Restriction endonucleases are used to cut the DNA into many small fragments, to isolate specific genes. They usually leave a staggered cut, leaving a sticky end.

Example of restriction endonuclease: EcoR1 from E.coli, this breaks DNA where guanine is next to adenine. Leaves a sticky end. The sticky ends on each strand are in reverse order of each other (palindrome)

Drawbacks of using endonuclease:

If the base sequence that the enzyme cuts is in the gene that needs to be isolated, then the gene will be broke into fragments that have no function
Eukaryotic genes contains introns, normally intons are removed before mRNA moves to the cytoplasm. Bacteria do not have introns so may not have the appropriate enzymes to remove them, any protein translated will include the introns and won’t be functional

Reverse Transcriptase

Though cells only have 2 copies of the insulin gene, there may be many copies of the mRNA strand, especially in those cells that secrete insulin. This mRNA can be extracted.

The cDNA produced by reverse transcriptase is complementary to the RNA, therefore many copies of cDNA for insulin can be made.

This doesn’t have the problem of introns as it is synthesised from the mRNA, where the introns have already been removed. DNA polymerase catalyses the synthesis of DNA that is complementary to the single stranded cDNA, making a double stranded molecule containing the gene for insulin.

	Created by Fauzia Ali about 7 years ago

A-Level Biology (Applications of Reproduction and Genetics) Note on Genetic Engineering, created by Fauzia Ali on 04/02/2017.