1. Producing DNA fragments eg for manufacturing insulin
Restriction endonucleases
and
Reverse transcriptase

2. Outline of gene transfer & cloning
1. Isolation
Produce DNA fragments that have the required gene for insulin – using reverse transcriptase or restriction endonucleases
2. Insertion
Insert DNA fragments into a vector (e.g. a plasmid) – using DNA ligase. It becomes a genetically modified organism (GMO)

4. Identify host cells that have taken up the DNA
Outline continued
3. Transformation
Introduce DNA fragment into suitable host cell (eg bacteria)
4. Identification
Identify host cells that have taken up the DNA
– using gene markers
5. Growth/cloning
Culture host cells containing the DNA to produce the protein on a large scale

7. Reverse transcriptase
Watch the animation

8. Isolating a gene: Reverse transcriptase
mRNA is extracted from a cell (eg Pancreas cell)
Reverse transcriptase is then used to make complimentary DNA (cDNA)
Reverse transcriptase catalyses the production of DNA from RNA – (reverse of the usual transcription of RNA from DNA in protein synthesis)
The single stranded DNA produced is cDNA – made of nucleotides with bases that are complementary to the mRNA

9. Producing cDNA DNA copy of gene Transcribed to mRNA
Reverse transcriptase
cDNA strand
mRNA strand is destroyed
Double-stranded DNA copy of requied gene

10. Reverse transcriptase
Another enzyme, DNA polymerase, is used to make the other strand of DNA
DNA polymerase builds up the complementary nucleotides on the cDNA template
The double strand of DNA produced is the required gene
See Fig 2, page 247 of AQA A2 text book

11. Isolating a gene: Restriction endonucleases
There are many different restriction endonuclease enzymes
Each restriction endonuclease recognises and cuts DNA at a specific sequence of bases – a recognition sequence
This produces DNA fragments that contain the required gene

13. /tmp/PreviewPasteboardItems/LigaseAnimation6-1 (dragged).tiff

14. Restriction enzymes are used to cut a segment of gene from a human DNA molecule.
Restriction enzymes can either produce sticky ends or blunt ends.
Sticky ends are produced by cutting the DNA in a staggered manner, within the recognition site, producing single-stranded DNA ends.
These ends have identical nucleotide sequence and are sticky because they can hydrogen-bond to complementary tails of other DNA fragments cut by the same restriction enzyme. 

15. Isolate the DNA gene you want by:
Summary
Isolate the DNA gene you want by:
Either:
Take pancreas (or other) cell mRNA
Use Reverse Transcriptase to make complementary cDNA
Use DNA Polymerase to make the next strand of DNA = double strand with gene
Or:
Use Restriction Endonuclease to cut DNA into pieces, including the required gene
Then:
Insert DNA (with gene) into a vector plasmid using DNA ligase
Put into a host cell (eg bacteria)
Identify host cells with required DNA by using markers
Culture and grow protein on large scale.

16. Restriction endonucleases
Watch the animations
Find out more by doing the work sheet!