting vectors into host cells
http://plantandsoil.unl.edu/croptechnology2005/crop_tech/animationOut.cgi?anim_name=bacteria_transformation.swfhttp://www.phschool.com/science/biology_place/labbench/lab6/images/trananim.gif
Microinjection
DNA Cloning
There are several steps involved in cloning a gene in a cell. The
specific steps in an individual procedure may vary, but most follow
these steps:
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
SomeSome possible ligation reaction products: possible ligation reaction products:
Recombinant No insert Fragments No ligation
To select host cells containing recombinant DNA
Phenotyping
Colony hybridization
To selectively kill cells, and select for successfully transformed To selectively kill cells, and select for successfully transformed bacterial cells, you must screen the original bacterial colonies bacterial cells, you must screen the original bacterial colonies from the original master plate, and make exact copies by from the original master plate, and make exact copies by replica platingreplica plating : :
• A sterile velveteen pad is placed on the plate, with cells A sterile velveteen pad is placed on the plate, with cells sticking to it. sticking to it.
• The pad is pressed on media in other plates, creating exact The pad is pressed on media in other plates, creating exact replicas of the original plate replicas of the original plate
• Antibiotics in the media in the new plates will kill any Antibiotics in the media in the new plates will kill any bacteria that do not have the recombinant plasmid inside of bacteria that do not have the recombinant plasmid inside of them. them.
• The original plate is then examined to determine which The original plate is then examined to determine which bacterial colonies were successfully transformed. bacterial colonies were successfully transformed.
Some possible products of the transformation reaction:Some possible products of the transformation reaction:
Plasmid with insert
Ampicillin resistant
Tetracycline sensitive
Plasmid w/o insert
Ampicillin resistant
Tetracycline resistance
No plasmid
No ampicillin resistance
No tetracycline resistance
Bacterial cell Genomic DNA
Finding the right clone Finding the right clone by colony hybridizationby colony hybridization
In the following scheme, bacterial containing recombinant plasmiIn the following scheme, bacterial containing recombinant plasmi
ds are grown as clones. ds are grown as clones.
The clones are blot transferred to a membrane sheet, and the DNA The clones are blot transferred to a membrane sheet, and the DNA
present denatured and fixed onto the surface.present denatured and fixed onto the surface.
Adding a radioactive "probe" or complementary fragment and allAdding a radioactive "probe" or complementary fragment and all
owing the DNA to hybridize followed by exposure to X-ray film identowing the DNA to hybridize followed by exposure to X-ray film ident
ifies the clone containing recombinant DNA with the correct insert.ifies the clone containing recombinant DNA with the correct insert.
Colony hybridization - to identify clone containing gene of interest
Propagation Once colonies are
identified, they are cultured in broth to increase numbers and therefore the amount of DNA
Samples are also prepared for storage at -80 degrees. They can be kept for many years this way.
A cloned DNA fragment can be replicated inside a bacterial cell
Bacterium
Bacterialchromosome
Plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene of interest
DNA ofchromosome
Recombinatebacterium
Protein harvested
Basic research on protein
Gene of interest
Copies of gene
Basic research on gene
Gene for pestresistance inserted into plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growth hormone treatsstunted growth
Protein expressedby gene of interest
3
Gene inserted into plasmid
1
Plasmid put into bacterial cell
2
Host cell grown in culture,to form a clone of cellscontaining the “cloned”gene of interest
3
Basic research and various applications
4