DNA Technology

1.Isolation – of the DNA containing the required gene 2.Insertion – of the DNA into a vector 3.Transformation – Transfer of DNA into a suitable host 4.Identification – finding those host organisms containing the vector and DNA (by use of gene markers) 5.Growth/Cloning – of the successful host cells

Learning Objectives: Stage 1 – Producing DNA fragments How is complementary DNA made using reverse transcriptase? How are restriction endonucleases used to cut DNA into fragments?

Reverse Transcriptase A group of viruses called retroviruses (e.g. HIV) contain an enzyme called reverse transcriptase. It is used to turn viral RNA into DNA so that it can be transcribed by the host cell into proteins.

Reverse Transcriptase Reverse Transcriptase makes DNA from an RNA template – it does the opposite of transcription. DNA polymerase

Using reverse transcriptase B-cells from Islets of Langerhans in the Human pancreas. Extract mature mRNA coding for Insulin. A single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase on the mRNA template. Single stranded cDNA is used to form double stranded DNA using DNA polymerase This forms a double stranded copy of the Human Insulin gene.

Restriction Enzymes Bacteria contain restriction enzymes in order to protect themselves from invading viruses. Restriction enzymes are used by bacteria to cut up the viral DNA. These enzymes cut DNA at specific sites – this property can be useful in gene technology.

Restriction Enzymes – “Blunt Ends” Some restriction enzymes cut straight across both chains forming blunt ends.

Restriction Enzymes – “Sticky Ends” Most restriction enzymes make a staggered cut in the two chains, forming sticky ends.

Sticky Ends… Sticky ends have a strand of single stranded DNA which are complementary to each other. They will join with another sticky end but only if it has been cut with the same restriction enzyme.

Restriction Enzymes Also called restriction endonucleases. Have highly specific active sites. Usually cut DNA at specific sites – about 4-8 base pairs long – these are called recognition sites. Recognition sites are usually palindronic, which means the sequence and its complement are the same but reversed. E.g. GAATTC and the complement CTTAAG

Learning Objectives Stage 2 – Insertion in to a vector What is the importance of “sticky ends”? How can a DNA fragment be inserted into a vector?

Importance of “sticky ends” DNA from different source can be joined together IF they have the same sticky ends – the same recognition site. In order to have the same sticky ends they must have been cut using the same restriction endonuclease. Sticky ends are joined together using DNA ligase to join the sugar-phosphate backbone together. The new DNA molecule is called recombinant DNA.

Insertion of DNA into a vector VECTOR – used to transport DNA into a host cell. PLASMID – the most commonly used vector. A circular piece of DNA found in bacteria. Plasmids are useful because the nearly always contain antibiotic resistance genes (see later).

The Plasmid One of the antibiotic resistant genes is disrupted when the restriction enzymes cuts open the plasmid. The other antibiotic resistant gene is used in selection of the correct host cells. (See later)

Insertion into plasmids What combinations of plasmid will form?