8.3 The Chemistry of Nucleic Acids

Duplex DNA and RNA can be denatured

Denaturation is the separation of complementary strands with loss of base pairing and stacking
Heat, extreme pH and chemical denaturants all reduce duplex stability
Phosphodiester bonds of the backbone stay intact, so denaturation and strand cleavage are different events
Loss of stacking raises absorbance at 260 nm, the hyperchromic effect
Tm is the temperature at which about half of the duplex is separated under the stated conditions
Example: A260 rising from 1.00 to 1.28 is a 28 percent increase, and the midpoint 1.14 marks Tm for that model
GC content, stacking energy, base order and ionic screening of phosphates all set Tm
Increasing ionic strength within a reasonable range tends to raise Tm
Renaturation and hybridisation depend on complementarity, strand concentration, length, repeats and cooling rate, and cooling alone does not guarantee complete re-pairing

표 8.3-1Variables that affect Tm

Figure 8-50Non-covalent base pairing separated from covalent backbone bonds during denaturation

Figure 8-47Change in A260 with temperature for two samples (simulated sigmoid curves)

Figure 8-49Simulated Tm change with GC content and ionic strength; the model shows trends only

Figure 8-48Teaching diagram of DNA denaturation and renaturation

Nucleotides and nucleic acids undergo non-enzymatic change

Depurination is spontaneous hydrolysis of the N-glycosidic bond between adenine or guanine and deoxyribose, releasing the base
The resulting AP site blocks replication and transcription and becomes a mutation if unrepaired
Deamination of cytosine gives uracil, and failure to remove it fixes a base pair change at the next replication
Deamination of 5-methylcytosine gives thymine, which is hard to distinguish from the normal base
This is why methylated CpG sites are mutational hotspots in genomes
Reactive oxygen species oxidise guanine to lesions such as 8-oxoguanine
Ultraviolet light creates photoproducts between adjacent pyrimidines and alkylation adds groups to particular base atoms
Damaged DNA can be repaired, but surviving damage becomes a fixed sequence change
Base damage, a chemically altered molecular state, is not the same as a mutation, a sequence change fixed by replication

표 8.3-2Non-enzymatic chemical changes and their consequences

Figure 8-51Representative non-enzymatic chemical changes in DNA, summarised by outcome

Figure 8-52Oxidative and ultraviolet DNA damage distinguished structurally

Some DNA bases are methylated

5-Methylcytosine carries a methyl group at C5 of cytosine, commonly at CpG dinucleotides in mammals
DNA methyltransferases use S-adenosylmethionine as the methyl donor
Methylated cytosine still pairs with guanine, so A, T, G, C notation alone cannot show methylation
Methylation is linked to gene regulation, maintenance of cell identity, genomic imprinting and control of transposable elements
Methylation does not always mean repression
A change in expression does not prove that methylation is the direct cause without further experiments
In bisulfite treatment unmethylated cytosine is converted to uracil and read as T while 5mC is relatively preserved
Incomplete conversion, sample damage and the distinction of 5-hydroxymethylcytosine need separate validation
Example: 72 C and 28 T reads out of 100 give an estimated methylation fraction of about 72 percent

Figure 8-53Biochemical meaning of CpG methylation and 5-methylcytosine, simplified

Figure 8-54Basic readout principle and cautions of bisulfite analysis

Chemical DNA synthesis is automated

The phosphoramidite method grows an oligonucleotide on a solid support by adding one protected monomer at a time
Synthesis starts at the support-bound 3-prime end and grows toward 5-prime, so assembly runs 3-prime to 5-prime
Biological DNA polymerases add nucleotides to a 3-prime OH and synthesise 5-prime to 3-prime, the opposite direction
The cycle is 5-prime DMT deprotection, coupling of the activated monomer, capping of unreacted 5-prime OH and oxidation of the phosphite
Capping prevents unreacted chains from being extended further
Work-up requires deprotection, cleavage from the support, purification and quality checking
With per-coupling success p, all n minus 1 couplings succeed with probability p to the power n minus 1
Example: 99.5 percent over 49 couplings for a 50 nt oligo gives 0.995 to the 49th, about 78.2 percent
That figure is an idealised upper bound that ignores losses in later steps

Figure 8-55Four representative steps of automated solid-phase DNA synthesis

Figure 8-56Fall in full-length product fraction with synthesis length under a coupling-probability model

Gene sequences can be amplified by PCR

PCR repeatedly amplifies a target region with two primers flanking it and a thermostable DNA polymerase
The reaction needs template, primers, dNTPs, Mg2+, buffer and polymerase
One cycle is denaturation, annealing and extension
Under ideal conditions Nn = N0 times 2 to the n, and with efficiency E the form N0(1+E) to the n applies
Real reactions run below 100 percent efficiency and approach a plateau as product accumulates
Example: 10 molecules over 20 cycles gives 10,485,760, and E of 0.80 gives about 1,274,823
A positive control checks that the reaction works and a no-template control detects contamination
A band of the expected size suggests product but does not confirm the exact sequence
Contamination control and, when needed, product purification and sequencing are essential

Figure 8-57Three steps of PCR thermal cycling; temperatures and times vary between instruments

Figure 8-58Ideal amplification compared with falling efficiency on a log plot, excluding the plateau

Figure 8-59Simulated agarose gel of PCR products; M is the size standard and NTC the no-template control

Long DNA strands can be sequenced

In Sanger sequencing a ddNTP lacks a 3-prime OH, so once incorporated the next phosphodiester bond cannot form and extension stops
Fluorescent fragments of different length are separated by capillary electrophoresis and read in signal order
NGS builds an adapter-ligated library and reads many surface-amplified clusters in parallel
Reversible terminators incorporate one base per cycle, measure fluorescence, then remove label and blocking group
Short reads struggle to assign position within long repeats
Long reads span a repeat and its unique flanks, which improves assembly
Longer reads are not automatically more accurate per base
The Phred score is Q = -10 log10 of the error probability, with Q20 at 0.01 and Q30 at 0.001
Example: at Q30 the expected number of misreads in 1,000 bases is one, though a given read need not contain exactly one error

표 8.3-3Characteristics of sequencing methods

Figure 8-60Simulated Sanger fluorescence chromatogram (teaching data, not measured)

Figure 8-61Basic cycle of reversible-terminator massively parallel sequencing

Figure 8-62Model of how short and long reads differ over a repeat region

Figure 8-63Quantitative relation between the Phred score and error probability

[Separate note 8-1] A powerful tool in forensic science

A short tandem repeat is a sequence repeated several times whose count varies between individuals and gives discriminating power
Forensic work analyses allele length by PCR amplification and capillary electrophoresis
A match at one locus or in some peaks cannot identify an individual
Mixtures and trace samples require attention to allele drop-out, stutter, contamination and secondary transfer
A matching genotype does not by itself show who did what
Courts and investigations must weigh method suitability, sample records, population allele frequencies and statistics

Figure 8-64Simulated STR electropherogram; mixed samples require cautious interpretation

Sequencing technology continues to advance

Scope has widened from single genes to whole genomes, transcriptomes, single cells and epigenetic marks
Short and long reads complement rather than replace one another and are chosen by aim
Long reads suit repeats and large structural variants while massively parallel short reads suit targeted variant screening across many samples
Method choice balances accuracy, read length, cost and sample amount

Figure 8-65Stepwise analysis from nucleic acid sample to a supportable conclusion

References

**표 8.3-1**Variables that affect Tm
Variable	Typical effect	Condition to check
Higher GC content	Tends to raise Tm at similar length and conditions	Neighbouring pair order still matters
Higher salt concentration	Screens phosphate repulsion and tends to raise Tm	Extreme salt and specific ions need separate assessment
Longer strand	Strong effect on stability for short duplexes	Do not mix equations for long DNA and oligos
Mismatched base pair	May lower local stability	Depends on the type and position of the mismatch
Extreme pH	Changes base state and pairing	Strand separation differs from backbone damage

**표 8.3-2**Non-enzymatic chemical changes and their consequences
Chemical change	Molecular change	Potential outcome
Depurination	Hydrolysis of the purine-sugar N-glycosidic bond	Formation of an AP site
Deamination	C to U, 5mC to T	Possible base substitution at replication
Oxidation	Formation of 8-oxoG and related lesions	Mispairing and greater repair burden
Ultraviolet damage	Photoproducts between adjacent pyrimidines	Blocks to replication and transcription
Alkylation	Alkyl group added to a base	Altered pairing or cross-linking

**표 8.3-3**Characteristics of sequencing methods
Method	Main information obtained	Typical strength	Caution in interpretation
PCR with a gel	An amplified product of a given size	Simple target detection	Exact sequence needs separate confirmation
Sanger	Base order of terminated fragments by length	Confirms a single target sequence	Limited for mixtures and long complex regions
Short-read NGS	Many short reads in parallel	Many samples and loci at once	Repeats, bias and mapping ambiguity
Long-read sequencing	Relatively long connectivity information	Links structural variants and repeat regions	Data quality and sample length must be managed
STR profile	Allele signals from repeat number	Statistical information for identification	Mixtures and the strength of evidence

**Figure 8-50**Non-covalent base pairing separated from covalent backbone bonds during denaturation

**Figure 8-47**Change in A260 with temperature for two samples (simulated sigmoid curves)

**Figure 8-49**Simulated Tm change with GC content and ionic strength; the model shows trends only

**Figure 8-48**Teaching diagram of DNA denaturation and renaturation

**Figure 8-51**Representative non-enzymatic chemical changes in DNA, summarised by outcome

**Figure 8-52**Oxidative and ultraviolet DNA damage distinguished structurally

**Figure 8-53**Biochemical meaning of CpG methylation and 5-methylcytosine, simplified

**Figure 8-54**Basic readout principle and cautions of bisulfite analysis

**Figure 8-55**Four representative steps of automated solid-phase DNA synthesis

**Figure 8-56**Fall in full-length product fraction with synthesis length under a coupling-probability model

**Figure 8-57**Three steps of PCR thermal cycling; temperatures and times vary between instruments

**Figure 8-58**Ideal amplification compared with falling efficiency on a log plot, excluding the plateau

**Figure 8-59**Simulated agarose gel of PCR products; M is the size standard and NTC the no-template control

**Figure 8-60**Simulated Sanger fluorescence chromatogram (teaching data, not measured)

**Figure 8-61**Basic cycle of reversible-terminator massively parallel sequencing

**Figure 8-62**Model of how short and long reads differ over a repeat region

**Figure 8-63**Quantitative relation between the Phred score and error probability

**Figure 8-64**Simulated STR electropherogram; mixed samples require cautious interpretation

**Figure 8-65**Stepwise analysis from nucleic acid sample to a supportable conclusion