Gene Sequencing Method: General overview of Sanger and Next-generation Sequencing

General overview of Sanger and Next-generation Sequencing

A gene sequence is the specific order of nucleotides (A, C, G, and T) that make up a gene, which is a segment of DNA that provides the instructions for making a specific protein or RNA molecule. Gene sequences can vary between different individuals and even within the same individual, leading to genetic diversity.

Here is a general overview of the process of gene sequencing:

1. DNA extraction: The first step is to extract the DNA from the sample (blood, saliva, tissue, etc.).
2. DNA fragmentation: Next, the DNA is fragmented into smaller pieces (typically 100-1000 bp) using specific enzymes or mechanical methods.
3. Library preparation: The fragmented DNA is then converted into a "library" of DNA fragments that can be read by the sequencing instrument. This step typically involves attaching adapters to the ends of the fragments and amplification of the library.
4. Sequencing: The DNA library is then loaded onto the sequencing instrument and the order of nucleotides is read.
5. Data analysis: The raw data obtained from the sequencing instrument is then processed and analyzed to obtain the final gene sequence.

It's worth noting that the specific process and steps may vary depending on the type of sequencing method used.

There are two main types of gene sequencing:

1. Sanger sequencing

Sanger sequencing, also known as dideoxy sequencing, is a method of DNA sequencing that was developed by Frederick Sanger in 1977. It is considered the traditional method of DNA sequencing and is still widely used today. The Sanger sequencing method relies on the use of specific enzymes and nucleotides to synthesize a new DNA strand complementary to the target gene. The new strand is then terminated at specific points (using "dideoxynucleotides") and the resulting fragments are separated by size and read to determine the order of nucleotides.

The basic principle of Sanger sequencing is the incorporation of a modified nucleotide, called dideoxynucleotides (ddNTPs), into a new DNA strand that is complementary to the template strand. ddNTPs differ from the regular nucleotides (dNTPs) in that they lack a 3' hydroxyl group, which is necessary for the next nucleotide to be added. This means that once a ddNTP is incorporated, the synthesis of the new strand stops at that point.

To perform

Sanger sequencing, a sample of DNA is first amplified using the polymerase chain reaction (PCR). The amplified DNA is then denatured, or separated into single strands, and the desired strand is selected as the template for sequencing. This is done by annealing specific primers, short sequences of nucleotides, to the template strand. The primers are then extended by a DNA polymerase, such as Taq polymerase, to generate a new complementary strand. Once the new strand is synthesized, the reaction is stopped and the DNA fragments are separated by size by gel electrophoresis. The gel is then stained with ethidium bromide, which causes the DNA to fluoresce when exposed to UV light. The different-sized DNA fragments show up as different colored bands on the gel, and the sequence of nucleotides can be read by reading the order of the bands from the bottom to the top of the gel.

Figure: Sanger Sequencing (Michel G Gauthier)

Sanger sequencing is a reliable and accurate method for DNA sequencing, and it has been used in many important research studies and projects, such as the Human Genome Project. However, it has some limitations, such as relatively low throughput and a relatively high cost.  

2. Next-generation sequencing (NGS)

Next-generation sequencing (NGS) is a term that refers to a set of high-throughput DNA sequencing technologies that have been developed in the last decade. These technologies allow for the rapid sequencing of large amounts of DNA in a single run, which has revolutionized the field of genomics. NGS methods have higher throughput and lower cost per base compared to traditional Sanger sequencing, making it possible to sequence whole genomes, transcriptomes, and epigenomes at an unprecedented scale and speed.

NGS technologies differ in how they read the DNA sequence, but they all involve breaking up the DNA into small fragments and reading them simultaneously in parallel. The image is taken from the website www.irepertoire.com

Figure: Next-generation Sequencing method overview (www.iRepertoire.com)

Some of the most widely used NGS methods include:

1- Illumina sequencing: This is the most widely used NGS method and is based on the use of reversible terminator chemistry. The DNA is fragmented into small pieces and adapters are attached to the ends. The adapters contain specific sequences that allow the DNA fragments to be attached to a solid surface, such as a bead or a flow cell. The DNA is then amplified and the nucleotides are added one by one in a controlled manner, allowing the sequencing process to be read in real-time.

2- PacBio sequencing: This method is based on single-molecule, real-time (SMRT) sequencing technology. The DNA is fragmented into very long pieces (typically 20-30 kb) and ligated to a hairpin adapter. The DNA is then loaded onto a zero-mode waveguide (ZMW) chip, which is a tiny well that allows for the observation of individual DNA polymerase molecules as they add nucleotides. This allows for the long-read sequencing of the DNA.

3- Nanopore sequencing: This method is based on the use of tiny pores, called nanopores, that are embedded in a thin membrane. The DNA is loaded onto the membrane and an electric current is applied, which causes the DNA to pass through the nanopore. As the DNA passes through the pore, the nucleotides change the electrical current in a way that is specific to each nucleotide, which allows for the sequencing of the DNA.

NGS has a wide range of applications in biomedical research, including the discovery of new genetic variants associated with diseases, understanding the genetic basis of cancer, and discovering new genetic markers for personalized medicine.

Additionally, NGS has been used to study the genetic diversity of different organisms, including the human gut microbiome and the evolution of species. With the advancements in NGS technologies, the cost has come down and it has become more accessible to researchers and clinicians.