Nucleic acid sequence determination.

the
sequence of DNA determines the characteristics of
gene
, DNA sequence analysis (sequencing, sequencing) is an important basic technique


molecular biology. Whether the cancer gene screened from the gene bank or amplified by
PCR
method, the final need for
nucleic acid
sequence analysis, in order to understand the fine structure of the gene, obtain its restrictive endoenzyme map, analyze the mutation of the gene and its effect on function, help artificial genes, design citations, and study the molecular pathogenesis of tumors. Sequencing is based on high-resolution denatured polyacrylamide gel electrophoresis technology. The most commonly used methods are Maxim-Gilbert's chemical degradation and Sanger's double deoxygenation method, which has been introduced in recent years with DNA sequence autometrics. Chemical degradation is the marking of nucleins at the 5' end of a fragment of DNA, and then the dna is specifically degraded by a single-sex
chemical reagent
, and after electrophoresis and self-development, fragments extending from the marker end can be obtained for reading sequences and comparisons. Generally can read 200-250
nucleotide
sequence. The double deoxygenation method is the use of nucleotide chain terminators, e.g. 2', 3'-double deoxynucleotide triphosphate ddNTP (e.g. ddTTP, ddTTP, ddGTP and ddCTP) mixed into the DNA chain to terminate the chain extension, with the addition of 4 The normal dNTP mixture is divided into four groups to react, so that a set of DNA fragments at the end of different ends of different specific nucleotide chain terminators can be obtained, by gel electrophoresis separation and radiation self-development, the synthetic DNA nucleotide sequence can be read out, according to the principle of base complementarity, the sequence of template DNA molecules can be deduced.
Chemical degradation method only needs a chemical
reagement
, good repeatability, easy to master, and the double deoxygenation method requires a single chain template, specific oligonucleotide quotations and high-quality DNA polymerase, then with the invention and application of M13 phage vector, synthetic citation easy to obtain, sequencing technology has been improved, so this method has been widely used. The automatic laser fluorescence sequencer of the base deoxygenation method makes the measurement work faster and easier, and ensures a high degree of repeatability. As for RNA sequencing, mRNA is now mostly reversed into cDNA after the same sequencing, and then pushed back the RNA sequence..