p.1
1. Denaturation (Tm), renaturation (Cot), hybridisation
Denaturation
- the double helix can be denatured in different ways: heat, pH (acid & alkali),
chemicals such as urea (poorly), formamide, dimethylsulfoxide, etc., and proteins
(single-strand binding proteins)
- dissociation by heating also named melting
- progress of denaturation can be monitored by UV absorption spectroscopy
- stability of helix:
- hydrogen bonds
- stacking (- interactions) between the planar rings
- cavitation energy
(the parts of the molecule that cannot form H-bonds to water tend to
form a single cavity => minimum cavitation energy)
- hyperchromicity effect : disruption of stacking
=> 30 to 40 % increase of UV (260 nm) absorption
- Tm : melting temperature
- position in melting profile where 50% is single-stranded
- pseudo-monomolecular reaction: strands are not physically separated
(but A-T rich zones 'melt' first) (dynamic equilibrium)
=> ' denaturation is concentration-independent'
- linear relationship between Tm and %G+C of a duplex
- physical separation requires temperatures far above Tm
- fast chilling (ice-water!) : nucleic acid remains single-stranded
- slow cooling is necessary to enable the base-pairs (and stacking) to rebuild
=> 'renaturation requires energy ! '
G. Volckaert
Denaturation, renaturation, hybridisation
2/16/2016
p.2
Renaturation
- renaturation is NOT simply the reverse of denaturation
- collision of complementary strands required
- nucleic acid strands are negatively charged in the phosphate moiety
-1 per nucleotide => repulsion
=> requires shielding to allow strands to approach one
another (use of Na+ or K+ salts)
- four parameters in renaturation kinetics
1) concentration of cations
2) incubation temperature (usually 20 to 25 °C below Tm)
3) DNA concentration (related to complexity of the DNA)
4) size of the fragments
- procedure
- purified DNA is sheared to uniform sizes (300 bp)
(controlled shearing can yield sizes from 100 to 100,000 bp)
- denaturing by brief heating at 100 °C (physical separation of the strands)
then quick chilling (ice-water)
- bring samples in buffer (e.g. 0.15 M of monovalent salt, around pH 7)
- follow extent of renaturation as a function of time
=> hypochromicity (instrumentation at high temperatures!)
=> sensitivity to nuclease S1
=> - TCA precipitation of ds-DNA,
- fragmented ss-DNA is not precipitated
- filtering precipitate
=> use of hydroxy-apatite (binds ds) or cellulose nitrate (binds ss)
G. Volckaert
Denaturation, renaturation, hybridisation
2/16/2016
p.3
- kinetics
- bimolecular second order reaction
- concentration of (single-stranded) DNA = C ; initial concentration = C0
- dC/dt = k C C' or = k C2 (if original, denatured DNA is used)
(minus sign because rate of disappearance of single strands)
hence: - dC/C2 = k dt
integration from the initial values C = C0 and t = 0 gives
1/C - 1/C0 = kt
and
C/C0 = 1/(1+kC0t)
C/C0 is the fraction which is still single-stranded
(hence a value between 1 and 0)
when half of the DNA is renatured, C/C0 is ½, and the formula becomes
C0t1/2 = 1/k
or
k = 1/C0t1/2
Thus, the value of k can be experimentally derived from a reassociation curve.
This value depends on cation concentration, temperature, fragment size, etc.
- C0t became pronounced Cot and Cot curve, and hence also written as Cot.
- Similarly, reassociation kinetics with RNA is known (analysed) as Rot curves.
- genomes (especially eukaryotic genomes) may contain
- unique sequences (single copy)
- moderately repeated sequences
- highly repetitive DNA
G. Volckaert
Denaturation, renaturation, hybridisation
2/16/2016
p.4
- Cot analysis allows characterisation of sequence complexity in terms of different
subclasses of sequences depending on degree of repetitivity, and fractionation of
the different subsets.
remember: 90% change (ss versus ds) between Cot – 1 and Cot + 1
- further notes
- renaturation between different molecules, RNA or DNA, is possible ;
originally, reassociation of an RNA strand to a (complementary) DNA strand
was named "hybridization" (forming the hybrid duplex), but this name
was soon used for any combination (DNA-RNA, RNA-RNA or DNA-DNA)
- analysis of reassociation kinetics is usually done in solution
- renaturation is also possible in a two-phase system with one strand
immobilised (target) and the other strand in solution. The strand in
solution is labelled radioactively (or tagged by chemical structures
specifically detectable) and is named the 'probe'.
(but not in DNA chip technology ! see later)
- qualitative and quantitative comparison of nucleic acid sequences is feasible.
=> the kinetics of renaturation may be quite different, but the Tm values still do apply.
Hybridisation
- DNA-DNA, RNA-RNA, DNA-RNA hybrids duplexes may form.
- complex fragment mixtures (e.g. after restriction digestion) can be analysed after
fractionation and immobilisation:
=> blotting techniques : Southern, northern, western, eastern, south-western, etc.
cellulose nitrate ("nitrocellulose"), nylon membrane
capillary transfer, electro-blotting, vacuum blotting, dot blot
- labelling of probe: P32, P33, S35
tagging: biotin, digoxigenin
G. Volckaert
Denaturation, renaturation, hybridisation
2/16/2016
p.5
- detection:
autoradiography (if radioactively labelled)
=> visualization: X-ray film
biotin-streptavidin alkaline phosphatase
peroxidase
=> substrate => product
color 1 => color 2
=> visualization: color (by sight), light emission (X-ray film,
polaroid film, other instrumentation)
digoxigenin => antibody => idem
- Tm
cfr. y = ax + b
where x is the G+C percentage and y is Tm
- the slope a depends on the kind of duplex
- the constant b depends on salt conditions a.o.
- Tm : can be used to define the stringency of hybridisation
- stringency expresses conditions that favours/disfavours formation of H-bonds
- solution of high stringency : disfavours H-bonds
- solution of low stringency : favours H-bonds
- at high stringency: high degree of base complementary is necessary to keep
association of probe and template
- at low stringency: association is obtained, even at a low degree of base
complementarity
- Tm as initially described by Marmur: (for G+C between 30 and 75%)
Tm = 0.41 [%GC] + 69.3 in 0.15 M NaCl + 0.015 M Na-citrate pH 7.0
= 0.41 [%GC] + 53.9 in 0.015 M NaCl + 0.015 M Na-citrate pH 7.0
(slope unchanged but value of the constant b decreases)
G. Volckaert
Denaturation, renaturation, hybridisation
2/16/2016
p.6
- the contribution of ionic concentration to the value of b was adapted by Wetmur
Tm = 0.41 [%GC] + 81.5 + 16.6 log[M+]
if [M+] < 0.4
and then to
Tm = 0.41 [%GC] + 81.5 + 16.6 log{[M+]/(1+0.7[M+])}
for [M+] up to 1M NaCl
- specific problems are created when not all positions in the DNA strands are
complementary to the opposite strand (named mismatch positions)
- mismatches destabilize the duplex: in first approximation 1% mismatch gives a
decrease in Tm of 1 °C.
- denaturing agents in the buffer also decreases Tm
=> formamide causes a decrease of 0.63 °C per percent
- for shorter strands, the size start influencing Tm : a factor of -500/n may be added
Thus : for DNA-DNA
Tm (C°) = 0.41 [%GC] + 81.5 + 16.6 log{[M+]/(1+0.7[M+])} – F – 500/n – P
where F is 0.63 x % formamide and P is % mismatch
n is the size of the double-stranded segment
For RNA-RNA, the formula becomes
Tm (C°) = 0.7 [%GC] + 78 + 16.6 log{[M+]/(1+0.7[M+])} – F – 500/n – P
where F is 0.35 x % formamide
For RNA-DNA hybrids, the formula becomes
Tm (C°) = 0.8 [%GC] + 67 + 16.6 log{[M+]/(1+0.7[M+])} – F – 500/n – P
where F is 0.5 x % formamide
=> the slopes are different, as well as the influence of formamide and other factors
than ionic conditions.
Exercise : when is an RNA-DNA duplex more stable
than the corresponding DNA-DNA duplex?
G. Volckaert
Denaturation, renaturation, hybridisation
2/16/2016
p.7
With oligonucleotides, these formulas are not applicable because Tm is now a
measure of a bimolecular reaction (denaturation dissociates the oligonucleotide
from the template).
Tm is calculated from thermodynamic values and based on nearest neighbors.
Tm (C°) = T°H°/(H°-G°+RT ln[C]) – 269.3 + [salt formula as above]
(H° and G° are summed up from a number of separate contributions)
(an oligonucleotide of N nucleotides consists of N-1 nearest neighbors)
(each mismatch reduces this number by 2)
For a long period of time, in the absence of better alternatives, the rule of Wallace
was very often applied:
Td = (2 x nAT) + (4 x nGC)
but this rule is severely restricted to sizes around 17 (note that it is cumulative)
and hence has been abused very often.
This formula should now be considered as obsolete.
G. Volckaert
Denaturation, renaturation, hybridisation
2/16/2016