recombinant DNA Technology Flashcards
What is recombinant DNA technology
Transfer of DNA fragments from one organism to another
Why is recombinant DNA technology possible
Genetic code is is universal
As are transcription and translation mechanism
Method 1- Conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase
1)mRNA isolated from a cell that codes for a protein for the desired gene
2)Mix mRNA with DNA nucleotides and reverse transcriptase—> reverse transcriptase uses mRNA as a template to synthesise a single strand of cDNA
3) DNA polymerase forms second strand of DNA using cDNA as template
Method 2- Using restriction enzymes to cut a fragment containing the desired gene from DNA
1)Different restriction endonucleases cut DNA at specific sequences of bases called recognition sequence. Shape of recognition site complementary to active site.
Some restriction enzymes cut in a staggered end which forms the sticky end which is complementary to each other
Method 3- creating the gene in a gene machine
Synthesises fragments of DNA from scratch without the need for a pre-existing DNA template.
DNA fragments produced quickly/ accurately
Free om introns so can be transcribed by prokaryote who can’t remove introns
Advantages of using mRNA to make DNA fragment rather than restriction enzymes to cut genes from DNA
More mRNA in cell than DNA–> easily extracted
Introns removed by splicing whereas DNA contains introns
Bacteria cannot remove introns
The use of restriction enzymes and ligases to insert fragments of DNA into vectors
-Vector transports DNA into host cell e.g., plasmids or bacteriophages
-Vector DNA and DNA fragment is cut using the same restriction enzyme so they have complementary sticky ends
-DNA ligase forms phosphodiester bonds between adjacent nucelotides on sticky ends
The use of marker genes to detect genetically modified cells/ organisms
Not all cells will take up vectors.
Marker genes, are added in order to identify which cells have the desired gene
Gene markers can be:
- Resistant to an antibiotic
-Fluorescent protein
-Enzymes whose action can be identified
