Recombinant DNA (RDNA) Technology - EASY NOTES

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Definition:

-it is a type of genetic engineering technique in which a gene of one species is transferred to another species by artificial means.

Technique of RDNA technology involves:

a)SPLICING OF DNA BY RESTRICTION ENDONUCLEASES.

-- selection of parent DNA and selectively splitting using restriction endocucleases also called as molecular scissors.

-- these restriction endonucleases are isolated from bacteria.e.g - EcoRI from Escherichia coli.

-- Function of restriction endonucleases is to identify or recognize specific sequences called as palindrome sequence 

(palindromic sequences are inverted repeat sequences i.e the nucleotide sequence is same in both the strands when read in 5' to 3' direction)

--  the restriction endonuclease enzymes cut the DNA at the palindromic sequence.

--some restriction enzymes cut the DNA fragments in such a manner that there are no unpaired bases on either end  and they are called as blunt ends.

--some restriction enzymes cut the DNA fragments in a staggered manner on  the two strands of DNA so that 2-4 nucleotides of one strand are left unpaired and they are called as sticky ends. 

-- the so produced DNA fragments are called as restriction fragments.

-- these restriction fragments are 4-7 nucleotide base pairs long

2)ISOLATION OF SPECIFIC HUMAN DNA

--the so produced restriction fragment of interest is separated by electrophoresis/HPLC.

3)INSERTION OF ISOLATED HUMAN DNA INTO VECTOR:

--The specifically selected restriction fragment of DNA is inserted into a vector(carrier).

--Vectors can be plasmids,cosmids & bacteriophages.

--Most commonly used vectors are plasmids.

--both the vector DNA and human DNA are curt by same restriction endonuclease enzyme so that the cut pieces with sticky ends generated have a same sequence

4)JOINING OF TWO DIFFERENT FRAGMENTS BY DNA LIGASES

--the vector DNA & human cut piece DNA are incubated together and the complementary sequence on the sticky ends come in contact with each other.

-- DNA ligase enzyme is added and its function is to introduce phosphate link between vector and human DNA fragment.

-- after the formation of  phosphate links,finally the chimeric or recombinant DNA molecule is produced.

-- after the formation of chimeric DNA, amplification of the DNA copy is done in vivo by a process called as cloning.

Cloning has two steps

a)Introduction of RDNA into appropriate host cell - 

Introduction of RDNA into host cell(bacterial cell) is called as transfection & the cells are called as transformed cells.

b)Amplification of RDNA - 

-- the host cells containing RDNA are incubated and the conditions are provided such that in addition to the host DNA, the chimeric DNA also replicates.