Isolating of DNA

BIOL2004 Most biological material contains DNA.

Fundamentals of Molecular (even hair, although not mature red blood cells)
DNA has chemical properties that enable it
Biology
to be purified from other cellular
components.
Lecture 3: Manipulating DNA The purification process is usually called
Mark Thomas DNA extraction.

DNA extraction methods Reagents for DNA extraction

All that is required of a DNA extraction method is that it
effectively separates DNA from other cellular
components.
This is necessary because other cellular components
can:
interfere with the DNA manipulation / analysis techniques to
be used after DNA extraction.
cause the breakdown of DNA.
encourage the growth of microorganisms that can breakdown
DNA.
The main cellular components that need to be removed
are proteins, lipids (fats) and metal ions.
Phenol: denatures (strongly) protein, dissolves lipids, does not mix with water or
aqueous solutions, does not interact with DNA.
Chloroform: denatures (weakly) protein, dissolves lipids, does not mix with
water or aqueous solutions, does not interact with DNA.
Detergents (various): denatures (strongly) protein, dissolves lipids, soluble in
aqueous solutions, does not interact with DNA.
Proteinase (various): digests protein, does not interact with DNA.
Guanidine thiocyanate: denatures (strongly) protein, chaotropic agent (causes
DNA to bind reversibly to silica).
Ethylenediaminetetraacetic acid (EDTA): binds to and effectively removes metal
ions (which are used by DNAses).
pH buffers: stop the pH going too high or too low.
Alcohols (various): in combination with various salts will cause DNA to form a
solid precipitate, which may be spun down.
Commercial resins (various): selectively bind DNA or protein
DNA manipulation with enzymes
Most DNA manipulation is done with naturally occurring
or genetically modified enzymes.
These enzymes do a range of things including:
Cutting DNA (nucleases)
Joining DNA (ligases)
Copying DNA (polymerases)
Chemically modifying DNA (various)
Restriction endonucleases
Restriction endonucleases recognise certain DNA
sequences, e.g:
and cut the DNA in a predictable way.
Nucleases
Exonucleases: break off nucleotides from
the end of the DNA or RNA molecule.
Endonucleases: cleave the DNA or RNA
molecule internally.
Some cleave indiscriminately.
Some cleave at specific base-sequences only
(restriction endonucleases).
Restriction endonucleases
What makes restriction endonucleases special is that
they only cut DNA molecules at defined sequences.
There are several classes of restriction
endonucleases.
The ones used in molecular biology are all Type II.
These are enzymes that recognise a specific
sequence and then cut at a defined site within that
sequence.
For the molecular biologist this is priceless - it
provides the key to just about everything we do
with DNA.
 Restriction endonucleases
Restriction endonucleases are enzymes made by bacteria
that serve as a defense against viral attack (we think).
The bacterial DNA is protected from digestion by having
its restriction endonuclease recognition sequences
chemically modified (by methylases).
Restriction endonuclease nomenclature
Many different RE's have been isolated from
different organisms.
They are named after the organism from which
they've been isolated.
1st letter of the genus + 1st two letters from the
species + (sometimes) a number or letter
denoting the strain or the plasmid + a number
denoting the specific restriction/modification
system (sometimes more than one per strain).
Restriction endonucleases and
methylases
In nature, Type II REs form one half of a
restriction/modification system.
This is composed of two enzymes.
The 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 or adenine ! 6-methyladenine).
Methylation prevents the sequence being cut by the
restriction enzyme - it is essential to prevent the
bacterium from self-destructing!
Restriction endonuclease
nomenclature
e.g:
BamHI = Bacillus amyloliquefaciens H
R/M system No. I
BglIII = Bacillus globigii
R/M system No.II
HinDIII = Haemophilus influenzae D
R/M system No.III
 Target sequences - RE types Target sequences
REs can be classified as Type I, Type II or Type III Different restriction endonucleases recognise
depending on how they act.
different target sequences, e.g:
Type I's: Recognise a specific site, but then cut at a random
site at least 1000 bases away.
Type II's: Recognise and cut specific sites.
Type III's: Cut 24-26 bp away from recognition site.

Type II's cut within the recognition site - which makes them
by far the most useful for molecular biology.

Target sequences are usually Cleavage sites

palindromic Restriction enzymes can cut the DNA in three different ways:
Because the enzymes act as homodimers, antiparallel pairs of 1. Blunt: e.g. BalI
identical subunits. 5'......TGG/CCA.... ...TGG CCA...
........ACC/GGT....5' ...ACC GGT...

2. With an overhang at the 5' end e.g. Asp718

5'....G/GTACC.... ...G GTACC...
....CCATG/G....5' ...CCATG G..

The symmetry of the recognition site matches the symmetry of the 3. With an overhang at the 3' end e.g. KpnI
enzyme: this ensures that both strands will be cut. 5'....GGTAC/C... ....GGTAC C...
Sometimes different enzymes from different organisms recognise ....C/CATGG...5' ....C CATGG...
the same sequence - are called isoschizomers
 How often do REs cut?
Overhangs The number of times a RE would cut random DNA sequence is
determined by the target sequence length.
If an REs target sequence is 4 bp long, we would expect that
Overhangs may be 1, 2, 3, 4 or 5 bases long sequence to occur one in every 4 x 4 x 4 x 4 bp (= 44
(depends how close to the centre of the = 256bp).
recognition sequence the enzyme cuts). If an REs target sequence is 6 bp long, we would expect that
sequence to occur one in every 4x4x4x4x4x4
Note that nearly all enzymes cut to leave a bp (= 46 = 4096bp).
5' terminal phosphate. If an REs target sequence is 8 bp long, we would expect that
sequence to occur one in every 4x4x4x4x4x4x
8
4 x 4 bp (= 4 = 65,536bp).

Joining DNA molecules Joining DNA molecules

Don't always have to use the same enzyme on both molecules: some enzymes
have different recognition sequences, but nevertheless produce compatible
Enzymes that join DNA molecules are called DNA ligases. sticky ends:
Joining DNA molecules is called ligation. e.g. BamH1, BglII and Sau3A

Most REs leave sticky ends, e.g: BamHI 5 ....G GATCC...3'

5 ..G AATTC 3 3'....CCTAG G...5'
3 ..CTTAA G 5
These are much easier to ligate than blunt ends, e.g: BglII 5 ...A GATCT....3'
5 ..GAA TTC 3 3'...TCTAG A....5'
3 ..CTT AAG 5
Sau3A 5'.. GATC....3'
3'..CTAG ....5'
 Joining blunt ends
Sometimes, it's not convenient to join DNA molecules this way
(maybe there aren't suitable sites for the same enzyme in the right
region of both molecules).
In this case, can use blunt ends.
Harder to join together (need to use special conditions, and very
concentrated DNA mixtures, but can be done using T4 DNA
ligase.
To get blunt ends:
Use a blunt cutter (e.g. Bal1 or SmaI)
Or: use an enzyme which digests the single-stranded overhang (S1
nuclease) will be discussed later.
Mode of action of restriction
enzymes
Type II RE's function as a homodimer - one subunit binding each
strand of the DNA and cutting it.
The hydrolysis of the sugar-phosphate bonds provides the free
energy required.
Nearly all type II's cut to leave a 5' phosphate
Most restriction enzymes are highly specific for double-stranded
DNA.
Some will cut single-stranded DNA very slowly, some not at all.
Some enzymes show a preference for relaxed DNA rather than
supercoiled (eg SalI)

Requirements for RE's

What conditions do you need to provide to make sure your Storage
enzymes will do their job?
Usually stored in a sterile buffered solution
Need Mg2+ to work.
Usually pH 7-8 (so buffer the reaction mix)
containing glycerol, at -20C.
Different enzymes have different requirements for NaCl (ionic Soon lose activity due to denaturation and
strength) - could use low, medium or high salt buffers. oxidation if allowed to sit around at room
Usually work best around 37C (some exception are RE's temperature.
isolated from thermophilic bacteria, eg TaqI has an optimum
around 65C) This goes for most enzymes used in
Also some thermolabile enzymes (eg SmaI, optimum is about molecular biology.
25C).
 Units of activity
One unit is the amount of enzyme required to cut 1g of lambda
DNA to completion in 1 hour at 37C. BIOL2004
Definition breaks down:
in the case of enzymes which have optimum efficiencies at temperatures Fundamentals of Molecular
other than 37 C (thermolabile / thermostable enzymes)
with some enzymes which don't have a recognition site in lambda (eg NotI Biology
is assayed using adenovirus 2 DNA).
Enzymes affected by dam and dcm methylation (depends which E.coli
strain the lambda has been isolated from).
Lecture 4: Manipulating DNA
Mark Thomas