Topic K. Genetic Engineering Flashcards
What are the basic steps of cloning a human gene into a bacterium?
- purify plasmid
- amplify target gene with PCR
- cut gene & plasmid with restriction enzymes
- ligate gene into plasmid
- transform cells
- induce gene expression
- purify product
Why can’t RNA be used directly for PCR in cloning?
PCR requires DNA so RNA must be converted to cDNA (complementary DNA)
What is the role of restriction enzymes in cloning?
They cut DNA at specific sequences, creating sticky ends that allow the gene and plasmid to be joined
What is the purpose of DNA ligase in the cloning process?
DNA ligase seals the cut plasmid and gene by joining the sugar-phosphate backbones
What are two components of a restriction-modification (R-M) system?
- restriction enzymes
- methylase
How do restriction enzymes protect bacteria from bacteriophage infection?
they cut viral DNA at specific sequences –> prevents phage replication
How does methylation protect bacterial DNA from its own restriction enzymes?
methylation modifies the DNA at recognition sites –> blocks the restriction enzyme from cutting it
What is the role of methylation in bacterial DNA replication?
methylation protects bacterial DNA from being cut by restriction enzymes and aids in DNA repair
How do endonucleases and exonucleases differ in function?
- endonucleases cut WITHIN a DNA strand
- exonucleases remove nucleotides from the ENDS of DNA strands
What is the role of endonucleases in bacterial immunity?
endonucleases (like restriction enzymes) cut foreign DNA (ex: from viruses) –> protecting the bacteria
What are the key components of the CRISPR-Cas system?
- CRISPR array
- crRNA
- tracrRNA
- Cas9 nuclease
How does the CRISPR-Cas system protect bacteria from bacteriophage?
crRNA guides Cas9 to the viral DNA where Cas9 cuts the viral genome –> prevents phage replication
What is the role of Cas9 in the CRISPR-Cas system?
Cas9 creates double-strand breaks in the viral DNA –> disables phages
