Manipulating DNA: Enzymes & Reagents
DNA Extraction
In order to manipulate DNA, it has to be purified – separated from other
cellular components: proteins, lipids, metal ions (especially divalent ions)
Cellular components can interfere with manipulation/analysis techniques,
cause breakdown of DNA, encourage growth of microorganisms than can
breakdown DNA
Reagents used for DNA Extraction
Phenol: Strongly denatures protein, dissolves lipids, does not mix with water or
aqueous solutions, does not interact with DNA
Chloroform: Weakly denatures protein, dissolves lipids, does not mix with water
or aqueous solutions, does not interact with DNA
Detergent: Strongly denatures protein, dissolves lipids, soluble in aqueous
solutions, does not interact with DNA
Various detergents - SDS
Proteinase (K): Digests protein, does not interact with DNA
Naturally occurring (eg. pineapple, papaya)
Placed at room temperature for long enough – will digest itself
Guanidine Thiocyanate: Strongly denatures protein, chaotropic agent (causes
DNA to bind reversibly to silica)
Silica can form salt bridges with DNA
If acid is added, liberates large amounts of cyanide
Method
Ethylenediaminetetraacetic Acid (EDTA): Binds to and effectively removes
metal ions (which are used DNAses)
, Forms a “cage” around metal ions: binds/collates strongly to divalent ions
pH buffers: Tris.HCL
DNA is pH sensitive – can separate strands, too acidic can cause bases to
be snapped off (depurination)
Alcohols (Ethanol, Isopropanol): In combination with various salts will cause
DNA to form a solid precipitate, which can be spun
Commercial Resins: Selectively bind DNA or proteins
Silica spin columns etc.
DNA Manipulation with Enzymes
Usually stored in sterile buffered solution containing glycerol at -20 oC
Soon lose activity due to denaturation and oxidation if placed at room
temperature for long periods of time
Nucleases
Exonucleases: break off nucleotides from the end of DNA/RNA molecule
Endonucleases: cleave the DNA/RNA molecule internally – some cleave
indiscriminately, some cleave at specific base-sequences only (restriction
endonucleases)
Restriction Endonucleases
REs recognise certain DNA sequences and
cut them in a predictable way (energetically
favourable – energy contained in phosphate
bonds)
Several classes of REs – Type II commonly used: recognise a specific
sequence and cut at a defined site at that sequence
REs are enzymes made by bacteria and are hypothesised to be defences
against viral attack
The bacteria prevents self-digestion by having its restriction
endonuclease recognition sequences chemically modified (by methylases)
, Restriction/Modification System
In nature, Type II REs form one half of a restriction/modification
system – composed of two enzymes
Restriction enzyme recognises a specific DNA sequence and cuts within it
The modification enzyme recognises the same sequence and methylates
bases within it
Cytosine 5-methylcytosine
Adenine 6-methyladenine
Methylation prevents the sequence being cut by the restriction enzyme –
it is essential to prevent the bacterium from self-destriction
Restriction endonuclease nomenclature
1st letter of the genus + 1st two letters from species + number/letter
denoting strain/plasmid + number denoting specific
restriction/modification system (sometimes more than one per strain)
BamHI = Bacillus amyloliquefaciens H – R/M System I
BglIII = Bacillus globigii – R/M system II
HinDIII = Haemophilus influenzae D = R/M system No. III
Target Sequences – RE Types
Type Function
I Recognise a specific site, then cuts at a random site at least 1000 bases away
II Recognise and cut specific sites
III Cut 24-26bp away from recognition site
Target sequences are usually palindromic –
enzymes act as homodimers, antiparallel pairs of
identical subunits
1. Type II REs function as a homodimer = one
subunit binding each strand of the DNA and
cutting it
2. Hydrolysis of the sugar-phosphate bonds
provides the free energy required
Particularly useful – Different DNA
fragments cut by different REs can be
joined together, as long as the 5’ overhangs are complementary
DNA Extraction
In order to manipulate DNA, it has to be purified – separated from other
cellular components: proteins, lipids, metal ions (especially divalent ions)
Cellular components can interfere with manipulation/analysis techniques,
cause breakdown of DNA, encourage growth of microorganisms than can
breakdown DNA
Reagents used for DNA Extraction
Phenol: Strongly denatures protein, dissolves lipids, does not mix with water or
aqueous solutions, does not interact with DNA
Chloroform: Weakly denatures protein, dissolves lipids, does not mix with water
or aqueous solutions, does not interact with DNA
Detergent: Strongly denatures protein, dissolves lipids, soluble in aqueous
solutions, does not interact with DNA
Various detergents - SDS
Proteinase (K): Digests protein, does not interact with DNA
Naturally occurring (eg. pineapple, papaya)
Placed at room temperature for long enough – will digest itself
Guanidine Thiocyanate: Strongly denatures protein, chaotropic agent (causes
DNA to bind reversibly to silica)
Silica can form salt bridges with DNA
If acid is added, liberates large amounts of cyanide
Method
Ethylenediaminetetraacetic Acid (EDTA): Binds to and effectively removes
metal ions (which are used DNAses)
, Forms a “cage” around metal ions: binds/collates strongly to divalent ions
pH buffers: Tris.HCL
DNA is pH sensitive – can separate strands, too acidic can cause bases to
be snapped off (depurination)
Alcohols (Ethanol, Isopropanol): In combination with various salts will cause
DNA to form a solid precipitate, which can be spun
Commercial Resins: Selectively bind DNA or proteins
Silica spin columns etc.
DNA Manipulation with Enzymes
Usually stored in sterile buffered solution containing glycerol at -20 oC
Soon lose activity due to denaturation and oxidation if placed at room
temperature for long periods of time
Nucleases
Exonucleases: break off nucleotides from the end of DNA/RNA molecule
Endonucleases: cleave the DNA/RNA molecule internally – some cleave
indiscriminately, some cleave at specific base-sequences only (restriction
endonucleases)
Restriction Endonucleases
REs recognise certain DNA sequences and
cut them in a predictable way (energetically
favourable – energy contained in phosphate
bonds)
Several classes of REs – Type II commonly used: recognise a specific
sequence and cut at a defined site at that sequence
REs are enzymes made by bacteria and are hypothesised to be defences
against viral attack
The bacteria prevents self-digestion by having its restriction
endonuclease recognition sequences chemically modified (by methylases)
, Restriction/Modification System
In nature, Type II REs form one half of a restriction/modification
system – composed of two enzymes
Restriction enzyme recognises a specific DNA sequence and cuts within it
The modification enzyme recognises the same sequence and methylates
bases within it
Cytosine 5-methylcytosine
Adenine 6-methyladenine
Methylation prevents the sequence being cut by the restriction enzyme –
it is essential to prevent the bacterium from self-destriction
Restriction endonuclease nomenclature
1st letter of the genus + 1st two letters from species + number/letter
denoting strain/plasmid + number denoting specific
restriction/modification system (sometimes more than one per strain)
BamHI = Bacillus amyloliquefaciens H – R/M System I
BglIII = Bacillus globigii – R/M system II
HinDIII = Haemophilus influenzae D = R/M system No. III
Target Sequences – RE Types
Type Function
I Recognise a specific site, then cuts at a random site at least 1000 bases away
II Recognise and cut specific sites
III Cut 24-26bp away from recognition site
Target sequences are usually palindromic –
enzymes act as homodimers, antiparallel pairs of
identical subunits
1. Type II REs function as a homodimer = one
subunit binding each strand of the DNA and
cutting it
2. Hydrolysis of the sugar-phosphate bonds
provides the free energy required
Particularly useful – Different DNA
fragments cut by different REs can be
joined together, as long as the 5’ overhangs are complementary
