Control of gene expression

What are the 5 stages of producing DNA fragments

Isolation

Insertion

Transformation

Identification

Cloning

What is the isolation stage of producing DNA fragments

Isolating the DNA fragments that have the genes for the desired protein (eg. cells from the pancreas for insulin.

What is the insertion phase of the production of a DNA fragment

Insert the DNA fragment into a vector (eg. a plasmid for a bacteria) using enzymes

What is the transformation phase of the production of a DNA fragment

Transfer of DNA into a suitable host cell - DNA is universal so it is not recognised as non-self

Describe the identification phase of the production of DNA fragments

Identification of host cells that have successfully taken up the gene by using gene markers.

What is the growth phase of producing DNA fragments

Growth and cloning of the host cells into a colony

Describe the action of reverse transcriptase and what happens after in production of DNA fragments

Takes a pretranscribed mRNA strand and makes a complimentary single stand of cDNA

The cDNA and mRNA are bonded together, so cDNA is isolated by the hydrolysis of the mRNA strand with sn enzyme

DNA polymerase is used to form a double stranded DNA molecule

Describe the action of restriction endonucleases

Restriction endonucleases hydrolyse phosphodiester bonds.

If cuts are are made between 2 opposite pairs this leaves blunt ends which cannot recombine

So, it cuts the DNA in a staggered fashion to exposes DNA and vector palindromes (sticky ends), if they are complimentary, they will recombine with one another.