Gene Tech Flashcards
Why is it harder to translate the genome of complex organisms to identify their proteome?
Because they contain large sections of non-coding DNA.
Also contain complex regulatory genes, which determine when the genes for particular proteins should be switched on and off.
What does recombinant DNA technology include?
Transferring a fragment of DNA from one organism to another; can be done due to the universal genetic code, and very similar transcription and translation mechanisms.
What are the three ways in which DNA fragments can be produced?
Using reverse transcriptase, using restriction endonuclease enzymes, and using a gene machine.
Outline the use of reverse transcriptase.
- A cell that readily produces the protein is selected (the b cells of islets of langerhans), as these have large quantities of the relevant mRNA, so it more easily extracted.
- Reverse transcriptase used to make RNA to DNA - now known as cDNA (because it’s made from nucleotides that are complementary to the mRNA).
- To make the other strand of DNA, DNA polymerase builds up the complementary nucleotides on the cDNA template.
Outline the use of restriction endonuclease enzymes.
- Restriction endonuclease are enzymes that recognise specific palindromic sequences (recognition sites) and cut (digest the DNA here.
- Different REs cut at diff recognition sequences, because the shape of the recognition sequence is complementary to the enzyme’s active site.
- The DNA sample is incubated with the specific RE, which cuts the DNA fragment out via a hydrolysis reaction.
- Sometimes the cut leaves sticky ends, which can be used to bind the DNA fragment to another complementary sequence.
Outline the gene machine
- The sequence is designed.
- The first nucleotide in the sequence is then fixed onto a form of support (e.g. bead).
- Nucleotides are added step by step, in a cycle that includes adding protecting groups.
- Oligonucleotides are produced. Once these are complete, they’re broken off from the support and all protecting groups are removed. These oligonucleotides can then be joined together to form longer DNA fragments.
What is a protecting group?
These make sure that the nucleotides are joined at the right points, to prevent unwanted branching.
What is an oligonucleotide?
Around 20 nucleotides long, a short section of DNA.
What are sticky ends?
Small tails of unpaired bases at each end of the DNA fragment. These can then be used to bind (anneal) the fragment to another sticky end with complementary sequences.
Outline how PCR works
- A DNA mixture (containing free nucleotides, primers and DNA polymerase) is heated to 95*C to break the H bonds between the two strands of DNA.
- The mixture is cooled to 50*C so that primers can anneal to the strands.
- The reaction mixture is heated to 72*C for DNA polymerase.
- The DNA polymerase lines up free nucleotides alongside each template strand. Specific base pairing.
- Two new copies of the fragment of DNA are formed.
- Cycle starts again with all 4 strands, so each PCR cycle doubles the amount of DNA.
What are the thee temperatures used in PCR?
95C, 50C and 72*C
What is a primer?
Short sequence of nucleotides that have a set of bases complementary to those at one end of each DNA fragment
What is a thermocycler?
A computer controlled machine that varies temperatures precisely over a period of time.
Advantages of in vitro cloning?
- extremely rapid
- doesn’t require living cells
Advantages of in vivo cloning?
- v accurate
- almost no risk of contamination; because the gene that’s been cut by the same restriction endonuclease can match the sticky ends of the opened up plasmid.
