SAC 1 Long Defintions Flashcards
(11 cards)
Outline the four stages of PCR
- Denaturing - Heat DNA to 90oC to separate the DNA into single strands by breaking hydrogen bonds between complementary bases.
- DNA Primer annealing - Cool to approximately 50oC to attach/anneal DNA primers to the single-stranded DNA.
- Extension- Heated to approximately 72oC - DNA Taq polymerase copies the single DNA strands in a 3’ to 5’ direction using complementary base pairing..
- Repeats - Process is repeated 35 times.
Describe how to create a DNA profile
- Cut the DNA sample with an endonuclease
Place DNA sample in a well at the negative end of a gel - Turn on the electricity and the DNA fragments will move towards the positive end.
- They will separate based on size and charge.
- Compare with standards which are of know size, that were also placed in the first well, to estimate the size of the other fragments
Outline the steps taken to produce human insulin from bacteria.
- Isolate gene for insulin chain A
- Use reverse transcriptase to get c.DNA from isolated mRNA so that it contains no introns.
- Cut gene and plasmid using the same endonuclease.
- Stick insulin gene into plasmid using DNA ligase which joins the sugar phosphate backbone.
- Transform bacteria using heat or electroporation and check for successful transformation by growing on an antibiotic plate with ampicillin and then an antibiotic plate with tetracycline
- Bacteria with no plasmid will not grow on the ampicillin plate, successfully transformed bacteria with the non-recombinant plasmid will grow on both plates and the successfully transformed bacteria with the recombinant plasmid will only grow on the ampicillin plate.
- Purify plasmids, cut with an endonuclease and stick β-galactosidase gene into plasmid using DNA ligase
- Transform bacteria using heat or electroporation and check for successful transformation by growing on X-gal agar plate
- Bacteria with no β-galactosidase gene will appear white and bacteria with the β-galactosidase gene will appear blue, collect blue colonies
- Repeat for insulin chain B
- Purify insulin chains A and B and join them together to create a functional insulin
Describe the role of CRISPR in bacteria.
- Primitive adaptive immune system this means that
- Invading viral DNA is cut and stored as spacers in the CRISPR array providing a memory of viral infections
- If the same virus reinfects the bacteria, gRNA is transcribed and attached to a Cas9 endonuclease forming a gRNA-Cas9 complex
- gRNA guides Cas9 to the viral DNA and Cas9 recognises the PAM sequence and then cuts the viral DNA, preventing destruction of the bacteria.
Describe the steps in using CRISPR technology to edit genomes in other organisms
- Identify the nucleotide sequence of the ______ target gene.
- Make single guide RNA which is complementary to the ____ target DNA.
- This is joined with Cas9 to form the sg.RNA Cas9 complex.
- Sg.RNA guides Cas9 to the _____ gene and Cas9 cuts the DNA.
- Transcription/no transcription of the _____ gene will occur and a functional ____ protein/non-functional _____protein is produced.
Transcription
Transcription this means that DNA unwinds and RNA Polymerase attaches to the promotor region for the _______ gene.
1. DNA template strand is copied into pre-mRNA via complementary base pairing using RNA polymerase.
2. pre mRNA undergoes RNA processing. Introns are removed, a 5’ methyl G cap and 3’ poly-A tail are added to form mRNA.
3. mRNA for _______ leaves the nucleus and goes to a ribosome.
Translation
Translation this means that
1. Ribosomes read the mRNA code for _____.
2. tRNA brings the correct and specific amino acid to the ribosome.
3. tRNA anticodons are complementary and specific to mRNA codons.
4. Amino acids are joined together by a peptide bond, in a condensation polymerisation reaction, and a ______ polypeptide is formed.
Repression does not occur (Trp not present in cytosol)
- Regulatory gene codes for trp repressor protein
When there is no trp present it can’t bind to the trp repressor protein. - The repressor protein does not change its 3D shape and is not specific and complimentary to the operator region, therefore it can’t bind to the operator region. The promoter region is left free.
- RNA polymerase binds to the promoter region and transcribes the structural genes.
- Transcription, translation and gene expression of the structural genes occurs and enzymes to make trp are produced.
Repression occurs (Trp present in cytosol)
- Regulatory gene codes for trp repressor protein
- When trp is present, 2x trp binds to trp repressor protein.
- The repressor protein 3D shape is changed making it specific and complimentary to the operator region, where it attaches and overhangs the promoter region.
- RNA polymerase cannot bind to the promoter region
- No transcription, translation and gene expression of the structural genes occurs and enzymes to make trp are not produced.
Attenuation occurs (Trp present on tRNA)
- The leader region is transcribed.
- When tryptophan is present on tRNA, the ribosome translates the two tryptophan codons and stops at the stop codon, blocking domain 2
- This leads to domain 3 and 4 forming a hairpin loop.
- That puts tension on the attenuator region and mRNA pulls away from the DNA, causing RNA Polymerase to detach
- Preventing transcription of the structural genes and no enzymes for the synthesis of tryptophan are produced.
Attenuation does not occur (Trp not present on tRNA)
- The leader region is transcribed.
- When there is no Tryptophan in the cytosol (no repression occurs) and none on tRNA (no attenuation occurs) then the ribosome stalls at the two trp codons
- This leads to domain 2 and 3 forming a hairpin loop
- That does not cause the attenuator region to pull away from the DNA, allowing RNA Polymerase to continue and transcribe the structural genes
- The enzymes for the synthesis of tryptophan are produced
