BIO 208L Chapter 6: Ligation

what is a plasmid?

a small circular, double-stranded DNA molecule that is replicated independently of chromosomes

what are 3 features found in plasmid vectors?

• origin of replication

• selectable marker gene

• multiple cloning site

what is a restriction enzyme

enzymes that recognize and cut specific sequences in double-stranded DNA

sticky ends

overhanging single-stranded ends that base-pair w/ complementary DNA

blunt ends

straight cut w/ no overhangs

what is ligation?

joining together two DNA fragments

what is DNA ligase?

enzyme that seals DNA together (T4 DNA ligase)

sticky end ligation

complementary overhangs pair makes for easier ligation

blunt end ligation

no overhangs makes for harder ligation

proofreading DNA polymerase

enzyme that corrects mismatches and removing 3' overhangs, it creates precise, clean, blunt-ended DNA fragments necessary for efficient ligation

pJET1.2 Vector

contains amp^r (ampicillin resistance gene), pMB1 origin of replication, eco47IR lethal gene

what is the ampicillin resistance gene necessary for

allows selection of transformed bacteria— only cells containing the plasmid survive

what is the origin of replication

enables plasmid to replicate inside of E. coli

what is the eco47IR gene for

it is a negative selection marker, if the vector does not receive an insert the gene remains active and kills the cell

• only cells w/ the insert survive

what is the cloning site (MCS)

designated location for inserting a foreign DNA fragment

characteristics of a good vector

• Ability to accept foreign DNA

  • Has a multiple cloning site (MCS) with many restriction enzyme sites

  • Allows easy insertion of DNA fragments

• Compatible ends for insertion

  • Can be linearized (cut open)

  • May have blunt ends or sticky ends for ligation

  • Your example: designed for blunt-end cloning

• Selectable marker gene

  • Usually an antibiotic resistance gene (like ampᴿ)

  • Lets you identify bacteria that took up the vector (they survive on antibiotic plates)

• Screening system (distinguish correct inserts)

  • Helps identify cells that actually contain inserted DNA

  • Example: eco47IR gene in your vector

    • If disrupted by insertion → cells survive → positive selection

• Origin of replication (ori)

  • Allows the vector to replicate inside the host cell

  • Ensures DNA gets copied

• High copy number

  • Produces many copies of the plasmid per cell

  • Makes DNA easier to isolate and study

• Small size

  • Smaller plasmids are easier to manipulate and enter cells efficiently

• Stable in host cells

  • Maintains inserted DNA without rearrangement or loss