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

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)

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

Reverse transcriptase Watch the animation http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

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

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

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

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

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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. 

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.

Restriction endonucleases Watch the animations http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf http://trc.ucdavis.edu/biosci10v/bis10v/media/ch11/restriction_enzyme.html Find out more by doing the work sheet!