Cloning
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Cloning. Guanqun Yuan 12-13-2004 R.A.Scott Group Meeting. Outline. What is cloning? How to do cloning? Validation Application. What is Cloning?. Generating identical copies of organisms, cells, or replicating nucleic acid sequences from organisms involving human intervention.

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Cloning

Cloning

Guanqun Yuan

12-13-2004

R.A.Scott Group Meeting


Outline

Outline

  • What is cloning?

  • How to do cloning?

  • Validation

  • Application


What is cloning

What is Cloning?

  • Generating identical copies of organisms, cells, or replicating nucleic acid sequences from organisms

  • involving human intervention.

  • Giving rise to new generation

  • Dolly (the sheep) is a clone, but a natural identical twin is not a clone.

  • DNA sequence amplified by growth is a clone, Identical DNA molecules produced in vitro (a PCR rxn) is not a clone.


Outline1

Outline

  • What is cloning?

  • How to do cloning?

  • Validation

  • Application


Cloning

How?

ORI

Cells that do not take up plasmids die on ampicillin plates

Amp R.

ORI

Amp R.

Plasmid vector

Enzymatically insert DNA into plasmid vector

Mix E.coli cells with plasmids in presence of CaCl2 Culture on nutrient agar plates containing ampicillin

+

Recombinant plasmid

Transformed E.coli cell survives

DNA fragment to be cloned

Bacterial chromosome

Independent plasmid replication

Cell multiplication


Composition

Composition

  • Vectors

    Plasmids, or phage

  • The cell

    E.coli, yeast

  • Inserted sequence

Plasmid vector

DNA fragment to be cloned


Vectors

Vectors

The substance that can serve as carriers to allow replication of recombinant DNAs.

  • Plasmids

  • Phage λ

  • Plasmid phage hybrids


Plasmids

Plasmids

MCS

Ab. Resis

ORI

ds circles of DNA that can replicate autonomously.

Three features of the plasmid cloning vectors:

  • Multiple cloning site. The place where foreign DNA fragments can be inserted.

  • An origin of replication. The replication origin is a specific DNA sequence of 50-100 base pairs that must be present in a plasmid for it to replicate. Host-cell enzymes bind to ORI, initiating replication of the circular DNA.

  • A gene specifying resistance to an Antibiotic. This permits selective growth of the host cell.

    Most often used: Resistance to ampicillin, penicillin, tetracycline, kanamycin, and chloramphenicol.


Phage

Phage λ

  • A phage λ virion has a head, which contains the viral DNA genome, and a tail, which functions in infecting E.coli host cells.

  • Advantages over plasmids: They infects cells much more efficiently than plasmids transform cells. The yield of clones with vectors usually higher.

  • Because of its efficiency, phage λ is often used in library construction.

Viral Genome


The cell

The Cell

E.coli:

  • Normal E. coli cells cannot take up plasmid DNA from the medium. Exposure to high concentration of certain divalent cations, CaCl2, makes a small fraction of cells permeable to foreign DNA.

  • Each component cell incorporates a single plasmid DNA molecule, which carries an antibiotic-resistance gene. When the cells are treated wit antibiotics on plates, only a few of the transformed cells containing the antibiotics-resistance gene on the plasmid vector will survive.


Inserted sequence

Inserted Sequence

  • Source of Nucleic acid to be cloned:

    -DNA directly from organism

    -DNA synthesized or amplified in vitro (cDNA or PCR reactions).

    -Previously cloned DNA. Generally a specific sequence.

  • Quality of DNA can be crucial

    -Its purity, being free of contaminants

    -its size is crucial when cloning very large pieces


Two important enzymes

Two Important Enzymes

  • Restriction Enzymes:

    cuts the DNA from any organism at specific sequences of a few nucleotides, generating a reproducible set of fragments.

  • DNA Ligases:

    insert DNA restriction fragments into replicating DNA molecules producing recombinant DNA.


Cloning

Mechanism

Restriction Enzyme: EcoRI

5` G A A T T C 3`

5` G A A T T C 3`

3` C T T A A G 5`

3` C T T A A G 5`

Cleavage

DNA Ligases

Sticky ends

OH

5` OH

5` A A T T 3`

Unpaired B and C

+

P

P

3` T T A A P

3` T T A A 5`

+

OH

Complementary ends base-pair

5` OH

A

3` T T A A P

DNA ligases

5` OH

B

3` C G P

5` A A T T 3`

5` OH

C

3` T T A A 5`

3` T A C G P


Outline2

Outline

  • What is cloning?

  • How to do cloning?

  • Validation

  • Application


Validation

Validation

Because introducing DNA into an organism is usually not very efficient, we need to do validation.

  • Selection- A technique that isolates only a particular type of cell or organism.

  • Screen- A technique that allows identifying a particular type of cell or organism but does not isolate it from other types.


Selection

Selection

pBR322

Amp. R

Tet. R

Tet. R

EcoRI

Amp. R

Cell

Ampicillin


Screen

Screen

Cell

pBR322

EcoRI

EcoRI

Tetracycline

Amp R.

Tet. R

EcoRI

Tet. R

Replica plating process

Add Amp.

The cells we want


Other validation methods

Other validation methods

About 100 bp

U.P.

Promoter

Multi. cloning site

BamHI

EcoRI

Ori.

SalI

L.P.

PCR

Ab. Resis.

+

EcoRI

EcoRI

Restriction Enzyme

Inserted Gene

About 500bp-5kb

Marker

With Prod.

Without Prod.

Marker

With Prod.

Without Prod.


Sequencing

Sequencing

Dideoxy: (Sanger) Manual

  • Primer extension reactions in four separate tubes.

  • Using a dideoxy nucleotide as the chain terminator.

  • Each tube contains different dideoxy nucleotide (ddATP, ddCTP, ddGTP, ddTTP).

  • Radioactive dATP is also included in all the tubes so the DNA products will be radioactive.

  • The results is a series of fragments of different lengths.

  • Finally, autoradiography is performed to visualize the DNA fragments.


Dideoxy sanger manual

Dideoxy: (Sanger) Manual

a) Primer extension reaction

c) Electrophoresis of the Protein

ddA ddC ddG ddT

TACTATGCCAGA

TCTGGCATAGTA

20-base primer

Replication with ddTTP

TACTATGCCAGA

ATGA

T

25-base primer

b) Product of the four reactions

Product of ddA rxn

Product of ddC rxn

Template:

TACTATGCCAGA

Template:

TACTATGCCAGA

(21)

A

(27)

ATGATAC

(24)

ATGA

(31)

ATGATACGGTC

(26)

ATGATA

Product of ddA rxn

Product of ddG rxn

Template:

TACTATGCCAGA

Template:

TACTATGCCAGA

(22)

AT

(23)

ATG

(25)

ATGAT

(28)

ATGATACG

(30)

ATGATACGGT

(29)

ATGATACGG

(32)

ATGATACGGTCT


Sequencing1

Sequencing

Dideoxy: (Sanger) automated

  • The “manual” sequencing technique is powerful but slow, thus Rapid automated sequencing methods are required.

  • Still based on the procedure using dideoxy nucleotides, but tagged with a different fluorescent molecule, so the product from each tube will emit a different color fluorescence when excited by light

  • After extension reaction and chain termination, all 4 solutions are mixed and electrophoresed together in the same lane on gel analyzed by laser beam

  • The color of the fluorescent light emitted from each oligonucleotide is detected electronically


Outline3

Outline

  • What is cloning?

  • How to do cloning?

  • Validation

  • Application


Application

Application

  • Expression

  • Library


Expression

Expression

Why?

You want the cloned gene to make its product, normally a protein.

  • Identifying gene from library requires expression.

  • To overproduce the protein and purify it.

  • For in vivo studies of the protein.


Expression1

Expression

Expression Vectors:

Vectors that can yield the protein products of the cloned genes.

Two elements that are required for active gene expression: a strong promoter and a ribosome binding site near an initiating ATG codon.

The main function of an expression vector is to yield the product of a gene, therefore a strong promoter is necessary. The more mRNA is produced, the more protein product is made.


Inducible expression vectors

Inducible Expression Vectors

  • Protein produced in a large quantity in bacteria can be toxic, so it is advantageous to keep a cloned gene repressed before expressing it.

  • Solution: keep the cloned gene turned off by placing it downstream of an inducible promoter that can be turned off.

  • IPTG strongly induce lac promoter


Expression2

Expression

Expression Vector that Produce Fusion Proteins

  • Fusion proteins: Gene (or part of a gene) for one protein fused to part or all of a gene for a second protein.

    Major uses for generating fusion proteins:

  • The ‘tag’ of the fusion protein can greatly aid biochemical purification. If the tag binds a particular substance, a column prepared containing that bound substance can be used to purify the tagged protein from virtually all other proteins.

(His)6

Oligohistidine regions like this have a high affinity for metals like nickel, so proteins that have such regions can be purified using nickel affinity chromatography

MCS

ATG


Fusion protein

Fusion Protein

  • The ‘tag’ can serve as a convenient way for identification of the tagged protein in cells or extracts. For example, a short peptides can be sufficient to use as a tag for antibody binding. In this way, a common antibody can be used, eliminating the need to develop a novel reagent specific to the protein of interest.

  • The ‘tag’ can be used as a surrogate in the quantification of the tagged protein. Again, a routine assay of the activity of the tag can be used to monitor amounts of a protein of interest that may have no means of assay otherwise.


Library construction

Library Construction

A library is a collection of different cloned DNAs from a single source that are present in different copies of a particular cloning vector.

  • Genomic library – for genome sequencing

  • cDNA library – derived from mRNA of a particular tissue, for isolating specific genes


Library construction1

Library Construction

The principle of library construction is basically quite simple.

  • Cut a DNA vector at a unique restriction site and ligate into it the DNA that you want to make a library out of.

  • If you want a library of human genomic DNA, you use fragmented human DNA.

  • The ligation mix is not yet considered the library. The library comes after the generation of E. coli cells carrying the cloned DNA.

  • To generate a library with a million clones for example, you need to recover a million independent colonies carrying plasmids or a million independent phage plaques. By pooling together all the independent clones you get the library.


Library construction2

Library Construction

Though simple in principle, libraries are difficult to make well.

  • Partly this is just a matter of scale. While in routine cloning, you generally just need to recover a single type of clone, a library has to generate very large numbers of independent DNA inserts.

  • While used pretty frequently, libraries are seldom made. Few people have much experience.

  • The best advice for making a library is to not do it unless you really have to. Get a library from someone else that has already made one. Some are commercially available.


Thanks

Thanks

Dr. Robert A. Scott

All the members in the group

Xiaoming Wang


Reference

Reference

  • Manuscript of Course Genetics 8920

  • Molecular Biology

    Robert F. Weaver

  • Molecular Cell Biology

    Lodish, Berk, Zipursky, Matsudaira,

    Baltimore, Darnell


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