Topic 8 A Level Biology Flashcards
(29 cards)
What is recombinant DNA?
- The transfer of DNA fragments from one species to another.
How is it possible for recombinant DNA to be replicated inside another organism?
- Due to the genetic code being universal, the transferred DNA can be translated within the organism that recieves the DNA.
How can we use reverse transcriptase to produce fragments?
1) mRNA for a polypeptide is isolated from a cell.
2) It is mixed with free DNA nucleotides and reverse transcriptase.
3) Reverse transcriptase uses the mRNA as a template to produce complementary DNA, which is a double stranded copy of the required gene.
Why is mRNA useful in recombinant DNA?
- there is lots of the mRNA on each gene.
- mRNA has no introns when matured.
How do we use restriction endonucleases to produce fragments?
- Breaking phosphodiester bonds between DNA bases.
- Binding to recognition sites through complementary pairing.
- Once binded together, there is cutting of the DNA.
What ends to restriction endonucleases form?
- Sticky ends
What are sticky ends?
- Staggered cuts, revealing unpaired base sequences at either end of the fragment.
What are vectors?
- DNA mocleucles that carry DNA fragments into a cell.
What are 2 types of vectors?
- Plasmids
- Bacteriaphages
What enzyme joins sticky ends of the vector and fragments?
- DNA ligase (forms phosphodiester bonds)
What is the gene machine?
- The gene machine determines the amino acid sequence of a specific polypeptide.
- This tells us the mRNA base sequence, from which we can determine the base sequence which acted as a template.
Why is amplification necessary?
- To produce numerous copies to work with.
What are 2 methods of amplification?
- invitro and invivo
What happens during transformation (invivo)
- There is insertion of recombinant DNA into a host cell
How does transformation differ between bacteriophages and plasmids?
- In bacteriophages, they inject their DNA into the bacterium.
- In plasmids, host cells are stimulated to take in the plasmid.
How are cells that take up recombinant DNA identified?
- Marker genes are inserted into the vector together with the required gene.
- Marker genes may code for antibiotic resistance so when cells take up recombinant DNA, they survive, and are seen.
- Flourescent dye can be used.
Why are promoter and terminator regions essential?
- They determine the rate of translation through stop and start codons.
What is PCR?
- PCR is invitro
- widely used to make millions to billions of copies of a specific DNA sample rapidly
How does PCR work?
1) Reaction mixture should contain DNA polymerase, primers, DNA fragments and free DNA nucleotides.
2) Heat to 95 degrees celsius so the DNA denatures and hydrogen bonds break.
3) Cool to 50-60 degrees celsius, allowing the primers to bind to complementary bases at the ends of fragments.
4) Heat again to 72 degrees celsius so that DNA polymerase carries out the extension of the strands (new complementary strands will form). REPEAT
What are DNA primers?
- Primers are short, single stranded DNA base sequences that are complementary to the bases at the start of the desired DNA fragment.
- primers allow DNA polymerase to bind to the DNA fragment.
What are DNA probes?
- Short-single stranded sequence of DNA whose bases are complementary to a specific DNA sequences.
- If DNA sequence is present, then the DNA probe will bind via complementary base pairing.
What are gene probes?
- Probes that test for the presence of a mutated allele.
Why is identification important?
- Helps prevent the worsening of diseases.
How do gene probes get made?
1) Sequence the allele.
2) Produce the probe using a gene machine.
3) PCR will produce many probes.
