Thursday, June 20, 2013

Vectors for cloning

Cloning vectors:

A vector is a molecule of DNA where DNA of interest can be integrated. Essential properties of a vector include: 1) It must be capable of autonomous replication within a host cell; 2) it must contain at least one restriction endonuclease site; 3) it must carry at least one gene that can be used to select the cloned vector, such as an antibiotic resistance gene (selectable markers) Bacterial Plasmids:

These are small circular duplex DNA whose natural function is to confer antibiotic resistance to the host cell. These are used in recombinant technology because,
  1. They  exist  as  single  of  multiple  copies  within  bacterium  and  have  independent replication.
  2. Complete DNA sequences of many plasmids are known hence precise location of restriction enzyme cleavage site for inserting foreign DNA is available.
  3. They are small and are easily separated from host chromosome.
  4. The inserted DNA can be easily removed by using original restriction endonuclease from the site of insert.
  5. These can accept DNA fragment 6-10 kb long.
  6. They can be introduced into bacterial cells by transformation: Plasmid vector pBR322 can accept 0.01-10 kb DNA.
Phages (bacterial virus):

They have linear DNA molecules into which foreign DNA can be inserted at multiple restriction enzyme sites. Since one third of phage DNA is non essential and can be replaced with foreign DNA without disruption its function. Chimeric DNA is packaged into phage particle by adding them to crude bacterial cell extracts that contain all the proteins needed to assemble a complete phage. This is called invitro packaging. All viable phage particles will contain foreign DNA fragment in their head. Then subsequently they are transformed into bacteria. Inserted DNA is collected after the completion of lytic cycle where new phages are produced. These can accept DNA fragments 10-20 kb long, a limitation imposed by the amount of DNA that can be packed into the phage head. E.g. bacteriophage ʎ.

Phagemid vectors:

These are constructed by inserting the DNA of phage and plasmid. These are single stranded.

Cosmids:

These are plasmids that contain the DNA sequence called cos sites required for packaging lambda DNA into phage particle. These vectors grow in plasmid form in bacteria and these have many plasmid features like plasmid origin of replication, gene for drug resistance. It can accept 35-50 kb long DNA.

Bacterial Artificial Chromosome (BAC), Yeast Artificial Chromosome, E. coli bacteriophage P1- based (PAC) vectors:

BAC, are the plasmids designed for cloning of very long segment (100 – 300 kb) of DNA. These include selectable markers like resistance to chloramphenicol, gene that produce enzyme beta galactosidase as well as stable origin of replication that maintains plasmid. After recombination, the circular plasmid (BAC) containing large target DNA (plasmid DNA + inserted DNA) is inserted to host bacteria by electroporation. Recombinant DNAs are screened for resistance to antibiotic chloramphenicol to which they are resistant. Plates also contain substrate for beta galactosidase that yields colored product. Colonies with active beta galactosidase and hence no DNA insert in BAC vector turn blue; colonies without this enzyme activity-and thus with the desired DNA inserts are white. P1 can accept 50-250 kb.

YAC can accept 500-3000 kb DNA. Due to their greater capacity they have replaced former ones. YAC contain all elements needed to maintain a eukaryotic chromosome in yeast nucleus: a yeast origin of replication, selectable markers, and sequences needed for stability and proper segregation of chromosomes at cell division. It is propagated as circular bacterial plasmid. The entire sequence of yeast is known. It is easy to grow and maintain in large scale in laboratory. Here plasmids vectors are constructed that can be transferred to yeast.

CEN is centromere, TEL is telomere, X and Y are selectable markers.


A large segment of DNA upto 2 x 106 bp from human genome is produced by partial digestion by endonuclease and is ligated to the two arms to create YAC. This YAC transforms yeast cell.

After culture only those yeast cell will grow that contain a artificial chromosome with large insert.

Shuttle vectors: 

Plasmids that can be propagated in cells of two or more different species are called shuttle vectors.

Expression vector:

A vector which synthesizes and expresses the protein coded by inserted DNA is called expression vector. These are used to produce proteins by genetic engineering techniques. A expression vector contains all the machinery needed for protein synthesis.
Fig. Expression Vector
This figure shows the DNA sequences in typical E. coli expression vector. The gene to be expressed is inserted into one of the restriction site in the polylinker region near promoter (P). This also contain the gene for ribosome binding site for mRNA.

Nowadays chemical method of DNA synthesis allows the synthesis of cDNAs segments that contain the sequence of gene of interest (100-150 nt segments)

(Source: Lehninger's Textbook of Biochemistry and Harper's Illustrated Biochemistry)
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