DNA Sequencing Techniques to Know for General Genetics

DNA sequencing techniques are essential tools in genetics, allowing scientists to read and analyze genetic information. From Sanger sequencing's accuracy to the speed of next-generation sequencing, these methods have transformed our understanding of DNA and its applications in research and medicine.

1. Sanger sequencing (chain termination method)

   • Utilizes dideoxynucleotides (ddNTPs) to terminate DNA strand elongation.
   • Produces fragments of varying lengths that are separated by capillary electrophoresis.
   • Ideal for sequencing short DNA fragments (up to 1000 base pairs).
   • High accuracy and reliability, often used for validating NGS results.
   • Pioneered by Frederick Sanger in the 1970s, it remains a foundational technique in genetics.

2. Next-generation sequencing (NGS)

   • Allows for massively parallel sequencing, generating millions of sequences simultaneously.
   • Significantly reduces the time and cost of sequencing compared to Sanger sequencing.
   • Enables whole-genome sequencing, transcriptome analysis, and targeted sequencing.
   • Data analysis requires sophisticated bioinformatics tools due to the large volume of data produced.
   • Revolutionized genomics, making it accessible for various applications in research and medicine.

3. Polymerase chain reaction (PCR)

   • Amplifies specific DNA sequences, making millions of copies from a small initial sample.
   • Involves repeated cycles of denaturation, annealing, and extension using DNA polymerase.
   • Essential for preparing samples for sequencing and other genetic analyses.
   • Highly sensitive, allowing detection of minute amounts of DNA.
   • Widely used in diagnostics, forensics, and research applications.

4. Maxam-Gilbert sequencing

   • A chemical method of sequencing DNA that involves cleavage of DNA at specific bases.
   • Requires radioactively labeled DNA and is less commonly used today due to complexity.
   • Produces fragments that are analyzed by gel electrophoresis to determine the sequence.
   • Developed in the late 1970s, it was one of the first methods for DNA sequencing.
   • Primarily of historical interest, as Sanger sequencing and NGS have largely replaced it.

5. Shotgun sequencing

   • Involves randomly breaking DNA into small fragments and sequencing them.
   • Requires computational methods to assemble overlapping sequences into a complete genome.
   • Effective for large genomes, such as those of plants and animals.
   • Pioneered the sequencing of the human genome, enabling large-scale genomic projects.
   • Often used in conjunction with NGS for efficient genome assembly.

6. Illumina sequencing

   • A type of NGS that uses reversible dye terminators for sequencing by synthesis.
   • Highly scalable, allowing for sequencing of multiple samples in a single run.
   • Produces short reads (typically 50-300 base pairs) with high throughput and accuracy.
   • Widely used in genomics, transcriptomics, and epigenomics research.
   • Cost-effective and suitable for large-scale projects, including population genomics.

7. Ion torrent sequencing

   • A semiconductor-based sequencing technology that detects changes in pH as nucleotides are added.
   • Offers rapid sequencing with shorter run times compared to other NGS methods.
   • Produces medium-length reads (up to 400 base pairs) and is relatively low-cost.
   • Useful for targeted sequencing and small genome projects.
   • Less commonly used for large-scale genomic studies compared to Illumina.

8. Pyrosequencing

   • A sequencing method based on the detection of pyrophosphate release during nucleotide incorporation.
   • Produces real-time sequencing data and is suitable for short reads (up to 300 base pairs).
   • Allows for direct quantification of DNA sequences and is useful for SNP analysis.
   • Less widely adopted than other NGS methods but valuable for specific applications.
   • Combines aspects of both sequencing and quantitative analysis.

9. Single-molecule real-time (SMRT) sequencing

   • A technology that allows for the observation of DNA synthesis in real-time at the single-molecule level.
   • Produces long reads (up to 30,000 base pairs or more), facilitating the assembly of complex genomes.
   • Reduces the need for amplification, minimizing biases and errors in sequencing.
   • Useful for studying structural variants and repetitive regions in genomes.
   • Developed by Pacific Biosciences, it has applications in de novo genome assembly and transcriptome analysis.

10. Nanopore sequencing

    • A portable sequencing technology that detects changes in ionic current as DNA passes through a nanopore.
    • Capable of producing ultra-long reads (up to millions of base pairs), useful for complex genomic regions.
    • Real-time sequencing allows for immediate data analysis and feedback.
    • Minimal sample preparation and no amplification required, preserving original DNA.
    • Applications include field-based sequencing, metagenomics, and real-time pathogen detection.