DNA synthesis and sequencing

Solid-phase phosphoramidite synthesis

Oligonucleotides are assembled on controlled-pore glass or polystyrene, so excess reagents are simply washed away. Unlike polymerases, chemical synthesis runs 3′→5′: the first nucleoside is anchored by its 3′-O.

The monomers are nucleoside phosphoramidites, carrying:

• a 4,4′-dimethoxytrityl (DMT) group on the 5′-OH, acid-labile, preventing multiple additions;
• base-labile acyl groups (e.g. benzoyl, isobutyryl) on the exocyclic amines of A, C and G, preventing side reactions at the bases;
• a 2-cyanoethyl group on the phosphorus oxygen, removed by \beta-elimination at the end;
• a diisopropylamino group on P(III), which becomes a leaving group when protonated by a weakly acidic activator such as tetrazole or a substituted analogue.

Each cycle has four steps:

1. Detritylation with dichloro- or trichloroacetic acid frees the 5′-OH. The orange DMT cation absorbs strongly and is used to monitor coupling yields. 2. Coupling: the activated phosphoramidite reacts with the 5′-OH to form a phosphite triester. 3. Capping: acetic anhydride acetylates any unreacted 5′-OH so failure sequences cannot grow further. 4. Oxidation with iodine and water converts P(III) to the stable P(V) phosphate triester (or a sulfurising reagent gives phosphorothioates).

Finally, aqueous ammonia cleaves the chain from the support and removes the base and cyanoethyl protecting groups. Because the overall yield is the product of the stepwise yields, coupling efficiency is critical: at 99% per step a 50-mer is formed in only about 61% yield (0.99^{49}), and at 98% about 37%. Longer genes are assembled from overlapping oligonucleotides by enzymatic methods such as assembly PCR.

PCR

The polymerase chain reaction amplifies a target defined by two primers. Each cycle: denature (about 95 °C) to separate strands, anneal primers (typically 50–65 °C), extend with a thermostable DNA polymerase (Taq) at about 72 °C using dNTPs. Ideally each cycle doubles the target, so n cycles give up to 2^n copies. Quantitative (real-time) PCR follows the product by fluorescence; the cycle at which signal crosses a threshold reflects starting amount.

Sequencing

Sanger (chain-termination) sequencing uses a primer, polymerase and dNTPs plus a small proportion of fluorescently labelled 2′,3′-dideoxynucleotides (ddNTPs). A ddNTP lacks the 3′-OH needed for the next phosphodiester bond, so incorporation stops the chain. The result is a set of fragments ending at every position, separated by capillary electrophoresis; the dye colour at each length reads off the base. Reads are long (several hundred bases) and very accurate.

Next-generation (sequencing-by-synthesis) methods fragment DNA, ligate adapters, amplify fragments into clusters on a flow cell, and add fluorescent reversible terminators one base per cycle; imaging, then chemical removal of the dye and blocking group, lets billions of short reads be collected in parallel.

Oligonucleotide chemistry now underpins a class of medicines. Antisense oligonucleotides and siRNAs rely on backbone and sugar modifications introduced during phosphoramidite synthesis: phosphorothioates for nuclease resistance and protein binding, 2′-O-methyl, 2′-fluoro and 2′-O-methoxyethyl sugars, and locked nucleic acids. Phosphorothioate linkages are stereogenic at phosphorus, and stereocontrolled synthesis of defined P-configurations is an active area. Conjugation to N-acetylgalactosamine directs siRNA to liver cells. Manufacturing large quantities sustainably is a challenge, since the process uses large solvent volumes, and liquid-phase and enzymatic ligation routes are under development. mRNA vaccines, by contrast, are made enzymatically by in vitro transcription, with modified uridines such as N1-methylpseudouridine reducing innate immune activation.

Enzymatic DNA synthesis using engineered terminal deoxynucleotidyl transferase with reversibly blocked nucleotides promises longer, greener template-free synthesis and is being explored for DNA data storage.

In sequencing, third-generation, single-molecule methods give long reads. Nanopore sequencing measures ionic current changes as a strand threads through a protein pore, reading tens to hundreds of kilobases and detecting base modifications such as 5-methylcytosine directly. Single-molecule real-time sequencing watches a polymerase incorporate fluorescent nucleotides in a zero-mode waveguide, giving accurate long reads by repeatedly reading circular templates. These have enabled telomere-to-telomere human genome assemblies, resolution of repetitive regions and structural variants, and direct RNA sequencing. Short-read platforms remain dominant for counting applications (RNA-seq, single-cell methods, clinical panels) because of their cost and accuracy per base.