Recombinant DNA technology Flashcards
(11 cards)
What is recombinant DNA technology?
Recombinant DNA technology involves the transfer of fragments of DNA from one organism or species to another. Because the genetic code is universal, the transferred DNA can be translated within cells of the recipient organism.
What are the 3 key ways of producing DNA fragments?
Using reverse transcriptase, using restriction endonucleases, and using a gene machine.
How can DNA fragments be produced using reverse transcriptase?
When using reverse transcriptase, mRNA for a specific polypeptide is isolated from the cell and mixed with free DNA nucleotides and reverse transcriptase. The reverse transcriptase uses the mRNA as a template to produce complementary DNAA which is a double-stranded copy of the required gene.
What is the role of restriction endonucleases?
Restriction endonucleases break phosphodiester bonds between DNA bases.
How can DNA fragments be produced using restriction endonucleases?
As the active site of the restriction enzyme is complementary to a specific recognition site on the DNA, the restriction endonucleases bind to the recognition site and cut the DNA at either side of the gene. Most restriction endonucleases form what is known as ‘sticky ends’.
What are ‘sticky ends’?
Sticky ends are staggered cuts. revealing unpaired base sequences at either end of the fragment (or plasmid)
How can DNA fragments be produced using a gene machine?
The AA sequence of a specific polypeptide is determined which tells us the mRNA base sequence from which we can determine the DNA base sequence which acted as a template for it. A machine assembles and joins together nucleotides in the desired sequence.
What are two ways of amplifying DNA fragments?
In vivo amplification, and in vitro amplification.
What is in vivo amplification?
The vector DNA and DNA fragment are combined to produce recombinant DNA.
Transformation involves the insertion of recombinant DNA into a host hell. If a plasmid is the vector, host cells can be stimulated to take in the plasmid. If a bacteriophage is the vector, bacteriophage injects its DNA into the bacterium.
Identification is when only very few host cells will take up the recombinant DNA. In order to identify which have taken up the DNA, we use marker genes which are inserted into the vector together with the required gene.
Lastly, the transformed bacteria are allowed to reproduce (in vivo cloning).
What is the role of marker genes?
Marker genes can code for antibiotic resistance. The bacteria can be grown on an agar plate containing the antibiotic, and so only transformed cells will survive and produce colonies.
They can also code for fluorescence which can be identified using UV light.
What is in vitro amplification?
A reaction mixture is set up containing DNA fragments to be copied, primers, DNA polymerase, and free DNA nucleotides.
Heat to 95 degrees celsius which denatures the DNA as the hydrogen bonds between strands break.
Cool to 50-60 degrees, allowing primers to anneal (bind) to complementary bases at ends of fragments.
Heat to 72 degrees so that DNA polymerase can carry out extension of the strands meaning new complementary strands are formed.
