DNA sequencing refers to the analysis of the base sequence of a specific DNA fragment, that is, the adenine (A), thymine (T), cytosine © and guanine (G) arrangement. the way. The advent of rapid DNA sequencing methods has greatly facilitated research and discovery in biology and medicine.
In basic biology research, and in numerous fields of application such as diagnostics, biotechnology, forensic biology, and biosystematics, DNA sequence knowledge has become indispensable knowledge. Rapid sequencing speeds with modern DNA sequencing technology have helped to sequence complete DNA sequences, or multiple types of genome sequencing and life species, including the human genome and many other animal, plant and microbial species complete DNA sequences.
Sequencing purpose
Identify the direction and structure of recombinant DNA, locate, identify and compare mutations
Development History
In the late 1970s, Walter Gilbert invented the chemical method, Frederick Sanger invented the double deoxygenation termination manual sequencing, isotope labeling
In the mid-1980s, an automatic sequencer (using the principle of dideoxy termination), fluorescence instead of isotopes, computer image recognition
In the mid-1990s, major improvements in the sequencer and clustered capillary electrophoresis replaced gel electrophoresis.
Completed the Human Genome Framework in 2001
Sequencing principle
Chemical modification sequencing principle
The chemical reagents process the final DNA fragment, causing specific cleavage of the base, producing a set of reaction mixtures of DNA strands of various lengths, which are separated by gel electrophoresis. Chemical cleavage reaction: including the modification of a base, the modified base is transferred from its sugar ring and the DNA is broken at the sugar ring of the lost base.
Principle of Sanger sequencing
It is the use of a DNA polymerase to extend the primers bound to the template of the sequence to be determined. Until a chain termination nucleotide is incorporated. Each sequence consists of a set of four separate reactions, each containing all four deoxynucleotide triphosphates (dNTPs) and spiked with a different amount of a different dideoxynucleoside triphosphate (ddNTP). Since the ddNTP lacks the 3-OH group required for extension, the extended oligonucleotide is selectively terminated at G, A, T or C. The termination point is determined by the corresponding dideoxy in the reaction. The relative concentrations of each of the dNTPs and ddNTPs can be adjusted to provide a set of chain termination products ranging from a few hundred to several kilobases in length. They have a common starting point, but terminate in different nucleotides. High-resolution denaturing gel electrophoresis can be used to separate fragments of different sizes. After gel treatment, X-ray film can be autoradiographed or non-isotopically labeled. Detection.
Sequencing rule
Several sets of radiolabeled oligonucleotides are generated that are independent of each other, each set of oligonucleotides having a fixed origin, but randomly terminating on a particular one or more residues.
Since each base on the DNA has equal chance of appearing at the variable terminus, each of the above products is a mixture of oligonucleotides whose length is from a specific base in the original DNA fragment. The position on the top is determined.
Electrophoretic analysis of each set of oligonucleotides can be performed under conditions that distinguish different DNA molecules that differ by only one nucleotide in length, as long as several sets of oligonucleotides are loaded into several adjacent lanes in the sequencing gel. On top, the nucleotide sequence on the DNA can be read directly from the film from the emission of the gel.
Sequencing technology
High-throughput sequencing, also known as “Next-generation” sequencing technology, enables sequencing and general reading of hundreds of thousands to millions of DNA molecules in parallel at a time. The shorter length is the sign.
According to the history of development, influence, sequencing principles and technology, there are mainly the following: Massively Parallel Signature Sequencing (MPSS), Plonony Sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, ABI SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, and the like.
MPSS
Massively Parallel Signature Sequencing, developed by Lynx Therapeutics in the 1990s, is a pioneer in the development of “next generation” sequencing technologies. MPSS is a complex technique based on the connection and decoding of beads and adaptors. The results are short and are used for transcriptome sequencing to determine gene expression. MPSS assay results have sequence preference and are easy to lose certain specific sequences in DNA, and the operation is complicated, and has gradually faded out and replaced by new methods.
Polony Sequencing
A sequencing method based on techniques such as emulsion PCR and automated microscopy developed at Harvard George Church in 2005. Related technologies have been integrated into ABI’s SOLiD sequencing technology platform
454 pyrosequencing
A parallel pyrosequencing method developed by the 454 company. The method amplifies DNA (ie emulsion PCR) in water droplets wrapped in an oil solution, each water droplet initially comprising only one magnetic bead coated with a large number of primers and one DNA template molecule linked to the microbead (controlling the concentration of DNA) Large probability event)
The emlusion PCR product is loaded onto a special PTP plate with millions of wells, each containing only one magnetic bead. DNA Polymerase releases a PPi (pyrophosphate molecule) when a dNTP is polymerized onto the template; PPi and APS generate an ATP molecule catalyzed by ATP-Sulfurylase (ATP Sulfate); ATP molecule is in Luciferase (fluorescence) Under the action of the enzyme, luciferin is oxidized to oxy luciferin, and the visible light is captured by the CCD optical system to obtain a specific detection signal. The signal intensity is proportional to the corresponding number of bases. DNA sequence determination is achieved by sequentially adding four dNTPs in sequence and reading the signal intensity and time of occurrence. The read length and cost per base of this technique are between the Sanger method and the Solexa and SOLiD methods. The 454 company is now part of Roche.
Illumina (Solexa) sequencing
Solexa has developed a reversible dye termination method. The DNA template is first ligated to a primer that crosslinks with a solid medium, such as a glass plate, and amplified to form a local microclones. Four ddNTPs were added to the system in turn, and unbound ddNTPs were washed away before adding the next nucleotide. Unlike pyrosequencing of 454, this method extends only one base at a time.
Sequencing method
Automatic sequencing
The gene analyzer (ie, DNA sequencer) replaces the traditional polyacrylamide plate electrophoresis with capillary electrophoresis technology, and uses the company’s patented four-color fluorescent dye-labeled ddNTP (labeled terminator method), so the single primer PCR sequencing reaction, The generated PCR product is a single-stranded DNA mixture of 4 different fluorescent dyes at the 3' end of 1 base, so that the sequencing PCR products of the four fluorescent dyes can be electrophoresed in one capillary, thereby avoiding migration between lanes. The effect of rate differences greatly improves the accuracy of sequencing.
Due to the different molecular sizes, the mobility in capillary electrophoresis is also different. When passing through the capillary reading window segment, the CCD (charge-coupled device) camera detector in the laser detector window can detect the fluorescent molecules one by one. The fluorescence is spectrally split to distinguish the fluorescence of different colors representing different base information, and is simultaneously imaged on a CCD camera. The analysis software can automatically convert different fluorescence into DNA sequences for DNA sequencing purposes. The analysis results can be output in various forms such as gel electrophoresis pattern, fluorescence absorption peak map or base arrangement order.
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