Topic K. Genetic Engineering Flashcards

1
Q

What are the basic steps of cloning a human gene into a bacterium?

A
  1. purify plasmid
  2. amplify target gene with PCR
  3. cut gene & plasmid with restriction enzymes
  4. ligate gene into plasmid
  5. transform cells
  6. induce gene expression
  7. purify product
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2
Q

Why can’t RNA be used directly for PCR in cloning?

A

PCR requires DNA so RNA must be converted to cDNA (complementary DNA)

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3
Q

What is the role of restriction enzymes in cloning?

A

They cut DNA at specific sequences, creating sticky ends that allow the gene and plasmid to be joined

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4
Q

What is the purpose of DNA ligase in the cloning process?

A

DNA ligase seals the cut plasmid and gene by joining the sugar-phosphate backbones

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5
Q

What are two components of a restriction-modification (R-M) system?

A
  1. restriction enzymes
  2. methylase
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6
Q

How do restriction enzymes protect bacteria from bacteriophage infection?

A

they cut viral DNA at specific sequences –> prevents phage replication

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7
Q

How does methylation protect bacterial DNA from its own restriction enzymes?

A

methylation modifies the DNA at recognition sites –> blocks the restriction enzyme from cutting it

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8
Q

What is the role of methylation in bacterial DNA replication?

A

methylation protects bacterial DNA from being cut by restriction enzymes and aids in DNA repair

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9
Q

How do endonucleases and exonucleases differ in function?

A
  • endonucleases cut WITHIN a DNA strand
  • exonucleases remove nucleotides from the ENDS of DNA strands
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10
Q

What is the role of endonucleases in bacterial immunity?

A

endonucleases (like restriction enzymes) cut foreign DNA (ex: from viruses) –> protecting the bacteria

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11
Q

What are the key components of the CRISPR-Cas system?

A
  1. CRISPR array
  2. crRNA
  3. tracrRNA
  4. Cas9 nuclease
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12
Q

How does the CRISPR-Cas system protect bacteria from bacteriophage?

A

crRNA guides Cas9 to the viral DNA where Cas9 cuts the viral genome –> prevents phage replication

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13
Q

What is the role of Cas9 in the CRISPR-Cas system?

A

Cas9 creates double-strand breaks in the viral DNA –> disables phages

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