Week 3 - Enzymes

Nucleases

Break PO bonds in nucleic acids

5’ end vs 3’ end

Phosphate vs OH

How Nucleases Break PO Bonds

Hydrolysis (or lyase)

Exonucleases

Cut from ends

Endonucleases

Cut internal bonds

How to Prevent Nucleases

EDTA - chelate Mg2+

DNase I

ss or dsDNA

Endonuclease

Produce small fragments

DNase - Mg2+ Present

Cut strands independently

Random fragments

DNase - Mn2+ present

Cut strands close together

1-2nt overhangs

DNase I - Sourced From

Bovine pancreas

Exonuclease III

dsDNA

Remove bases sequentially 3’ to 5’

Exonuclease III - Sourced From

E coli

Nuclease S1

Exonuclease

Remove ss overhangs

Produce blunt ends

Nuclease S1 - Sourced From

Aspergillus

Bacterial Restriction Modification System (RMS) - 2 Components

RE - cut unmethylated foreign DNA

Methyltransferase - add methyl groups to own DNA

Type I & III vs Type II

Asymmetrical + cut away from site vs symmetrical + cut within site

Nomenclature of RE

Genus

Species

Strain

Order

Most Common DNA Ligase

T4

E coli DNA Pol I - 3 Parts

5 to 3 pol

5 to 3 exonuclease - remove bases ahead

3 to 5 exonuclease - proofreading

Klenow Fragment

DNA Pol I without 5 to 3 exonuclease

2 Ways Klenow is Used in Lab

Fill in 5’ overhangs

Remove 3’ overhangs

T4/T7 DNA Pol - Uses

Modify ends

DNA sequencing

T4 vs T7

Strong proofreading - modify ends

High processivity - DNA sequencing

Processivity/Speed

No of nucleotides added before DNA Pol falls off

Terminal Transferase

No template - add bases

Primers

Cofactor - Co

Terminal Transferase - Use

Create overhangs for cloning

DNA Polymerases - 4

E coli

T4/T7

Terminal transferase

Thermostable

Nucleases - 3

DNase I

Exonuclease III

Nuclease S1