Gene Expression, Transcription, and DNA Sequencing Methods

Gene Expression & Transcription of mRNA

Overview of Gene Structure

Genes are composed of exons and introns. Exons are coding regions that remain in the mature mRNA, while introns are non-coding regions removed during RNA processing.

• Exons: Segments of a gene that code for proteins.

• Introns: Non-coding sequences that are spliced out during mRNA maturation.

• Example: The adenovirus hexon gene contains both exons and introns, as shown in the diagram (see image A).

Additional info: Exon-intron structure is typical of eukaryotic genes and is essential for alternative splicing.

Hybridization of DNA and RNA

Hybridization experiments can reveal the structure of genes and the presence of introns and exons.

• Single-stranded DNA can hybridize with mRNA to form regions of double-stranded nucleic acid.

• Loops in the DNA (as seen in electron micrographs) indicate the presence of introns, which are absent in the mature mRNA.

• Example: In the adenovirus gene, regions labeled A, B, and C correspond to exons, while loops represent introns (see image B).

Dideoxy DNA Sequencing (Sanger Sequencing)

Principle of Chain-Terminating Nucleotides

Dideoxy DNA sequencing relies on the incorporation of chain-terminating nucleotides (ddNTPs) to determine the sequence of DNA.

• Deoxynucleotide triphosphates (dNTPs): Standard nucleotides used for DNA synthesis.

• Dideoxynucleotide triphosphates (ddNTPs): Modified nucleotides lacking a 3' hydroxyl group, preventing further chain elongation.

• When a ddNTP is incorporated, DNA synthesis stops, producing fragments of varying lengths.

• Example: The absence of the 3' OH group in ddNTPs is crucial for chain termination (see image 2).

Equation:

$\text{DNA synthesis:} \quad (dNTP) + \text{DNA polymerase} \rightarrow \text{elongated DNA}$

$\text{Chain termination:} \quad (ddNTP) + \text{DNA polymerase} \rightarrow \text{terminated DNA fragment}$

Fluorescent Chain-Terminating Nucleotides

Modern sequencing reactions use fluorescently labeled ddNTPs to identify the terminal nucleotide of each fragment.

• Each ddNTP is tagged with a unique fluorescent dye.

• Fragments are separated by size using capillary electrophoresis.

• The sequence is determined by detecting the color of each fragment as it passes a detector.

• Chain-terminating nucleotides are present at a lower concentration to ensure random incorporation.

• Example: The resulting chromatogram displays colored peaks corresponding to each base (see image 3).

Next Generation Sequencing (NGS)

Limitations of Sanger Sequencing

Sanger sequencing is accurate but limited in throughput and cost-effectiveness for large genomes.

• Can sequence hundreds to thousands of base pairs per reaction.

• Cost per base pair is relatively high ($\sim$ $4 per 1000 bp at UCI).

• Sequencing the human genome ($\sim 10^9$ bp) would be prohibitively expensive using Sanger methods.

Sequencing by Synthesis (NGS Technologies)

Next generation sequencing technologies enable rapid, high-throughput sequencing of entire genomes.

• Sequencing by synthesis: DNA is fragmented, adapters are ligated, and fragments are amplified in parallel.

• Fluorescent nucleotides are incorporated one at a time, and the sequence is read by imaging the fluorescence.

• NGS platforms (e.g., Illumina) can sequence billions of base pairs in a single run.

• Current cost is ~$2000 for $10^8$ bp, but the goal is a $1000 human genome.

• Example: Cyclic array sequencing involves repeated cycles of nucleotide incorporation and imaging (see image 6).

Additional info: NGS has revolutionized genomics, enabling applications in medicine, research, and biotechnology.

Comparison of Sequencing Methods

Method	Read Length	Throughput	Cost per Genome
Sanger (Dideoxy)	100-1000 bp	Low	High
NGS (Illumina, etc.)	50-300 bp	High	Low

Additional info: NGS methods are preferred for large-scale sequencing projects due to their efficiency and scalability.

Key Terms and Definitions

• Exon: Coding region of a gene that remains in mature mRNA.

• Intron: Non-coding region removed during RNA splicing.

• dNTP: Deoxynucleotide triphosphate, standard building block for DNA synthesis.

• ddNTP: Dideoxynucleotide triphosphate, chain-terminating nucleotide used in Sanger sequencing.

• Sequencing by synthesis: NGS method where DNA is sequenced by incorporating nucleotides and detecting fluorescence.