Controlling Gene Expression - Recombinant DNA Technology Flashcards
How is complementary DNA made using reverse transcriptase?
- Reverse transcriptase makes DNA copies from mRNA and naturally occurs in viruses such as HIV
- A cell that naturally produces the protein of interest is selected
- This cell should have large amounts of mRNA for the protein
- Reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence
- Single stranded complementary DNA is made
- This is made double stranded with DNA polymerase enzyme
How are restriction endonucleases used to cut the DNA fragments?
- Naturally occur in bacteria as a defence mechanism
- Each cut the DNA double strand at a specific base sequence being the recognition site so target specific base sequences
- When the cut occurs at two opposite base pairs, then blunt ends are produced
E.g. when cutting:
TT|AA
AA|TT - Some restriction enzymes cut the dna to form sticky ends where each end is palindromic As they can join to DNA by forming CBPs
Why are sticky ends important?
Because DNA from different sources that form sticky ends by being cut with the same restriction enzyme are can be joined together by DNA ligase to form a recombinant DNA molecule
What is a promoter region and terminator region
When preparing for insertion:
Promoter region: sequence of DNA added to the start of a DNA fragment which is the binding site of RNA polymerase to allow transcription to occur
Terminator region: sequence of DNA added to the end of a gene which causes RNA polymerase to detach so transcription stops
How is a DNA fragment inserted into a vector?
- bacterial cell with no plasmids is non transformed
- bacterial plasmid (the vector) is cut open using the same restriction endonuclease that cut the DNA fragment
- DNA fragment sticky ends are complementary to the sticky ends on the plasmid
- DNA fragment and open plasmid are combined with ligase which catalysed the condensation reaction to form phosphodiester bonds between nucleotides to join together the sticky ends of the dna fragments with some of the open plasmids
- some of the open plasmids have closed to some plasmids don’t have recombinant DNA and some of the plasmids do have recombinant DNA
How is the DNA of the vector introduced into the host cell?
- host cell being bacterial cell, to insert the vector being the plasmid into it, the bacterial cell has to be made more permeable by mixing it with Ca2+ and feeling heat shocked
- Mix the bacterial cells with the mixture of plasmids
- Some bacterial cells remain non-transformed where the plasmids don’t get into the cell
- Other bacterial cells are transformed as they contain plasmids but some contain recombinant DNA and others contain non recombinant DNA
- Marker genes are used to differentiate between the bacterial cells
- Once the transformed bacterial cells with recombinant DNA are identified, they are fermented to grow multiple copies of the recombinant plasmid and these can then produce the protein coded for by the inserted DNA fragment (e.g. insulin)
What are the three different marker genes used in genetic engineering to differentiate between the bacterial cells?
- antibiotic resistance genes
- genes coding for fluorescent proteins
- genes coding for enzymes
How does antibiotic resistant marker genes work?
- All the plasmids are resistant to both tetracycline gene and ampicillin gene
- The plasmids with recombinant DNA are not resistant to tetracycline but still resistant to ampicillin
- The bacterial colonies are grown on an agar plate
- They are then transferred to an agar plate laced with ampicillin antibiotic so here, only the transformed bacteria will grow
- These are then transferred to an agar plate laced with tetracycline antibiotic so that only the bacteria with the tetracycline resistance will grow which are the ones with the non recombinant DNA
- The bacteria that grew on the ampicillin and not on tetracycline are the ones with recombinant DNA
How do fluorescent marker genes work?
Gene that codes for a fluorescent protein made by jelly fish is inserted into the plasmids and the plasmids with recombinant DNA have the fluorescent gene disrupted so when the bacterial cells are transferred onto a Petri dish, the non glowing colonies are the ones that contain the recombinant plasmid
How do enzyme marker genes work?
- Lactase can turn a certain substance from blue to colourless
- Lactase inserted into plasmid
- If DNA fragment is in the plasmid, it disrupts the lactase
- Bacteria are then grown on the agar plate that contains the colourless substance
- Colonies that remain white as they couldn’t turn the colourless substance blue contain the recombinant plasmid
What is PCR?
Polymerase Chain Reaction: amplifies trace amounts of DNA to create a larger quantity of that DNA which can now be manipulated
How does PCR work?
- Using restriction enzymes, cut the DNA into fragments
- Select the target DNA section
- Heat the DNA at 95°C so the h bonds between CBPs are broken allowing the two strands to separate and expose the nucleotide bases of the target section
- Reduce the temp so specific primers complementary to the dna bases and form h bonds with CBPs
- The temp is raised again to 70°C so TAQ DNA polymerase binds to the base sequence after the primers to form phosphodiester bonds between free DNA nucleotides that have formed h bonds through CBPairing with the target strand
- The first cycle is complete and this cycle continues until the desired number of DNA strands are reached
What is a DNA probe and what does it do?
A single stranded molecule of DNA that’s marked, 15-20 single nucleotides long and is COMPLEMENTARY to the DNA sample being tested. It identifies a mutated sequence in a DNA strand
How do DNA probes work?
- First separate the relevant pieces of DNA by heating the strands to break h bonds between CBPS
- One strand is normal base sequence and the other is the mutated base sequence
- The DNA probe will only CBP with the mutated base sequence where it sticks to it in DNA hybridisation
- The sample is washed to remove unbound DNA probe
- The hybridised DNA probe that’s left indicates the mutation present
What are mini satellites?
Sections of repeating DNA present in everyone but have variable combinations of lengths so are unique to each individual
