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