An Overview of Gene Sequencing and Microfluidic Chip Applicationss in Life Sciences
What is Gene Sequencing?
Gene sequencing is the process of determining the exact order of nucleotides—adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U)—within nucleic acid molecules. It provides a foundational understanding of genetic information by decoding the nucleotide sequence of DNA or RNA.
What is High-Throughput Gene Sequencing (HTS)?
Traditional Sanger sequencing, also referred to as dideoxy sequencing, is limited to producing sequences of 700–1000 base pairs per reaction.
High-throughput gene sequencing (HTS), also known as Next-Generation Sequencing (NGS), revolutionized the field by enabling the parallel sequencing of millions of nucleic acid molecules. A single reaction can generate data exceeding 100 megabases (Mb). This capability allows for comprehensive analysis of genomes, transcriptomes, and epigenomes at unprecedented scales.
Key technological platforms for second-generation sequencing include:
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Roche/454 GS FLX
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Illumina/Solexa Genome Analyzer
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Applied Biosystems/SOLiD™ System
The core principle involves the cyclic array method, where DNA samples are immobilized on a chip and undergo repeated extension reactions. Fluorescently labeled nucleotides are incorporated, and optical signals generated during these cycles are recorded to determine the DNA sequence.
What is Single-Cell Sequencing?
Single-cell sequencing is an advanced technique for analyzing the genome, transcriptome, and epigenome at the level of individual cells. This process involves isolating single cells, extracting RNA, performing reverse transcription, constructing libraries, and conducting sequencing.
Unlike bulk sequencing, which averages the signals from a cell population and masks cellular heterogeneity, single-cell sequencing preserves information on individual cell differences. It provides critical insights into cellular diversity and dynamics, especially in complex biological systems.
Key technological platforms include:
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Fluidigm C1
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10x Genomics Single-Cell Sequencing System
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BD Rhapsody Platform
Microfluidic Chips in High-Throughput Sequencing
Microfluidic chips play an integral role in high-throughput sequencing by enabling precise manipulation of small fluid volumes, reducing reagent consumption, and enhancing reaction efficiency.
(1) 454 Sequencing Chip
The 454 sequencing chip consists of optical fibers configured into a plate-topped polymer (PTP) platform. It features wells 55 µm deep and 44 µm wide, each capable of holding a single bead.
Each well contains the enzymes and substrates necessary for chemiluminescent reactions. During sequencing, one type of deoxynucleotide triphosphate (dNTP) is added per cycle. The emitted fluorescence signals are captured, allowing the nucleotide sequence to be determined.
(2) Illumina Sequencing Chip
The Illumina Flowcell, a glass slide with embedded nano-scale wells, is coated with oligonucleotides that hybridize to DNA fragments. The chip facilitates cyclic incorporation of nucleotides complementary to the DNA template. Each nucleotide emits a unique fluorescent signal upon incorporation, which is detected to determine the sequence.
Microfluidic Chips in Single-Cell Sequencing
Microfluidic chips enhance the efficiency and resolution of single-cell sequencing by facilitating the isolation and barcoding of individual cells.
10x Genomics Platform
The 10x Genomics system encapsulates single cells within nanodroplets, along with barcoded beads and library preparation enzymes. Each bead carries a unique barcode, which tags the transcripts originating from the corresponding cell. This allows for high-throughput gene expression analysis at single-cell resolution.
References
Rothberg, J. M., & Leamon, J. H. (2008). The development and impact of 454 sequencing. Nature Biotechnology, 26(10), 1117–1124.