DNA Technology

DNA Technology

1. Isolation – of the DNA containing the required gene

2. Insertion – of the DNA into a vector3. Transformation – Transfer of DNA into a

suitable host4. 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) isformed using reverse transcriptase on the mRNA template.

Single stranded cDNA is used to form double strandedDNA using DNA polymerase

This forms a double stranded copy of the Human Insulingene.

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?