DNA SEQUENCING

BY: SREE LAKSHMI (MSIWM012)

Maxam-Gilbert sequence:

In 1976-1977, Allan Maxam and Walter Gilbert developed DNA sequences based on chemical reactions and subsequent purification. It depends on the associated chemical bond of different nucleotide bonds. Chemical degradation is also known. Chain jumping method is the most widely used method due to its speed and ease. This process requires the installation of radiation labels on one side and the cleaning of the DNA fragment to be followed. The following chemical treatments are:

• G reaction: Dimethyl Sulfate + Piperidine treatment

• A + G Response: Dimethyl Sulfate + Piperidine + Formic Acid Treatment

• T + C reaction: Hydrazine + Piperidine

• C reaction: Hydrazine + Piperidine + 1.5M Sodium Chloride

• Dimethyl sulfate attacks the purine ring (A & G)

• Attack of Hydrazine ring pyrimidine (C&T)

• Piperidine restores the binding capacity of the phosphodiester where the base is removed

 Chemical therapy creates breaks with a small amount of one or two four nucleotides based on each of the four reactions (G, A + G, C, C + T). So a series of labelled pieces were made, from the end of the radio beams to the first ‘cut’ site in each molecule. The four reaction fragments were arranged separately on the gel electrophoresis for size separation. These fragments are shown using a gel shown on an X-ray film of autoradiography showing a series of black bands attached to each radiolabelled DNA fragment, respectively.

Benefits

• Pure DNA can be read directly
• Homopolymeric DNA run is best followed as a unique DNA sequence
• It can be used to analyze the interaction of DNA proteins
• Can be used to analyze the formation of nucleic acids and epigenetic changes in DNA

Disadvantages:

• Requires extensive use of hazardous chemicals.
• He has advanced technology.
• It is difficult to “grow” and cannot be used to analyze more than 500 basic pairs.
• Learn reading length decreases from incomplete response to completion.
• It is difficult to make Maxam-Gilbert’s DNA kits based on sequence.

Sanger sequence

Also known as chain termination method or video sequencing method. The terminator process or video sequencing process of DNA sequences in two layers of DNA polymerases:

• Their ability to faithfully integrate a complimentary copy of a single-tailed DNA template.
• Their ability to use 2 ‘, 3’-dideoxynucleotide as substrates

 When the analog is embedded in a growing DNA sequence, the 3 ‘end has no hydroxyl group and is no longer a substrate for chain expansion. Thus, the growing DNA sequence is broken, meaning that dideoxynucleotide acts as terminators. In practice, the Klenow piece of DNA polymerase is used because this does not have 5 ‘→ 3’ remission functions associated with an incomplete enzyme. DNA synthesis regeneration needs to be done first and this is usually the chemical oligonucleotide bound to the sequence of analyzes. dideoxynucleoside triphosphate. Thus, in each reaction there are fragments of active DNA fragments that are slightly divided, each with the same end of 5, but each varies in length to the end of a particular 3. After a good incubation period, the DNA in each structure was shown to be electronically converted into a successive gel. The terminator chain method works very well and uses fewer toxic chemicals and a lower amount of radioactivity than the Maxam and Gilbert method. The main purpose of the Sanger method was to use dideoxynucleotide triphosphates (ddNTPs) as breakers in DNA chains.

WORKING

This chain-breaking process requires a single-stranded DNA template, DNA primer, DNA polymerase, radiotide or shiny nucleotides, and modified nucleotides that complete the DNA strand expansion. The DNA samples was divided into four sequences, which included all four deoxynucleotides dATP, dGTP, dCTP, dTTP and also DNA polymerase. In each reaction only dideoxynucleotide (ddATP, ddGTP, ddCTP, and ddTTP) is added to the nucleotide terminals, lacking the 3′-OH group needed to form a phosphodiester bond between the two nucleotides, thus ending DNA expansion strand and lead to DNA fragments of various lengths. Freshly formed and transcribed DNA fragments are burned, and measured with gel electrophoresis in a gel that explains the polyacrylamide-urea gel for each of these four processes is performed in one of four ways (lines A, T, G, C). DNA strands are then detected by autoradiography or UV light, and DNA sequences can be read directly from an X-ray film or gel image. The black band on the track shows a DNA strip that leads to the removal of chains after the introduction of dideoxynucleotide (ddATP, ddGP, ddCTP, or ddTTP). chain fragmentation involves the marking of nucleotides containing radios with a phosphorus label, or the use of a labeled primer at the end of 5 in a fluorescent color.Dye-primer sequences help optical system study to speed up analysis and cost.