Dna Cloning&cloning Vector

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 Cloning 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

 How is the DNA removed from the cells?  How is the DNA cut into pieces?

Purification of DNA from living cells Total cell DNA (DNA of interest) Genomic DNA library cDNA library

Vector DNA 

Plasmid DNA

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Phage 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 the genome, Collectively contain all the genes and are called a library.

Construction of a human genomic DNA library

Genomic DNA Libraries Foreign genome cut up with restriction enzyme

or

Recombinant plasmids

Bacterial clones

(a) Plasmid library

Recombinant phage DNA

Phage clones

(b) Phage library

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

• 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 pH 12-12.5 Supercoiled plasmids

Single-stranded linear DNA pH 7

Tangled mass of linear DNA

Ethidium bromide-Caesium chloride density gradient centrifugation

CsCl

1.6 1.65 1.7 1.75 1.8

Protein DNA

RNA

CsCl+EtBr Protein Linear DNA Supercolied DNA RNA

How are the pieces of DNA put back together?

Restriction enzyme 

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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 can be separated and viewed.

Restriction enzymes and recombinant DNA Restriction site DNA 5′ 3′ 1 Restriction enzyme cuts the sugar-phosphate backbones at each arrow

AATTC

G CTTAA

G

Sticky end

2 DNA fragment from

another source is added. Base pairing of sticky ends produces various combinations.

AATTC

G

G CTTAA

Fragment from different DNA molecule cut by the same restriction enzyme G AATT C C TTAA

3 DNA ligase

3′ 5′

GAATTC CTTAAG

G

G AATTC CTTAA G

One possible combination

seals the strands.

Recombinant DNA molecule

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 into the host cell. The vector needs to have the following characteristics : Have an origin of replication so that the DNA can be replicated within a host cell.  Be small enough to be isolated without being degraded during purification.  Have several unique restriction sites for cloning a DNA fragment (called a “multiple cloning site,” or “MCS”) so that the vector will be cut only once to open it.  Have selectable markers for determining whether the cloning vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector. 

Cloning Vectors A. Bacterial Vectors Plasmids Bacteriophage Cosmids A. Vectors for Other Organisms Yeast Artificial Chromosomes (YACs) Bacterial Artificial Chromosomes (BACs) Plant Cloning Vectors Mammalian Cell Vectors

Bacterial 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

A bacterial plasmid used as a cloning vector

Plasmid Cloning Vectors 

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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 lacZ+ gene

Ampicillin resistance gene

Origin sequence

Fig. 7.5 *Cut with same restriction enzyme

*DNA ligase

Bacteriophage cloning vector A virus that infects bacteria, and whose DNA can be engineered into a cloning vector. Kills bacteria by two pathways: Lytic pathway: Bacteriophage DNA is inserted into the bacteria, and phage DNA and phage proteins are made, assembled, and burst out of the bacteria, causing the bacteria to burst (called “lysis”). Lysogenic pathway: The bacteriophage genome is integrated into the bacterial DNA, replicating along with the bacterial cell genome.

Phage Cloning Vectors    

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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)  

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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 plasmidYeast 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.

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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.

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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 produce two sticky ends. A sample of human DNA is also digested with EcoRI to produce pieces with the same sticky ends. The two samples are mixed and allowed to hybridize, some molecules will form with pieces of human DNA inserted into the p lasmid vector at the EcoRI site. DNA ligase is used to covalently link the fragments.

Chemical Reaction of DNA Ligase

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Phosphodiester bond (covalent bond)

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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

Inserting vectors into host cells Transformation: cell made competent to take up DNA Transduction: when the cloning vector used has aspects of a virus, the host cell can be infected (transfected) to insert the recombinant molecule Electroporation: the cell is placed in an electric field such that small pores are temporarily opened in the membrane. Added DNA can enter through these pores. Microinjection of DNA in the cell nucleus, such as an animal egg, is needed to introduce DNA into an entire animal. The DNA integrates into the animal chromosomes, the egg is implanted, and the animal is born with the desired traits.

http://plantandsoil.unl.edu/croptechnology2005/crop_tech/animationOut.cgi?anim_name=bacteria_transformation.swf http://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

Some 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 bacterial cells, you must screen the original bacterial colonies from the original master plate, and make exact copies by replica plating : • • • •

A sterile velveteen pad is placed on the plate, with cells sticking to it. The pad is pressed on media in other plates, creating exact replicas of the original plate Antibiotics in the media in the new plates will kill any bacteria that do not have the recombinant plasmid inside of them. The original plate is then examined to determine which bacterial colonies were successfully transformed.

Some possible products of the transformation reaction: Bacterial cell

Genomic DNA

Plasmid with insert

Plasmid w/o insert

No plasmid

Ampicillin resistant

Ampicillin resistant

Tetracycline sensitive

Tetracycline resistance

No ampicillin resistance No tetracycline resistance

Finding the right clone by colony hybridization In the following scheme, bacterial containing recombinant plasmids are grown as clones. The clones are blot transferred to a membrane sheet, and the DNA present denatured and fixed onto the surface. Adding a radioactive "probe" or complementary fragment and allowing the DNA to hybridize followed by exposure to X-ray film id entifies the clone containing recombinant DNA with the correct inser t.

Colony hybridization - to identify clone containing gene of interest

Propagation 

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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

Plasmid

Bacterial chromosome

Cell containing gene of interest

1 Gene inserted into plasmid

Recombinant DNA (plasmid)

Gene of interest 2 Plasmid put into bacterial cell

Recombinate bacterium

3

Gene of interest

Gene for pest resistance inserted into plants

3 Host cell grown in culture, to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest

Copies of gene Basic research on gene

DNA of chromosome

Protein harvested

4 Basic research and various applications

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolves blood clots in heart attack therapy

Basic research on protein

Human growth hormone treats stunted growth