Chapter 8: DNA Sequencing

Dideoxy (Chain Termination) Sequencing

A method of sequencing DNA that uses dideoxynucleotides (ddNTPs) to terminate synthesis of DNA strands. Also known as Sanger sequencing or cycle sequencing

How does chain termination sequencing differ from normal DNA replication?

Chain termination uses ddNTPs that lack a 3'-OH group, which halts DNA synthesis upon incorporation. In contrast, normal replication uses dNTPs, allowing continuous elongation

Difference Between ddNTPs and dNTPs

ddNTPs lack a 3'-hydroxyl group, preventing further nucleotide addition and causing chain termination. dNTPs have a 3'-OH, allowing chain elongation through phosphodiester bond formation

How Does a ddNTP Terminate DNA Synthesis?

When DNA polymerase incorporates a ddNTP, the absence of a 3’-OH group means no phosphodiester bond can form, so synthesis stops at that point

Advantage of Sequenase Over Klenow Polymerase

Sequenase has high processivity, rapid reaction rate, negligible exonuclease activity, and can incorporate modified nucleotides, making it ideal for sequencing. Klenow has low processivity and lacks 5'→3' exonuclease activity

How Are DNA Fragments Separated and Detected After Chain Termination Sequencing?

Fragments of different lengths are separated by size via capillary electrophoresis. A laser detects the fluorescently labeled 3' ends, revealing the DNA sequence base-by-base

Adapters

Short double-stranded DNA fragments with known sequences added to the ends of genomic DNA for next-generation sequencing

Next-Generation Sequencing (NGS)

Experimental techniques that decode the base order of millions of DNA fragments simultaneously

Tagmentation

A DNA fragmentation method using a transposase enzyme to both cut DNA and insert adapter sequences at breakpoints for NGS

Barcode (Index) Sequence

A unique DNA sequence within adapters that distinguishes DNA samples in multiplexed NGS runs

Consensus Sequence

An idealized DNA sequence composed of the most frequent bases at each position across aligned reads

Contig

A continuous stretch of DNA sequence without gaps, assembled from overlapping reads

Read Depth

The number of sequencing reads that overlap a specific nucleotide location in the genome

Emulsion PCR

PCR performed in isolated water droplets within oil, enabling millions of separate reactions in parallel

Third-Generation Sequencing

Sequencing method that reads a single DNA strand without needing amplification or multiple copies

Two Methods of Fragmenting Genomic DNA for NGS

• Tagmentation: Transposase cuts DNA and adds adapters in a single step.

• Ultrasonic Disruption: Sound waves generate shock waves that shear DNA by collapsing microbubbles

Illumina Sequencing (Steps)

• Fragment DNA

• Add adapters

• Bind to glass slide and amplify into clusters

• Add fluorescent nucleotides one at a time

• Capture image of incorporated base

• Repeat to read entire sequence

Ion Torrent Sequencing (Steps)

• Fragment DNA and add adapters

• Attach DNA to beads and amplify

• Place beads into wells

• Add nucleotides sequentially

• Detect H⁺ release if a base is incorporated

• pH sensor reveals the base

Nanopore Sequencing (Steps)

• Thread single DNA strand through a nanopore

• Each base alters electrical current

• Current change interpreted to identify the base

SMRT Sequencing (Steps)

• Place DNA in a zero-mode waveguide (ZMW)

• Add fluorescent nucleotides

• Real-time observation of DNA synthesis via light signals

• Flash color indicates the added base

Two Methods for Targeted Sequencing Library Prep

• PCR Amplification: Design primers → amplify target region → add adapters

• Biotinylated Probes: Fragment DNA → hybridize with biotin probes → isolate with beads → add adapters