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Enzymes in Genetics Engineering. Restriction Enzymes & Ligase. These are used to make recombinant DNA. Two important first steps in cloning require two enzymes. 1. Restriction Enzymes Bacterial enzymes that cut at specific restriction site sequences Cut DNA by breaking phosphodiester bonds

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restriction enzymes ligase
Restriction Enzymes & Ligase

These are used to make recombinant DNA. Two important first steps in cloning require two enzymes.

1. Restriction Enzymes

  • Bacterial enzymes that cut at specific restriction site sequences
  • Cut DNA by breaking phosphodiester bonds
  • Yield restriction fragments that can be used to clone

2. DNA Ligase

  • Enzyme reforms phosphodiester bond between adjacent nucleotides
types of restriction enzymes
Types of restriction enzymes
  • Type I  Recognize specific sequences·but then track along DNA (~1000-5000 bases) before cutting one of the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA
    • e.g. EcoK.
    • Require Mg2+, ATP and SAM (S-adenosyl methionine) cofactors for function
  • Type II  Recognize a specific target sequence in DNA, and then break the DNA (both strands), within or close to, the recognition site
    • e.g. EcoRI
    • Usually require Mg2+
  • Type III  Intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site
    • e.g. HgaI
    • Require Mg2+ and ATP
restriction enzyme type ii
Restriction enzyme type II
  • Type II is the most frequent used in biology molecular techniques.
  • Hundreds of restriction enzymes have been isolated and characterised
  • Enables DNA to be cut into discrete, manageable fragments
  • Many are now commercially available
  • Each restriction enzyme will recognize its own particular site
  • Some recognize more than one sequence
  • Many Type II restriction endonucleases recognize PALINDROMIC sequences
  • Restriction enzymes do not discriminate between DNA from different organisms
slide6

5'-G A A T T C-3'

3'-C T T A A G-5'

Palindromic sequence

Generate 3' overhangs –

eg: PsfI

Generate 5' overhangs –

eg: EcoRI

Generate blunt end , eg: SmaI

slide12

Restriction endonucleases are a

natural part of the bacterial

defence system

  • Part of the restriction /

modification system found in

many bacteria

  • These enzymes RESTRICT the

ability of foreign DNA (such as

bacteriophage DNA) to infect /

invade the host bacterial cell by

cutting it up (degrading it)

  • The host DNA is MODIFIED by

METHYLATION of the sequences

these enzymes recognise

    • Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes
ligation
Ligation
  • When sticky ends are created on the vector and the rDNA, the ends are compatible and complementary
  • Can be added as linkers or adapters
  • DNA Ligase

Seals single stranded nicks between adjacent nucleotides in a duplex DNA chain (catalyze the formation of a phosphodiester bond between adjacent 3’ hydroxyl and 5’ phosphate termini in DNA)

t4 polymerase
T4 polymerase
  • DNA polymerase activity
  • A very active single-stranded 3'->5' exonuclease
  • Lacks a 5'->3' exonuclease activity
slide29

Other useful DNA modification enzymes

used for manipulating DNA