cloning

Cloning in biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as bacteria,

insects or plants reproduce asexually

Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning),

cells (cell cloning), or organisms.

cloning

Plasmid

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.[1] In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-

ring in Saccharomyces cerevisiae).

• Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp).[2][3][4] The number of identical plasmids within a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.

Plasmid Replication

vectors

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and

biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.[2] Many

plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing

genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites

allowing the easy insertion of DNA fragments at this location

Vector map

Yac vector

steps

• Cloning of any DNA fragment essentially involves four steps

• fragmentation - breaking apart a strand of DNA • ligation - gluing together pieces of DNA in a

desired sequence • transfection - inserting the newly formed pieces

of DNA into cells • screening/selection - selecting out the cells that

were successfully transfected

Isolation of insert

• Initially, the DNA fragment to be cloned needs to be isolated. Preparation of DNA fragments for cloning can be accomplished in a number of alternative ways.

• Insert preparation is frequently achieved by means of polymerase chain reaction, but it may also be accomplished by restriction enzyme digestion, DNA sonication and fractionation by agarose gel electrophoresis.

• Chemically synthesized oligonucleotides can also be used if the target sequence size does not exceed the limit of chemical synthesis. Isolation of insert can be done by using shotgun cloning, c-DNA clones, gene machines (artificial chemical synthesis).

Restriction/ligation cloning

• In the classical restriction and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps: DNA fragmentation with restriction endonucleases, ligation of DNA fragments to a vector, transfection, and screening/selection. Although these steps are invariable among cloning procedures a number of alternative routes can be selected at various points depending on the particular application; these are summarized as a ‘cloning strategy’.

Transformation

• Following ligation, the ligation product (plasmid) is transformed into bacteria for propagation.

Transformation• Following ligation, a portion of the ligation reaction, including

vector with insert in the desired orientation is transfected into cells. A number of alternative techniques are available, such as chemical sensitization of cells, electroporation and biolistics. Chemical sensitization of cells is frequently employed since this does not require specialized equipment and provides relatively high transformation efficiencies.

• Electroporation is used when extremely high transformation efficiencies are required, as in very inefficient cloning strategies. Biolistics are mainly utilized in plant cell transformations, where the cell wall is a major obstacle in DNA uptake by cells. The bacterial transformation is generally observed by blue white screening.

• The bacteria is then plated on selective agar to select for bacteria that have the plasmid of interest. Individual colonies are picked and tested for the wanted insert. Maxiprep can be done to obtain large quantity of the plasmid containing the inserted gene

Plating

Selection

• Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that contain the desired insert at the appropriate orientation and isolate these from those not successfully transformed. Modern cloning vectors include selectable markers (most frequently antibiotic resistance markers) that allow only cells in which the vector, but not necessarily the insert, has been transfected to grow. Additionally, the cloning vectors may contain colour selection markers which provide blue/white screening (via α-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells. Further investigation of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

Selection-2

In gene cloning, X-gal is used to indicate whether a bacterium expresses the β-galactosidase enzyme, which is encoded by the lacZ gene, in a

technique called blue/white screening.X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. Thus, if X-gal and an inducer of β-galactosidase (usually IPTG) is contained within an agar

medium on a culture plate, colonies which have a functional lacZ gene can easily be distinguished

Selection blue/white colony

TOPO Cloning

TOPO Cloning is a molecular biology technique in which DNA fragments amplified by either Taq or Pfu polymerases are cloned

into specific vectors without the requirement for DNA ligases

• The technique utilises the inherent biological activity of DNA topoisomerase I.

• The biological role of topoisomerase is to cleave and rejoin supercoiled DNA ends to facilitate replication. Vaccinia virus topoisomerase I specifically recognises DNA sequence 5´-(C/T)CCTT-3'. During replication, the enzyme digests DNA specifically at this sequence, unwinds the DNA and re-ligates it again at the 3' phosphate group of the thymidine base.

Topo cloning

• TOPO vectors are designed in such a way that they carry this specific sequence 5´-(C/T)CCTT-3' at the two linear ends. The linear vector DNA already has the topoisomerase enzyme covalently attached to both of its strands' free 3' ends. This is then mixed with PCR products. When the free 5' ends of the PCR product strands attack the topoisomerase/3' end of each vector strand, the strands are covalently linked by the already bound topoisomerase. This reaction proceeds efficiently when this solution is incubated at room temperature with required salt.[1] Different types of vectors are used for cloning fragments amplified by either Taq or Pfu polymerase as Taq polymerase (unlike Pfu) leaves an extra "A" nucleotide at the 3'end during amplification.

Topo vector