Top 3 Methods of Recombinant DNA Formation

The following points highlight the top three methods of recombinant DNA formation. The methods are: 1. Transformation 2. Transfection 3. Non-Bacterial Transformation.

Recombinant DNA Formation: Method # 1. Transformation:

The restriction enzyme which causes a break in foreign DNA also causes a staggered cut in the vector DNA at a specific cleavage site. The cohesive ends of vector DNA possess the sequences of nucleotides complementary to the cohesive ends of foreign DNA. This can be illustrated using example of enzyme Eco RI, similar sticky ends having same base sequences in single stranded ends will be produced.

When the two ends of the vector DNA meet the complementary ends of foreign DNA, the two will be held by H bonding. This process in which two separate strands of nucleic acid interact to form duplex molecule by interaction between complementary base pairs present in the individual strands is often referred to as annealing (Fig. 24.9).

Foreign DNA fragment with non-sticky ends are first made stricky by addition of a homopolymer tail to the 3′ hydroxyl end. Homopolymer tail is essentially a sequence of the few similar nucleotides, e.g., poly A (AAAAA…….) or poly T (TTTTT…..).

Now the complementary nucleotide sequences of DNA fragment and those of vector are covalently linked together with the help of enzyme terminal deoxynucleotidyl transferase (Figs. 24.10 and 24.11).

Enzyme DNA ligase seals and stabilizes the cut ends of two DNA molecules by catalyzing phosphodiester bonds between 3’0 H and 5′ phosphate of two adjacent nucleotides. The main advantage of this technique is that the foreign DNA segment can be easily retrieved from the cloned copies of chimeric DNA by recleavaging using the same enzyme.

At the same time there are two disadvantages of this technique:

(i) The two ends of the cleaved vector DNA or of foreign DNA may join end to end and they may be re-circularised before insertion,

(ii) Recognition site particularly in the segment to be cloned may not lie at a convenient position. In this process, the insert may contain a selectable marker which allows for identification recombinant molecules. An antibiotic marker is often used. So a host cell without a vector dies when exposed to a certain antibiotics and the host with the vector will live because resistant.

Transformation of Escherichia coli with relatively large pieces of chimeric DNA can be facilitated by treating the cells with CaCl₂ at low temperature to make them permeable. With this procedure the transforming DNA enters the cell intact and the host bacterium remains viable. The entry of rDNA into E.coli is also facilitated by suddenly raising the temperature to 42°C for 2-5 minutes.

At this temperature, the cell membrane of E. coli becomes permeable to macromolecules Strains of coli have now been evolved which are permeable to macro-molecules. Then during the growth of transformed E. coli cell, the recombinant DNA of plasmid replicates.

The bacterial cell contains several plasmids in it and some of them are recombinant type and the others just the plasmid vehicles. In such cases it may not be easy to determine whether a clone of transformed E. coli cell carries a recombinant DNA molecule or just the plasmid vehicle.

The selection and isolation of transformed host depends on their resistance to antibiotics, colour change or some other characteristics. Different vectors have different properties to make them suitable to different applications. Some properties can include symmetrical cloning sites, size and high copy number.

For selection of transformed cells on the basis of their resistance antibiotics, it is important to incubate the cells in a medium without antibiotic for about an hour to allow the plasmid antibiotic resistant genes to be expressed.

The cells can then be placed on solid medium containing antibiotic for the selection of colonies with recombinant DNA Plasmids possessing antibiotic resistance marker will form a colony and those without resistance marker will be killed. This procedure for detecting insertion is called intentional inactivation.

In strain pBR-322, there are two different antibiotic markers—tet-r gene responsible for resistance to tetracycline and amp-r gene for resistance to ampicillin. One of the two genes becomes inactivated during restriction enzyme cleaving and insertion of foreign DNA. Now if the cells are plated on a medium containing ampicillin, all surviving colonies must be amp-r resistant.

Recombinant DNA Formation: Method # 2. Phage Introduction or Transfection:

Phage introduction is the process of transfection which is equivalent to transformation excepting that a phage is used instead of bacterial plasmid. In vitro packaging, a vector uses lambda or M 13 phages to produce phage plaques which contain recombinant DNA. The recombinant DNA can be identified using various selection methods. For the first time bacteriophage was used to transfer the foreign DNA into E. coli cells.

If the vector is bacteriophage, its replication in bacterial host would result in phage particles, each carrying an identical copy of target gene. When phage vectors are used, a population of cells is infected with the viruses and virus replication proceeds spontaneously.

Eventually, the phage DNA containing the target gene is inserted into the bacterial chromosome where it will replicate as though it was a part of normal chromosome. Thus these vectors along with target genes are introduced into a bacterial host. This is usually achieved with the enzyme DNA ligase. It is important for ligation that vector DNA and the target DNA have been cut by the same restriction endonuclease.

Recombinant DNA Formation: Method # 3. Non-Bacterial Transformation:

This process is similar to transformation. The only different between the two is that non-bacterial transformation does not use bacteria such as E. coli as the host. In microinjection the DNA fragment is injected directly into the nucleus of the cell be| transformed.

In biolistics, the host cells are bombarded with high velocity micro-projectile gun such as particles of gold or tungsten that have been coated with DNA (Fig. 24.12).