Gene cloning lecture notes 5 for 2010

Gene Cloning

Lecture 5

Recombinant DNA Technology- Recap

• A set of techniques for recombining genes from different sources in vitro and transferring the recombinant DNA into cells where it may be expressed.

• Use of recombinant DNA techniques allows modern biotechnology to be more precise and systematic than earlier research results.

Recombinant DNA

• It allows genes to be moved across species barriers – hence a powerful tool

• Our understanding of eukaryotic molecular biology has been enhanced

• It has been applied in the Human Genome Project – to transcribe and translate the entire human genome in order to understand the human organism

• The ultimate goal is the improvement of human health

Gene (DNA) Cloning

• Recombinant DNA technology makes it possible to clone genes for basic research and commercial applications

• Recombinant DNA technology allows scientists to examine the structure and function of the eukaryotic genome because it contains: Biochemical tools for the construction of recombinant DNA Methods for purifying DNA molecules and proteins of interest Vectors for carrying recombinant DNA into cells for replication Techniques for determination of nucleotide sequences of DNA

molecules

Application of Recombinant DNA

Restriction enzymes are used to make Recombinant DNA

• Restriction enzymes were first discovered in the 1960s• They occur naturally in bacteria where they protect the

bacterium against foreign invading DNA from other organisms (e.g. Viruses or phages)

• The foreign DNA is restricted by it being cut into small segments – thus restriction is the process of cutting of foreign DNA into small pieces.

• Most restriction enzymes only recognise small short sequences (4-8 nt) called recognition sites and only cut at specific points within these sequences

Example of a recognition sequence

Restriction enzymes

• Recognition sequences are symmetric in that the same sequence of nucleotides is found on both strands, but run in opposite directions

• They usually cut phosphodiester bonds of both strands in a staggered manner, so that the resulting dsDNA fragments have single stranded ends, referred to as sticky ends

• The sticky ends form hydrogen-bonded base pairs with complementary sticky ends on other DNA molecules

• These unions are temporary since they are only held by a few hydrogen bonds

• The bonding is made permanent by DNA ligase, which catalyses the formation of covalent phosphodiester bonds

• This result is the same as natural genetic recombination, the production of recombinant DNA – a DNA molecule carrying a new combination of genes

Cloning Vectors

• A Cloning Vector is a DNA molecule that can carry foreign DNA into a cell and replicate in the cell

• The two most often used vectors are bacterial plasmids and viruses (phages)

Cloning with Plasmid Vectors• Plasmids are circular, dsDNA molecules that

separate from a cell’s chromosomal DNA. • They occur naturally in bacteria • Plasmids exist in parasitic or symbiotic

relationship with their host cells. Their sizes range from a few thousand bp to more that 100kb.

• They duplicate before every cell division. • During cell division, at least, one is segregated

into each daughter cell.

Cloning with Plasmid Vectors• Many plasmids also contain transfer genes coding for

proteins that transfer a copy of the plasmid to other host cells of the same or related bacteria species, by conjugation (E. coli plasmids can be engineered for use as cloning vectors)

• Plasmid most commonly used in recombinant DNA technology replicate in E. coli. These plasmids have been engineered to optimize their use as vectors in DNA cloning

• Their length (circumference) is reduced to range form 1.2 – 3 kb - much less than that occurring naturally in E coli plasmids

Cloning with Plasmid Vectors• Most plasmid vectors contain little more than the essential

nucleotide sequences required for their use in DNA cloning.

• They contain a replication origin (ORI), a drug-resistance gene, and a region in which exogenous DNA fragments can be inserted

• The ORI is a specific DNA sequence of 50-100 bp that must be present in a plasmid for it to replicate.

• Host enzymes bind to ORI, initiating replication of the circular plasmid

• Thus, any DNA sequence inserted into such a plasmid is replicated along with the rest of the plasmid DNA, the basis of molecular DNA cloning.

Cloning with Plasmid Vectors

• Steps of recombinant plasmid formationProduction of restriction fragmentsLigation of restriction fragments to plasmid DNATransformation of antibiotic-sensitive E. coli cells

with recombinant plasmids.

• Other cloning vectors are bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)

Cloning with Plasmid Vectors

Plasmid Cloning• Remember the Genetic code is the same in all

Organisms• Plasmids commonly used in recombinant DNA

technology replicate in E.coli• These plasmids have been engineered to

optimise their use as vectors for DNA cloning• Their length range from 1.2 to 3.0 kb• They contain a replication origin (ORI), a drug

resistance gene and a region for insertion of exogenous DNA

Transformation of the host cell

• Transformation is genetic alteration of a cell caused by the uptake and expression of foreign DNA regardless of the mechanism involved

• Transformation permits plasmid vectors to be introduced into and expressed in E. coli cells

• A plasmid vector must contain a drug-resistance gene coding for an enzyme that inactivates a specific antibiotic in order to be useful in DNA cloning.

Transformation of the host cell• The ability to select transformed cells is critical

to DNA cloning because the transformation of E.coli with isolated plasmid DNA is inefficient

• The cells must be exposed divalent cations such as Ca2+ to make the cells permeable

• E.coli are treated with CaCl2 and mixed with plasmid vecotrs

• Frequently, only 1 cell out of 10 000 or more cells beomes competent to take up foreign DNA

Transformation of the host cell

• Each cell takes up a single recombinant plasmid DNA molecule

• The treated cells are plated on a Petri dish of nutrient agar containing the antibiotic

• Only the transformed cells containing the antibiotic –resistance gene on the plasmid vector will survive

• Thus all the plasmids in such a colony of selected transformed cells are descended from a single plasmid taken up by cells that form the colony

Isolation of DNA Fragment From a Mixture

• The initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA since it can be isolated from the cloned cells

• DNA cloning allows a particular nucleotide sequence to be isolated from a complex mixture of fragments with many different sequences.

• For example, assume we have 4 different types of DNA fragments each with a unique sequence.

• Each fragment type is inserted alone into a plasmids vector.


• The resulting mixture of recombinant plasmids is incubated with E. coli cells treated with CaCl2.

• The cells are then cultured on antibiotic selective plates. (e.g. plates containing ampicillin).

• Each colony that develops arises from a single cell that took up one or the other recombinant plasmids.

• All the cells in a given colony thus carry the same DNA fragment.


• Overnight incubation of E. coli at 37°C produces visible colonies.

• These colonies are isolated from each other on the culture plate. Hence, copies of the DNA fragments in the original mixture are separated in the individual colonies.

• Therefore, DNA cloning is a powerful but simple method for purifying a particular DNA fragment from a complex mixture of fragments and producing large numbers of the fragment of interest.

• Each transformed cell contains multiple copies of a given plasmid.

Production of recombinant Plasmids• To clone specific DNA fragments in a plasmid (or any

other) vector, the fragments must be produced and then inserted into the DNA vector.

• Restriction enzymes and DNA ligases are utilized to produce such recombinant cloning vector.

• Restriction enzymes are bacterial enzymes that recognize specific 4 to 8 bp sequences (restriction sites), and then cleave both DNA strands at this site.

• These enzymes cut within the DNA molecule hence they are called endonucleases, to distinguish them from exonucleases, which digest nucleic acids from an end.

Production of recombinant Plasmids

• Many restriction sites are short inverted repeat sequences. That is, the restriction site sequence is the same on each DNA strand when read in the 5 ′→3 direction. ′

• The DNA digest (fragments) produced by restriction enzymes are called restriction fragments.

• Modification enzymes protect bacterial DNA from cleavage by restriction enzymes, by adding a methyl group to one or two bases usually within the restriction site.


• Many restriction enzymes generate fragments that have a single stranded tail at both ends.

• These tails are complementary to those on all other fragments generated by the same restriction enzyme.

• These tails are referred to as sticky ends (also called cohesive ends) and can transiently base pair at room temperature with those on other DNA fragments generated with the same restriction enzyme, regardless of the source of the DNA molecules.

Production of recombinant Plasmids• The base paring of sticky ends permits DNA from

widely different species to be ligated, forming chimeric molecules

• Purified DNA ligase is used to covalently join the ends of restriction fragments in vitro.

• DNA ligase can catalyze the formation of a 5 →3 ′ ′phosphodiester bond between 3 - OH end of one ′restriction fragment strand and the 5 - PO′ 4 end of another restriction fragment strand during the time that the sticky ends are transiently base-paired.


• Plasmids vectors containing a polylinker (or multiple cloning site sequence) are commonly used to produce recombinant plasmids carrying exogenous DNA fragments.

• Polylinkers are chemically synthesized and then introduced into the plasmid vector.


• Since the polylinker contains several different restriction sites, one of the restriction enzymes whose recognition sites is in the polylinker is used to cut both the plasmid molecules and genomic DNA.

• This generates singly cut plasmids and restriction fragments with complementary sticky ends.


• In the presence of DNA ligase, DNA fragments produced with the same restriction enzyme will be inserted into the plasmid.

• The ratio of DNA fragments to be inserted to cut vectors and other reaction conditions are chosen to maximize the insertion of one restriction fragment per plasmid vector.


• The recombinant plasmids produced in in vitro ligation reactions are then used to transform antibiotic sensitive E. coli cells.

• All cells in each antibiotic-resistant clone that remains after selection contain plasmids with the same inserted DNA fragment, but different clones carry different fragments.

Steps of transformation

• Production of restriction fragments• Ligation of restriction fragments to plasmid

DNA• Transformation of antibiotic-sensitive E. coli

cells with recombinant plasmids

Identification of clones

Membrane hybridizationExpression cloningSouthern blotting

Cloning with Bacteriophage) λ-Phage

• Most cloning done with E. coli plasmid because of the relative simplicity of the procedure

• However, the number of individual clones that can be obtained by this method is limited by: (1)the low efficiency of E. coli transformation and (2) the small number of individual colonies that be detected on a Petri dish.


• The limitations make plasmid cloning of all genomic DNA of higher organisms impractical

• For example, 1.5 x105 clones carry 25 kb DNA fragments are required to represent the total human genome

• Cloning vectors from bacteriopage λ have proved to be a more practical means for obtaining the required number of clones to represent large genomes


• Such a collection of λ clones that includes all the DNA sequences of a given species is called a genomic library.

• A genomic library can be screened for λ clones containing a sequence of interest.

• Bacteriophage λ can be modified for use as a cloning vector and assembled in vitro.


• A λ-phage virion has a head region, which contains the viral DNA.

• It also has a tail region, which enables the λ-phage to infect E. coli host cells.

• Only the λ DNA enters the cell when a λ virion infects a host cell.

• The viral DNA then undergoes either lytic or lysogenic growth.


• In lytic growth, the viral DNA is replicated and assembled into more than 100 progeny virions in each infected cell.

• This kills the cell in the process and releases the replicated virions.

• In lysogenic growth, the viral DNA inserts in the bacterial chromosome where it is passively replicated along with the host-cell chromosome as the cell grows and divides.


• In the Lysogenic Cycle:Viral DNA merges with Cell DNA and

does not destroy the cell.The Virus does not produce progeny.There are no symptoms of viral

infection.Temperate viral replication takes

place.


• In the Lytic Cycle:Viral DNA destroys Cell DNA, takes over cell

functions and destroys the cell.The Virus replicates and produces progeny

phages.There are symptoms of viral infection.Virulent viral infection takes place.

Life cycles of Viruses

http://upload.wikimedia.org/wikipedia/commons/5/5a/Phage2.JPG


• The λ genes coding for the head and tail proteins as well as various proteins involved in the lytic and lysogenic growth pathways are clustered in discrete regions of about 50kb viral genome.

• The genes in the lysogenic pathway are not relevant for use of bacteriophage λ as a vector.


The λ proteins, designated Nu1 and A, bind to COS sites and direct insertion of the DNA between 2 adjacent COS sites into a preassembled head.


• Genes in the lysogenic pathway are not relevant for use of bacteriophage λ as a vector.

• Hence, they are removed from the viral DNA and replaced with other DNA sequences of interest.

• Up to 25 kb of foreign DNA then can be inserted into λ genome, resulting in a recombinant DNA that can be packaged to form virions capable of replicating and forming plaques in E. coli host cells.


• The key to the high efficiency of λ-phage cloning is the ability to assemble λ virions in vitro.

• Viral heads and tails initially are assembled separately from multiple copies of the various proteins that compose these complex structures.

• λ DNA replication in the host cell generates concatemers - multimeric DNA molecules that consist of multiple copies of the viral genome linked end-to-end and separated at COS sites.

COS sites are protein-binding nucleotide sequences that occur once in each copy of the λ genome.


• The host-cell chromosomal DNA is not inserted into the λ heads because it does not contain copies of the COS sequence.

• Only one λ DNA is inserted into a preassembled λ head.

• After insertion of the λ DNA the preassembled tail is attached producing complete virions.

• Preparation of recombinant infectious λ virions:The phage-assembly process is carried out in

vitro. E. coli cells are infected with a mutant defective

in A protein (one of the 2 proteins required for packaging λ DNA into preassembled phage heads).

These cells then accumulate Empty headsPreassembled tails also accumulate since they

only attach to heads filled with DNA


• The cells are lysed experimentally and an extract containing high concentrations of heads and tails is prepared.

• When this extract is mixed with A protein and recombinant λ DNA containing a COS site, the DNA is packaged into the empty heads.

• The tails in the extract then combine with the filled heads, yielding complete virions carrying the recombinant λ DNA.


• The recombinant virions produced by this method are fully infectious and can efficiently infect E. coli cells.

• Each virion particle binds to receptor on the surface of a host cell and injects it packed recombinant DNA into the cell.

• This infection process is about 1000x more efficient than transformation with plasmid vector.

.


• ≈106 colonies per µg in plasmid transformation compared to ≈ 109 plaques representing λ-phage representing λ clones for λ-phage transformation per µg of recombinant DNA

• a collection of λ clones that includes all the DNA sequences of a given species is called a genomic library. A genomic library can be screened for λ clones containing a sequence of interest.

Identification of a specific clone from a λ phage library by membrane hybridization to a radiolabeled probe

• The position of the signal on the autoradiogram identifies the desired plaque on the plate.

• In practice, in the initial plating of a library the plaques are not allowed to develop to a visible size so that up to 50,000 recombinants can be analyzed on a single plate.

• Phage particles from the identified region of the plate are isolated and replated at low density so that the plaques are well separated.

• Then pure isolates can be obtained by repeating the plaque hybridization as shown in the figure.

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A7315&rendertype=def-item&id=A7618



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Gene cloning lecture notes 5 for 2010