Agriculture: Methods For Recombinant DNA molecule
07 January 2013
Chimeric DNA or recombinant DNA molecules are produced by inserting foreign DNA segment into the DNA molecule of the vector. There are three common methods for the preparation of chimeric DNA.
There are three methods for making recombinant DNA molecule i.e.
1) Palindromic and staggered cleavage
A palindromic sequence is one which has complementary sequence on the both ends of single strand e.g ATC………….GAT.
This is the most common method in which a restriction enzyme cause staggered cuts producing short complementary single stranded sticky ends(palindromic sequence). For example Eco RI, which cuts the the DNA molecule at specific position. When another DNA cuts with the same enzyme similar sticky ends will be produced on the single strand of the DNA molecule. When it is mixed with previous DNA molecule similarly treated the chimeric DNA will be produced. Enzyme DNA ligase helps to joinig the broken ends of either DNA molecules.
This technique has the advantage of regenerating two restriction sites (e.g., EcoRl site) in the chimeric DNA, so that the foreign DNA segment can be retrieved rather easily from the cloned copies of chimeric DNA by cleavage again with the same enzyme.
There are also two disadvantages with this technique.
(i) The two cleaved ends of a vector or of a foreign DNA may join end to end before getting inserted. Therefore, while isolating chimeric DNA, one will have to select chimeric molecules each having only a single insert. This can be achieved by separating molecules of different sizes by gel electrophoresis. (ii) The recognition site, particularly in the sequence to be cloned may not lie at a convenient position, so that sometimes only a part of the desired segment will be inserted.
2) Adding poly dA at the 3rd ends of the vector and poly dT at the 3rd of DNA to be cloned:
In this method DNA is cut at desired position both of vector and foreign DNA without staggered cleavage. By using precursor dATP poly dA is added at the both 3rd cut end of the vector with the help of an enzyme terminal transrerase. Similarly by using precursor dTTP, poly dT is added on the 3rd cut ends of the DNA sequence to be cloned. These two DNA molecules e.g vector and clone can be joined by annealing the poly dA and poly dT and then ligating by using DNA ligase.In this technique, there is no chance of reannealing between the two cut ends of the same DNA molecule, so that one of the disadvantages of the first method has been overcome in this case. However, in this technique it will not be easy to retrieve the cloned DNA, because the recognition site of the enzyme due to insertion of poly dA and poly dT has been lost in this case.
3) Blunt end ligation by T4 DNA ligase:
In this method double stranded DNA molecule is cut at same place in both DNA strand by using restriction enzyme. These broken ends then joined by foreign DNA molecule irrespective of the sequence present at the broken ends of two DNA molecules. The T4 DNA ligase is used for joining of these two broken ends. The disadvantage of this technique is that any sequence can be joined even belonging to same DNA molecule.These ‘linkers’ can be linked to the blunt ends of vector DNA or of an insert by blunt end ligation. This will allow the creation of an EcoRIsite in the linker region of the vector. Therefore, with this method it is now possible to insert a foreign DNA segment at a particular site in the linker region of the vector and then retrieve this foreign DNA segment whenever necessary.
This method make use of DNA duplexes (linkers) that contain EcoRI palindrome or some equivalent palindrome, which may small in size, can be synthesized chemically. These linkers can be linked to the blunt ends of the vector DNA or of insert by DNA ligation.
By: Waqas Shafqat Chatha, Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan.
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