4A-4B Flashcards
What is the purpose of restriction enzymes?
In the bacteria, A restriction endonucleus cuts at a specific recognition site by breaking the bonds in a polynucleotide chain
What is RNA polymerase used as part of?
Used in Transcription
What is DNA polymerase used as part of?
Used in the replication of DNA or the amplification of DNA
Describe Recognition sites
Base sequences where endonucleases are palindromic
What are sticky ends?
Staggered cut in the two strands
What are blunt ends?
Cut straight across both chains
What are Ligases?
Join DNA or RNA together
Catalyse the formation of phosphodiester bonds between the two fragments (sugar phosphate backbone) to merge them together
What is CRISPR?
Gene editing system, naturally found in bacteria, now used to edit genes in other organisms
Describe the CRISPR process in bacteria
- When a virus attacksa prokaryotic cell, bacteria collect small fragments of the viral DNA which they insert to its own genome (CRISPR loci).
- Throughout the whole CRISPR loci there is viral sequences (coming from previous exposure to viruses), spaced with repats of host bacterial DNA
- Then transcription of the CRISPR locus occurs in which a RNA strand is reproduced. (The RNA strand is complementary to the integrated fragments of DNA).
- The gRNA then forms a complex with the Cas9 proteins which will cleave and destroy the viral DNA.
Describe the Cas9 enzyme
An enzyme that when attached to gRNA can cut a sequence of DNA
Bacterial RNA guided endonuclease. Uses base pairing to recognise and cleave target DNA which is complementary to the gRNA.
Describe gRNA
guided RNA. has a specific sequence determined by CRIPSR to guide Cas9 to a specific site
Designed to find and bind to a specific sequence within the DNA
gRNA is complementary to the DNA sequence
What are spacers?
Short sequences of DNA obtained from the invading bacteriophages added into the CRISPR sequence
What are bacteriophages?
Virus that infects prokaryotic organisms
Describe PAM
2 to 6 nucleotides found right next to DNA being targeted by Cas9
Non Homologous End Joining -NHEJ
DNA breaks, nucleotides deleted or added, knockout of gene
Homology directed repair
Uses a homologous repair template to repair the broken DNA
Limitations of CRISPR technology
Recent technology - little success seen in human studies
substituting/ adding nucleotides to a gene is not always easy - unexpected mutations occur
Outline the CRISPR Gene editing steps
- Synthetic gRNA is created in a lab
- Cas9 and gRNA are binded together to create the CRISPR-Cas9 complex
- The Cas9 finds the target PAM sequence and checks whether the gRNA aligns with the DNA.
Cas9 cuts the selected sequence of DNA.
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