- The enzymes that cut DNA at specific sites into fragments.
- They belong to a class of enzymes called nucleases.
- In 1963, two enzymes responsible for restricting growth of bacteriophage in E. coli were isolated. One enzyme added methyl groups to DNA. The other (restriction endonuclease) cut DNA.
- More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.
Naming of the restriction enzymes:
- First letter indicates genus. The second two letters indicate species of prokaryotic cell from which they were isolated.
E.g. EcoRI comes from E. coli RY 13 (R = the strain. Roman numbers = the order in which the enzymes were isolated from that strain of bacteria).
Types of Restriction enzymes:
· Exonucleases: They remove nucleotides from the ends of the DNA.
- They cut at specific positions within the DNA. E.g. EcoRI.
- They bind to specific recognition sequence of the DNA and cut the two strands at specific points.
- The first restriction endonuclease is Hind II. It cuts DNA molecules by recognizing a specific sequence of 6 base pairs. This is called the recognition sequence for Hind II.
- Restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA. It is a sequence of base pairs that read the same on the two strands in 5' → 3' direction and in 3' → 5' direction. E.g. Palindromic nucleotide sequence for EcoRI is
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
Steps in formation of recombinant DNA by EcoRI
- Restriction enzymes cut the strand a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded overhanging stretches at the ends. They are called sticky ends. They form H-bonds with their complementary cut counterparts. This stickiness facilitates action of the enzyme DNA ligase.
- When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of sticky-ends and these are joined together by DNA ligases.
- It is a DNA molecule that can carry a foreign DNA segment and replicate inside the host cells.
E.g. Plasmids, bacteriophages etc.
- Plasmids are autonomously replicating circular extra-chromosomal DNA of bacteria. Some plasmids have only 1-2 copies per cell. Others have 15-100 copies per cell.
- Bacteriophages (high number per cell) have very high copy numbers of their genome within the bacterial cells.
- When the cloning vectors are multiplied in the host, the linked piece of DNA is also multiplied to the numbers equal to the copy number of the vectors.
Features required for cloning into a vector
a. Origin of replication (ori)
- This is a sequence where replication starts.
- A piece of DNA linked to ori can replicate within the host cells. This also controls the copy number of linked DNA. So, for getting many copies of the target DNA, it should be cloned in a vector whose origin support high copy number.
b. Selectable marker (marker gene)
- It is a gene that helps to select the transformants and eliminate the non-transformants.
- If a piece of DNA is introduced in a host bacterium, it is called transformation. Such bacterium is transformant. If transformation does not take place, it is non-transformant.
- Selectable markers of E. coli include the genes encoding resistance to antibiotics like ampicillin, chloramphenicol, tetracycline, kanamycin etc. Normal E. coli cells have no resistance against these antibiotics.
c. Cloning sites
- These are the recognition sites for restriction enzymes.
- To link the alien DNA, the vector needs a single or very few recognition sites.
- More than one recognition sites generate several fragments. It complicates the gene cloning.
- Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
E.g. In vector pBR322, foreign DNA is ligated at Bam H I site of tetracycline resistance gene. As a result, recombinant plasmid is formed. If ligation does not occur, it is called non-recombinant plasmid.
- When a foreign DNA is inserted within a gene of bacteria, that gene is inactivated. It is called insertional inactivation. Here, the recombinant plasmids lose tetracycline resistance due to insertion of foreign DNA.
- When the plasmids are introduced into E. coli cells, 3 types of cells are obtained:
o Non-transformants: They have no plasmid. So they are not resistant to either tetracycline or ampicillin.
o Transformants with non-recombinant plasmid: They are resistant to both tetracycline & ampicillin.
o Transformants with recombinant plasmid: They are resistant only to ampicillin.
- Recombinant plasmids can be selected out from non-recombinant ones by plating transformants on ampicillin medium. Then the transformants are transferred on tetracycline medium.
- The recombinants grow in ampicillin medium but not on tetracycline medium. But, non-recombinants grow on the medium containing both the antibiotics.
- Thus, one antibiotic resistance gene helps to select the transformants. The inactivated antibiotic resistance gene helps to select recombinants.
- But this type of selection of recombinants is a difficult procedure because it needs simultaneous plating on 2 plates having different antibiotics. So, alternative selectable markers have developed based on their ability to produce colour in presence of a chromogenic substrate.
- In this, a recombinant DNA is inserted into the coding sequence (gene) of an enzyme, b-galactosidase. So, the gene is inactivated (insertional inactivation). Such colonies do not produce any colour. These are identified as recombinant colonies.
- If the plasmid in bacteria have no an insert, it gives blue coloured colonies in presence of chromogenic substrate.
d. Vectors for cloning genes in plants & animals
Genetic tools of some pathogens can be transformed into useful vectors for delivering genes to plants & animals. E.g.
· Agrobacterium tumefaciens (a pathogen of many dicot plants) can deliver a piece of DNA (T-DNA) to transform normal plant cells into a tumor. These tumor cells produce the chemicals required by the pathogen.
The tumor inducing (Ti) plasmid of A. tumefaciens is modified into a cloning vector which is not pathogenic but can use mechanisms to deliver genes of interest into plants.
· Retroviruses in animals can transform normal cells into cancerous cells. So, they are used to deliver desirable genes into animal cells.
- Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. So bacterial cells are made ‘competent’ to take up alien DNA or plasmid as follows:
- Treat bacterial cells with a specific concentration of a divalent cation (e.g. calcium) → DNA enters the bacterium through pores in cell wall → Incubate the cells with recombinant DNA on ice → Place them briefly at 420C (heat shock) → Put them back on ice → Bacteria take up recombinant DNA.
Other methods to introduce alien DNA into host cells
· Micro-injection: In this, recombinant DNA is directly injected into the nucleus of an animal cell.
· Biolistics (gene gun): In this, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA. This method is suitable for plants.
· ‘Disarmed pathogen’ vectors: They infect the cell and transfer the recombinant DNA into the host. E.g. A. tumefaciens.