Cutting and joining DNA molecules by restriction enzymes and ligases, respectively. Selective amplification of genes of interest using PCR (Polymerase Chain Reaction). Attachment of fragments of DNA to suitable replicons called cloning vectors. Small plasmids and bacteriophages are most

suitable. Maintenance does not require integration into the host genome. Genetic transformation of Escherichia coli. Recombinant DNA can be isolated in intact form. Pre requisites to gene manipulation: The vector DNA must be purified and cut open.

The passenger DNA must be inserted into the vector molecule to create the recombinant DNA. Cutting and joining reactions are monitored by gel electrophoresis. The recombinant must be introduced into the host - E.coli. Separation of nucleic acids:

Agarose and polyacrylamide gels are used to separate nucleic acids (DNA, RNA). Northern blot: separation of RNA molecules on agarose gel, blotting and hybridizing with specific labeled probe or gene Southern blot: separation of DNA molecules on agarose or polyacrylamide ges, blotting to nylon or nitrocellulose membranes and hybridizing with

specific labeled probe or gene Gel retardation or band shift assay (EMSA). Reduction in electrophoretic mobility upon binding of proteins to DNA. Western Proteins from acryl amide gels to membranes.

DNA CLONING VECTORS Characterization of the DNA of any organism: DNA cloning: A desired DNA fragment is identified and selectively amplified so that its structure can be studied using a variety of different techniques such as Restriction enzyme analysis, DNA sequencing,

in vitro expression studies. Molecular Hybridization: The DNA fragment is not amplified but rather studied as it is found in a complex mixture of DNA fragments. The restriction analysis can be performed as well as the chromosomal location. RNA expression can also be studied in this way.

How to introduce DNA into cells: Vectors Vectors used to move DNA between species, or from the lab bench into a living cell, must meet three requirements: They must be autonomously replicating DNA molecules in the host cell. -The most common vectors are designed for replicating in bacteria or yeast, but there are

vectors for plants, animals and other species. They must contain a selectable marker so cells containing the recombinant DNA can be distinguished from those that do not. An example is drug resistance in bacteria. They must have an insertion site to accommodate foreign DNA.

-Usually a unique restriction cleavage site in a nonessential region of the vector DNA. -Later generation vectors have a set of about 15 or more unique restriction cleavage sites. How to clone DNA: 1.Cell based DNA cloning. 2. Cell free DNA cloning.

Cell based Cloning of DNA: Construction of a recombinant DNA library Ligation of the target DNA into a suitable vector. Transformation of the library into a host E.coli or yeast to amplify cloned DNA fragments. Selective propagation of the clones Plating of clones on selection media to allow

screening. Isolation of the recombinant clones Identification and expansion of clones of interest. PLASMID CLONING VECTORS Plasmids are autonomously replicating circular DNA molecules found in bacteria. They have their own origin of replication, and they

replicate independently of the origins on the "host" chromosome. A plasmid that was widely used in many recombinant DNA projects is pBR322. It replicates from an origin derived from a colicin-resistance plasmid (ColE1). This origin allows a fairly high copy number, about 100 copies of the plasmid per cell.

Plasmid pBR322 carries two antibiotic resistance genes, each derived from different transposons (jumping genes) that were initially found in R-factors, which are larger plasmids that confer antibiotic resistance. Plasmid pBR 322

(beta lactamase) Features of plasmid pBR322: The gene conferring resistance to ampicillin (ApR) can be interrupted by insertion of a DNA fragment into the PstI site, and the gene conferring resistance to tetracycline (TcR) can be interrupted by insertion of a DNA fragment into the BamHI site.

Use of the TcR and ApR genes allows for easy screening for recombinants carrying inserts of foreign DNA. For instance, insertion of a restriction fragment in the BamHI site of the TcR gene inactivates that gene. One can still select for ApR colonies, and then screen to see which ones have lost TcR .

Plasmid pUC19 with lacZ gene and Ampr gene and an E. coli plasmid replicon. (IPTG: lacZ inducer X-gal: substrate for beta galactosidase) Yeast vectors:

The yeast 2m plasmid: REP1 and REP2 are involved in replication of the plasmid, and FLP codes for a protein that can convert the A form of the plasmid (shown here) to the B form, in which the gene order has been rearranged by intramolecular recombination. The function of D is not exactly known. Development of cloning vectors for yeast has been

stimulated greatly by the discovery of the 2 m plasmid that is present in most strains of S. cerevisiae. The 2 m plasmid is an excellent basis for a cloning vector. It is 6 kb in size which is ideal for a vector, and exists in the yeast cell at a copy number of between 70 and 200. Replication: makes use of a plasmid origin, several enzymes provided by the host cell, and the proteins

coded by the REP1 and REP2 genes carried by the plasmid. Some yeast cloning vectors carry genes conferring resistance to inhibitors such as methotrexate and copper for selection. Most of the popular yeast vectors make use of a radically different type of selection system.

In practice a normal yeast gene is used, generally one that codes for an enzyme involved in amino acid biosynthesis. In order to use LEU2 as a selectable marker, a special kind of host organism is needed. The host must be an auxotrophic mutant that has a nonfunctional LEU2 gene. Such a leu2- yeast is unable to synthesize leucine and

can survive only if this amino acid is supplied as a nutrient in the growth medium. Selection is possible because transformants contain a plasmid-borne copy of the LEU2 gene, and so are able to grow in the absence of the amino acid. In a cloning experiment, cells are plated out onto minimal medium, which contains no added amino acids.

Only transformed cells are able to survive and form colonies. CLONING STRATEGY USING THE YEAST 2 m PLASMID LAMBDA AND COSMID VECTORS Bacteriophages (lambda and M13) can be used as

cloning vectors. Lamda phage has a genome of about 50 kb of linear DNA. Its life cycle is conducive to the use as a cloning vector. The lytic cycle can be supported by only a portion of the genes found in the lambda genome. This type of lambda vector is also called as the

lambda replacement vector (because the lambda DNA is removed and replaced with foreign DNA). Lambda life cycle: The lytic life cycle produces phage

particles immediately. The lysogenic life cycle requires genes in the middle of the genome, which can

be replaced. Cos sites: at either end of the lamda DNA molecules (double stranded), a short 12 nucleotide stretch, in which DNA is single stranded. two single strands are complementary and

base pair with one another to form a circular, completely double stranded molecule. act as recognition sequences for an endonuclease cleaves the catenane at the cos sites to produce individual genomes. LAMBDA INSERTION AND REPLACEMENT VECTORS

Only 37 to 52 kb DNA fragments can be packaged into the lambda head. This can be done in vitro. The middle portion of the lambda genome can be replaced (that region is non essential) and DNA can be cloned (up to 20 kb). These vectors are used for construction of genomic DNA libraries.

LAMBDA GENOME Lamda DNA transfection efficiency low; when packed more efficient. the bacterial chromosome integrated with lamda DNA-lysogen M13 AND PHAGEMID VECTORS

These vectors are used to produce DNA for sequencing. M13 is a virus with a genome of single stranded DNA. It has a nonessential region into which foreign genes can be inserted. It has been modified to carry a gene for galactosidase galactosidase (lacZ) as a way to screen for recombinants. The replicative form is duplex, allowing one to cleave

with restriction enzymes and insert foreign DNA. Some vectors are hybrids between plasmids and singlegalactosidase strand phage; these are called phagemids. One example is pBluescript.

Phagemids are plasmids (with the modified, highcopy number ColE1 origin) that also have an origin of replication of the phage f1 which is related to phage M 13. The vector has a multiple cloning site inserted into the lacZ gene and which is flanked by

two different phage RNA polymerase promoters. One can easily obtain either doublegalactosidase or singlegalactosidase stranded forms of these plasmids. The "blue" comes from the bluegalactosidase white screening for recombinants that can be done with the multiple cloning sites which are in the galactosidase galactosidase gene.

The "script" refers to the ability to make RNA copies of either strand in vitro with phage RNA polymerases. Infection of transformed bacteria (containing the phagemid) with a helper virus (e.g. derived from M13) will cause the M13 origin to be activated, and

progeny viruses carrying singlegalactosidase stranded copies of the phagemid can be obtained. COSMID VECTORS A cosmid is a hybrid between a lambda vector and a plasmid. The COS sites are the only thing that is necessary for lambda DNA packaging.

Therefore if one can ligate COS sites about 50 kb apart then the ligation products can be in vitro packaged. BACTERIOPHAGE P1 These vectors are like lambda and can hold up to 100 kb of DNA.

This DNA can then be packaged by the P1 phage protein coat. The use of T4 in vitro packaging systems can enable the recovery of 100 kb inserts. YEAST ARTIFICIAL CHROMOSOMES (YACs) YACs are yeast vectors with centromeres and

telomeres. They can carry about 200 kb or larger fragments (in principle up to 1000 kb = 1 Mb). Thus very large fragments of DNA can be cloned in yeast. In practice, chimeric clones with fragments from different regions of the genome are obtained fairly often, and some of the inserts are unstable.

Chromosome Structure Centromere is required for the chromosome to be distributed correctly to daughter cells during cell division. Two telomeres, the structures at the ends of a chromosome, which are needed in order for the ends to be replicated correctly and which also prevent the

chromosome from being nibbled away by exonucleases. The origins of replication, which are the positions along the chromosome at which DNA replication initiates, similar to the origin of replication of a plasmid. Chromosome Structure

The structure and use of a YAC vector Several YAC vectors have been developed but each one is constructed along the same lines, with pYAC3 being a typical example. pYAC3 is essentially a pBR322 plasmid into which a number of yeast genes have been inserted.

The genes, URA3 from YIp5 and TRP1-ori from YRp7 have been included in pYAC3. The DNA fragment containing TRP1 is extended even further to include the sequence called CEN4, which is the DNA from the centromere region

of chromosome 4. The TRP1originCEN4 fragment therefore contains two of the three components of the artificial chromosome. The third component, the telomeres, is provided by the two sequences called TEL. These are not themselves complete telomere sequences, but once inside the yeast nucleus they act as seeding sequences onto which

telomeres will be built. SUP4, is the selectable marker into which new DNA is inserted during the cloning experiment. The cloning strategy with pYAC3 is as follows : The vector is first digested with a combination of BamHI and SnaBI restriction enzymes, cutting the molecule into three fragments.

The fragment flanked by BamHI sites is discarded, leaving two arms, each bounded by one TEL sequence and one SnaBI site. The DNA to be cloned, which must have blunt ends (SnaBI is a blunt end cutter, recognizing the sequence TACGTA), is ligated between the two arms, producing

the artificial chromosome. Protoplast transformation is then used to introduce the artificial chromosome into S. cerevisiae. The yeast strain that is used is a double auxotrophic mutant, trp1- ura3-, which is converted to trp1+ ura3+ by the two markers on the artificial chromosome.

Any cell transformed with an incorrect artificial chromosome, containing two left or two right arms rather than one of each, is not able to grow on minimal medium as one of the markers is absent. The presence of the insert DNA in the vector can be checked by testing for insertional inactivation of SUP4, which is carried out by a simple colour test: red colonies are recombinants, white colonies are

not. A YAC vector and the way it is used to clone large pieces of DNA.

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