Aal. 16900) \ iat sa DNA sequencing DNA ‘sequencing is the process’of tetermining the sequence of nucleotide bases (As, Ts, Cs, and Gsyimapiece of DNA: Today, with the right equipment and materials, sequencing a short piece of DNA js relatively straightforward. Sequencing an entire genome (all of an organism's DNA) remains a complex task. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long "consensus." However, thanks to new methods that have been developed over the past two decades, genome sequencing is now much faster and less expensive than it was during the Human Genome Project. ‘Sanger sequencing: The chain termination method Regions of DNA up to about 900 base called Sanger sequencing or the chain termination method. SaivgerSeqUEhEIng Was developed’ by” the British biochemist Fred Sanger and his colleagues if 1977" length are routinely sequenced using a method In the Human Genome Project, Sanger sequencing was used to determine the sequences of many relatively small fragments of human DNA. (These fragments weren't necessarily 900 bp or less, = 4. but researchers, were able t@ "walk" along each fragment using multiple rounds of Sanger sequencing.) The fragments were aligned based on overlapping portions to assemble the sequences of larger regions of DNA and, eventually, entire chromosomes. Although genomes are now typically sequenced using other methods that are faster and less expensive, Sanger-sequericing is still ii Wide ise Tor the seque DNATSiich as fragments used in DNA” cloning or generated “through polymerase chain. - realiGA(PCR). 2 Of iadividual pieces ofUses and limitations + Samo sequeiGing wives Aighrquatty sequence forrlalively long streicheSer DNATp _EOrabout 900 base pairs) It's typically Used 16 sequence individual pieces ODA, such Es bacterial plasmids or DNA copied in PCR. * Sanger sowencing + BMC tenntsalaasesale-pojst Sera te sequencing of an entire genome or metagenome (the “collective genome” of a microbial os community). For tasks such as these, new, large-scale sequencing techniques are faster and less expensive Ingredients for Sanger sequencing Sanger sequencing involves making many copies of a target DNA region. Its ingredients are similar to those needed for DNA replication in an organism, or for polymerase chain reaction (PCR), which copies DNA in vitro. They include: A DNA polymerase enzyme A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts asa "starter" for the polymerase © The four DNA nucleotides (dATP, dTTP, (CTP, AGTP) goo The template DNA to be sequenced == Sanger sequencing reaction also contains a unique ingredient:Dideoxy, or chain-terminating, versions of all four nucleotides (A4ATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye. The dye molecule on a dideoxy nucleotide is linked to the nitrogenous base. Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one key difference: they lack a hydroxyl group on the 3° carbon of the sugar ring. In a regular nucleotide, the 3° hydroxyl group acts as a “hook," allowing a new nucleotide to be added to an existing chain.Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl available and no further nucleotides can be added, The chain ends with the dideoxy nucleotide, which is marked with a particular color of dye depending on the base (A, T, C or G) that it carries.Procedure of Sanger sequencing a * mixture is first heated to denature the template DNA (separate the son 8A so so seat tena STE that the primer can bind to the single-stranded templat the primer hi temperature is raised again, allowing DNA polymerase to s} new DNA start primer, DNA polymerase will continue adding nucleotides to the chain until it happens to add a ideoxy nucleotide instead of a normal one, At that point, no further nucleotides can be added, so th strand will end with the dideoxy nucleotide. (er the reaction is done, the fragments are run through a long, thin tube containing a gel matrix — a process called capillary gel electrophoresis. Short fragments move quickly through the po EE of the while long fragments move more slowly. As each fragment crosses the “finish line” at the end of the tubevit's illuminated by a laser, allowing the attached dye to be detected. - The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first, followed by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus, from the colors of dyes registered one after another on the detector, the sequence of GD original piece of DNA can be built up one nucleotide at atime. The data recorded by the detectol consist of a series of peaks in fluorescence intensity, as shown in the chromatogram above. The DNA sequence is read from the peaks in the chromatogram. 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