L12- Recombinant DNA tech

Nucleases

• enzymes that digest DNA

• Exonucleases→ cut from outside strand

• Endonucleases→ cut within strand

Major classes of endonucleases

• Type 1→ Cut randomly away from recognition site→ useless in lab

• Type 2→ Cut at recognition site

• Type 3→ Cut randomly near recognition site

Restriction enzymes recognize

• a specific nucleotide-pair sequence within DNA

  • “recognition site” (palindromic reads same 5’-3’ and 3’-5’)

  • Cuts phosphodiester bond

How do restriction enzymes function as a primitive immune system

• Bacteriums own DNA not cut bc they have mechanisms to protect their DNA through methylation which marks it, thus protecting their DNA from digestion

General cutting frequencies

• Nucleotide pairs in restriction site.. MINIMUM= 4 (bc 4 bps)

• Probability of occurence= (1/4)^nuc pairs

• 4 pairs= 1/4^4= 1 in 256 bp

• 5 pairs=(1/4)^5= 1 in 1024 bp

Blunt vs Cohesive Ends

1. Cleavage by enzymes that produce blunt ends (like squares easily joined together)

2. Cleavage by enzymes producing sticky ends (ss overhangs)

• like tetris blocks

Restriction mapping used to ___ basic procedure:

• determine the location of restriction enzyme cut sites within a section of DNA

• Isolate DNA, digest it w/ restriction enzyme combinations, seperate bands via electrophoresis

Gene cloning

Define recombinant DNA and transgenic organisms

• Recombinant DNA→ created by joining DNA from diff organisms

• Transgenic organisms→ contain genes from diff organisms

Basic cloning procedure

• Isolate DNA

• Digest DNA w/ approp enzymes

• select vector + digest w/ compatible restriction enzymes

• ligate DNA into vector

• Transform into organism

What are vectors

• (circular) Carrier of DNA molecules

  • can rep within host cell

Plasmid Cloning Vectors Have

• Ori sequence

  • allows for replication in E.coli (recognized by bacteria machinery, vector can be copied by host enzymes)

• Unique restriction site (cut sites)

  • open plasmid (vector) for DNA insertion

• Selectable marker(s)

  • Used to determine which..

    • E.coli cells contain plasmid

    • Which plasmids contain insert DNA (indicates if DNA entered bc vector could have closed back up w/ out)

• ex) Amp resistance gene→ if vector gets in bac cell then bacteria can survive in presence of antibiotic

Example of using selectable marker to see if plasmid contains insert DNA

• Blue-white selection

• Disruption of lacZ gene by insert DNA leads to white colonies in presence of X-gal

• Bc the lacZ gene encodes β-galactosidase, which cleaves X-gal to produce a blue product.

• If foreign DNA is inserted into the lacZ gene it disrupts the gene’s function.

Result:

• Functional lacZ (no insert) → β-galactosidase made → X-gal cleaved → blue colonies

• Disrupted lacZ (insert DNA present) → no functional enzyme → X-gal not cleaved → white colonies

Cloning into an expression vector allows…

• for efficient transcription and translation (in bac cell)

• cDNA is typically used instead of genomic DNA

  • bc bacteria dont have introns cannot remove them. cDNA has no introns so it can be directly translated into protein

• Cloning insulin cDNA into an expression vector lets bacteria efficiently make human insulin protein

Restriction mapping importance for cDNA orientation

• Restriction digestion done to det. orientation of insert in plasmid

• if cDNA goes in backwards→ read backwards→ wont get right protein

• Can prevent this from restriction mapping

  • for both pieces DNA and vector