Authors: Dr. Rajesh C. Jeeterwal*1 and Anju Nehra2
1Young Professsional- II, ICAR-AICRP on Pearl Millet, Mandor, Jodhpur 342304 (Rajasthan)
2Ph. D. Scholar, SKN Agriculture College, (SKN Agriculture University, Jobner), Jaipur 303329 (Rajasthan)
*Corresponding author email: jeeterwal.raj.pbg@gmail.com
A cloning vector may be a small piece of DNA, taken from a micro-organism or from the cells of eukaryotes, that may be stably maintained in an organism, and into that a foreign deoxyribonucleic acid fragment is inserted for generating multiple copies of Dna. The vector thus contains features that allow the convenient insertion or removal of a deoxyribonucleic acid fragment to or from cloning vector, with a restriction endonuclease that cuts the deoxyribonucleic acid. Dna segments so generated contain either flush or protruding ends, and vector and foreign dna with compatible ends will then be joined together by ligation. When a deoxyribonucleic acid fragment has been cloned into a cloning vector, it should be further sub cloned into another vector designed for a specific use.
The cloning vector could be plasmid (extra chromosomal DNA), a bacteriophage (lamda, baculo virus, adeno virus, retro virus), cosmid (a combination of plasmid and phage), yeast, phage M12, T1 plasmid, phasgemids, plants and fungi. The cloning in different vectors are decribed below.
1. Cloning in Bacterial Plasmids
The plasmid vector pBR322 was the workhorse of molecular biologists and was practically the only choice till recent years. However, much superior vector like pUC PGEM vectors are available today.
The vectors pBR322 or derivatives like PBR325, pBR327 or pBR328 have two antibodies markers campr and tetr, which render the host bacteria resistant to the antibiotics like Ampicilin and Tetracycline. These genes have unique restriction sites for Pst-I, pou-I, Sca-I etc. (on amp gene) and Bam H-I, Sal-I and Hind-III etc. (on tet gene). When a foreign DNA as well as this vector DNA are restricted with any one of these enzymes and fragments from both the sources are mixed, they get ligated in presence of the enzyme DNA ligase. Insertion of the foreign DNA in any one of these genes (depending upon the restriction enzymes used) renders these genes inactivated. Thus host becomes sensitive to the corresponding antibiotic. Upon transformation of the recombinant vector in E.coli, the recombinant clone is replica plating on two nutrient agar plates (in identical positions) one containing amplicillin and the other containing tetracycline. The recombinant clone will grow in one antibiotic plate but not the other because of gene inactivation. This clone is picked up from the plates where it has grown and cultivated.
The foreign DNA (passenger DNA), which is to be inserted, is restricted out from the source by one or two restriction endonucleases(s). Similarly the vector DNA is also restricted with the same one or two enzymes. The vector DBA is subjected to enzyme alkaline phosphatase treatment, which knocks off the 5’ phosphate preventing the vectors from rejoining by it. The foreign DNA and vector DNA are mixed in presence of the enzymes E.coli DNA ligase or T4 DNA ligates the two complementary DNA strands. Few of the restriction enzyme makes blunt end cuts, which can also be ligated by T4 DNA ligase under appropriate conditions.
The ligated vector is then passaged into an appropriate host bacteria like (E.coli). This process is called “Transformation”. The cells are rendered competent by treatment with CaCl2 or by electrical current (electroporation) or by Laser beam when it can accept the input DNA across its cell wall/membrane.
The transformed DNA after entry has any one of the following fates:
- It remains as a free entity and replicate either independently or along with the host v genome.
- Gets integrated with the host genome.
- It may survive but do not replicate so that only one cell becomes a partially diploid.
- The host cell enzymes degrade the transformed DNA (Host restriction).
- The DNA is progressively lost due to “Genetic incompatibility”.
Several plasmid vectors have been incorporated with promoters from bacteriophage like T3, T7, SP6 (e.g. pGEM vectors) adjacent to the polyelonal site. The promoter is placed in one or both strands so that DNA dependent RNA polymerase can transcribe large quantity of m-ma. The RNA can be used as hybridization probe, PCR and for in vitro translation.
- Cloning in non E.coli Bacteria
4. Cloning in Lambda Bacteriophase
Recombinant phage DNA molecules with upto 25kb is introduced into bacterial cell by the following two methods:
- Transfection: In this case the purified phage DNA or recombinant phage DNA is taken up by competent E.coli cells (as in the case of plasmid) by heat shock.
- In vitro packaging: This has developed as the method of choice over transfection because of higher efficiency of introduction of foreign DNA in to bacterial host. The lambda DNA (52kb) can be increased by only 5% thus only 3kb of new DNA can be added. However, a large portion of lambda genome (map position from 20-35) can without affecting its infectivity to E.coli cells. Thus about 18kb inserts DNA can be cloned in vectors. This recombinant phage however, cannot integrate into host (non lysogenic) but only enter lytic cycle, which is a desirable quantity in gene cloning. Additional restriction sites present in the vector DNA have been eliminated by natural selection. The lambda DNA can be used both as an insertion as well as a replacement vector. The insertion vectors (e.g. lambda gt 10, lambda charon 16) are generated by deleting a portion of non essential genes of the phage and ligating the two arms which carries at least one restriction site. Insertion upto 8kb in te unique Eco R-I site located in the el gene of lambda gives to clear (non turbid) plagues due to loss of cells on bacterial lawn. The recombinant lambda charon 16 however, provides clear instead of blue plagues on X-gal plates. The replacement vectors (e.g. lambda EMBL4, WES B’ have at least two restriction sites for deleting a portion of replaceable DNA fragments also called stuffer fragments of the phage DNA where the foreign DNA can be ligated. This type of vectors can carry larger DNA fragments for cloning. The lambda EMBL4 can carry 23kb of DNA insert and three pairs of restriction sites namely, Eco H-I and Sal-I flanks the arms of the vectors. The lambda WES lambda B’ has one pairs of ECO R-I sites and can accommodate 15kb inserts. The charon series of vectors extending upto charon 40 which can accommodate upto 24.2kb of DNA inserts. Each of the plaques formed by lysis of bacterial cells on agar plate represent a single transacted cell thus it contains identical phage particles. Cloning in lamda vector is similar to that in plasmid. However, this necessitates that the vector is a circular one with its cos sites tagged with each other by hydrogen bond. Transfection with ligated phage particles unlike plasmid transformation is not very efficient. Therefore, aan alternate approaches of in-vitro packaging of recombinant vector DNA have been developed. The digested vector gives a long left arm between which the insert DNA is to be ligated. The presence of the ‘cos’ site offers efficient in vitro packing of DNA with the commercially available head and tail of the vector. This makes a viable phage, which infect E.coli at a high efficiency and generate large number of recombinent plaques.
Cosmid (e.g. pJB-8) is a hybrid between a plasmid (replication origin and selectable marker) and a phage (the ‘cos’ site required for in vitro packaging of nucleic acid in the phage protein coat). It is possible to clone 45kb of DNA in the cosmid vector. The cosmid lacks all other genes of lambda phage and hence it cannot produce plagues but grow like plasmids. Partially digested DNA is ligated and the appropriate sized DNA is packaged after cleaving the ‘cos’ site by the in vitro packaging system and thus the recombinants cosmids are placed in natural phage particles. The phage carrying the recombinant cosmids, which also carries an antibiotics marker, is used to infect E.coli cells and colonies of antibiotic resistant cells are selected. All colonies are recombinants as non-recombinant linear cosmids are too small to be packaged into lamda heads.
6. Cloning in M13 Vector
The bacteriophage M13 unlike a plasmid carries gene, which codes for its own replication, phage specific DNA replicative enzymes and protein coat, which are most essential for M13. The 6.4kb vector has a single 507 nucleotides intergenic sequence, which can accommodate foreign DNA. The M13 vector has double stranded DNA as replicative from (required for cloning) where as the released vector is a single stranded one (required for use as DNA probe and in sequencing as well as in vitro mutagenesis. Several improvements have been made in M13 vectors. One great advantage of M13 mp7 is that any DNA inserted in to Bam H-I Sal-I, Pst-I sites can be restricted out from recombinant vector by single Eco R-I digestion.
The maximum size insert that can be accepted by M13 vector is a limiting factor. It conventionally accepts 1500 bp although upto 3kb DNA has been cloned. This problem has been resolved by developing a vector called pEMBL8 ehere the M13 genome has been cloned in pUC-8 plasmid. The signal sequence of M13 makes the pEMBL-8 to be secreted out as single standard but it need a helper p (normal) M13 phage which codes for the replicative enzyme and phage coat protein. By this can be cloned and selected in X-gal plates.
7. Cloning by Reverse Transcription
Some viruses are having RNA as their genetic material. Some of them (e.g. Retro virus) synthesis a complementary DNA (cDNA) by the help of the enzyme reverse transcriptase which later duplicates and integrates in the host genome upon infection. Once a m-RNA specific for a gene has been identified as per methods before it can be cloned and amplified.
8. Bifunctional Vectors
Bifunctional vectors (e.g. pHP-13) provides two methods of selection like antibiotic resistance and alpha-complementation (lac z’ expression).
- Shuttle Vectors
10. Cloning in Expression Vectors
Expression vector (pars-035 and 036, lamda gt Ii etc.) are used for cloning of genes, which need to be transcribed and translated into large quantity of protein. These vectors have strong promoters like that from bacteriophage T7 which express the gene to produce large quantity of protein.
11. Cloning in Phagemids
Phagemids contain a plasmid like pUC118 and 476bp from the intergenic region of wild type M13 inserted at the Nde-I site of pUC-118. infection along with a helper virus M13KO7 produces single strand copies of phagemid genome with lac z’ gene for selection on X-gal for selection on X-gal plates.
12. Cloning in Yeast and Plants
Yeasts are unicellular eukaryotic organism, which can be propagated stably as a haploid or diploid. The Bakers yeast Saccharomyces cervisiae, is most commonly used in cloning although the fission yeast Sachizaosachoromyces probe is also used. There are three advantages in choosing yeast as a cloning vector:
- Yeast being an eukaryotic, it offers a better host vector system than bacteria for cloning of human or other genes.
- Selection by complementation for the cloned gene and subsequent isolation of cloned gene virtually permit isolation of any yeast gene.
- Extra secretary function permits easy collection of the cloned product released outside into the media. Cloning vectors (e.g. Ylp, Yrp7 Yep, 2 microns plasmid vector) have antibiotic markers for selection. The YAC or yeast artificial chromosome is a new dimension in gene cloning. Its main use is in cloning large DNA fragments (more than the capacity of cosmids). Cloning in higher plants, following vectors are used:
- Ti plasmid of Agarobacterium tumefaciens
- Plant viruses like caulimo viruses and Gemini viruses
- Direct gene transfer without a cloning vector
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