Tools used in Recombinant DNA Technology

Enzymes

1. Restriction endonucleases are used for cutting DNA molecules at speciic points. They are called biological /molecular/ chemical scissors/knives/ scalpels.

2. DNA ligase,

3. Reverse transcriptase,

4. DNA polymerase,

5. Alkaline phosphatases

 

Restriction endonucleases

They are used for cutting DNA molecules at speciic points. They are called biological /molecular/ chemical scissors/knives/ scalpels.

 

Vectors

  • They are the DNA molecules used to transfer genetic material into another cell.
  • It is like the mosquito acts as an insect vector to transer the malarial parasite into human body.
  • It may be a plasmid or bacteriophage DNA or Artificial DNA or cosmid.
  • These are low molecular weight DNA molecules capable of multiplying independent of the genomic DNA.
  • They can be employed to make copies of desired DNA fragments

Properties of vectors

  • The cloning vectors must be able to replicate in the host cell.
  • Thus it must have a replication origin.
  • A replication origin is a sequence element which is recognized by the host cell' s replication machinery (i.e. hosts DNA enzymes).
  • the cloning vector can be amplified in the host cell.
  • A segment of foreign DNA which has been inserted into the vector's DNA is therefore ampliied along with vector.
  • Large amounts of the desired foreign DNA are therefore, produced by gene cloning.
  • Vectors need to have restriction endonuclease recognition sites and some marker gene which will express in the host cell.
  • Marker in vector helps in identification of transformed bacterial colony.

 

Introduction of Recombinant vector in host:

The efficiency of transformation is very low and only a few cells in the population take up and retain the exogenous DNA.

Insertion of a vector into the target cell is usually called

  1. Transformation for bacterial cells,
  2. Transfection for eukaryotic cells,
  3. Insertion of a viral vector is called transduction.

 

Some of the common vectors:

  • Bacteria are plasmids, cosmids, lambda phage.
  • Baculovirus is useful in insects.
  • Ti plamid is for plants and YAC (yeast artificial chromosome) is for yeast cells.