Sanger Sequencing
The Sanger Method is a method of sequencing (determining the nucleotide sequence) of DNA, also known as the strand termination method. This sequencing method was first proposed by Frederick Sanger in 1977, for which he was awarded the Nobel Prize in Chemistry in 1980. This method has been the most common for 40 years.
Method principle
In the classical version of the Sanger method, one of the strands of the analyzed DNA acts as a matrix for the synthesis of the complementary strand by the enzyme DNA polymerase. The reaction with the same matrix is carried out in four different test tubes, each of which contains:
- primer – a small single-stranded DNA molecule complementary to the beginning of the region to be sequenced. The primer is necessary because DNA polymerases cannot start the synthesis of a chain “from empty space”, they only attach the next nucleotide to the already existing 3′-hydroxyl (OH) group of the previous one. The primer is thus a “primer” for DNA synthesis;
- four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP);
- a small amount (at a concentration of 1 to 100) of one of the radioactively labeled deoxynucleotides (dideoxynucleotide) (for example, [32P] -dATP), which is incorporated into DNA during synthesis and allows subsequent visualization of the reaction products;
Dideoxyribonucleotides (ddATP, ddGTP, ddCTP, or ddTTP) lack a 3′-hydroxyl group; therefore, after their inclusion in the chain, further synthesis is terminated. Thus, in each test tube, a set of DNA fragments of different lengths is formed, which end in the same nucleotide (in accordance with the added dideoxynucleotide). After completion of the reaction, the contents of the tubes are separated by electrophoresis in polyacrylamide gel under denaturing conditions and the gels are autoradiographed. The products of four reactions form a “sequencing ladder” that allows you to “read” the nucleotide sequence of a DNA fragment.
Sanger’s method also allows you to determine the nucleotide sequence of RNA, but it must first be “rewritten” in the form of DNA using reverse transcription.
How are things going today?
Today, Sanger DNA sequencing is fully automated and is carried out on special devices, sequencers. The use of fluorescently labeled dideoxynucleotides with different emission wavelengths allows the reaction to be carried out in one test tube. The reaction mixture is separated by capillary electrophoresis in solution, DNA fragments emerging from the capillary column are recorded with a fluorescence detector. The results are analyzed using a computer and presented as a sequence of multi-colored peaks corresponding to four nucleotides. Sequencers of this type can “read” sequences of 500-1000 nucleotides in length at a time. For comparison, the pyrosequencing method, developed in 1996, allows one to determine the sequence of a much smaller number of nucleotides. Automation has significantly accelerated the sequencing process and made it possible to carry out the sequencing of entire genomes, including the human genome.
Pyrosequencing
Pyrosequencing is a DNA sequencing method (determining the sequence of nucleotides in a DNA molecule) based on the principle of “sequencing by synthesis”.
The “sequencing by synthesis” method allows one strand of DNA to be sequenced by synthesizing a complementary strand, and the attachment of each nucleotide is recorded. During the reaction, the DNA template is immobilized, solutions of nucleotides A, C, G and T are added and washed sequentially after each sequencing cycle.
The pyrosequencing method is based on the detection of the activity of the enzyme DNA polymerase with another chemiluminescent enzyme. The sequence of feeding the reagents into the reaction mixture, which give a chemiluminescent signal, makes it possible to determine the sequence of the analyzed DNA region.
Pyrosequencing includes the following steps:
- Stage 1. Amplification of a DNA fragment and biotinylation of a strand that will serve as a template for pyrosequencing. Denaturation of a biotinylated single-stranded PCR amplicon, isolation and hybridization with a sequencing primer;
- Stage 2. Incubation of primer and single-stranded template in the presence of DNA polymerase, ATP-sulfurylase, luciferase, apyrase, and substrates: adenosine-5′-phosphosulfate (APS) and luciferin;
- Stage 3. Adding the first deoxyribonucleotide triphosphate (dNTP) to the reaction. DNA polymerase catalyzes the addition of dNTP to a sequencing primer if it is complementary to the template DNA sequence. Each attachment to the chain is accompanied by the release of pyrophosphate (PPi) in an amount equimolar to the amount of incorporated nucleotides;
- Stage 4. ATP sulfurylase converts pyrophosphate to ATP in the presence of adenosine 5′-phosphosulfate. The resulting ATP triggers a luciferase-mediated oxidation reaction of luciferin to oxyluciferin, resulting in the formation of visible light in an amount proportional to the amount of ATP. The light is detected by the CCD camera and displayed as peaks on the pyrogram. The height of the peaks is proportional to the number of nucleotides inserted into the template;
- Stage 5. During the entire reaction, the apyrase enzyme degrades unbuilt nucleotides and excess ATP. After the completion of the degradation of nucleotides that have not been incorporated into the DNA chain, a new nucleotide is added;
- Stage 6. The addition of nucleotides is carried out sequentially. It should be noted that deoxyadenosine-alpha-thio-phosphate is used as a substitute for deoxyadenosine-3-phosphate (dATP), since it is efficiently recognized by DNA polymerase, but not recognized by luciferase. During the reaction, the complementary DNA strand is completed, and the nucleotide sequence is determined according to the peaks in the pyrogram.
Applications of pyrosequencing
Genetic testing
- Genetic certification and identification of genetic polymorphisms associated with the development of multifactorial diseases;
- Genetic analysis of “complex” regions of the genome (translocations, repeats, deletions);
- Verification and validation of genome-wide analysis results.
Oncology
- Identification of activating somatic mutations in the genes EGFR, RAS, BRAF, PI3K, etc.;
- Analysis of methylation of tumor suppressor genes: MGMT, MLH1, p16, etc.;
- Search for new genetic markers of malignant neoplasms.
Microbiology and genetic engineering
- Strain genotyping;
- Gene re-sequencing: detection of new genetic variants, islands of pathogenicity, virulence, etc.;
- Identification of mutations that make viruses resistant to antiretroviral therapy;
- Identification of mutations that determine the resistance of bacteria and fungi to antibiotics;
- Verification of cloning results: sequencing of PCR amplicons, plasmids, DNA cassettes;
- Development of unique PCR test systems.