Editing DNA sequencing (section)

== Basic methods ==

=== Maxam-Gilbert sequencing ===
{{Main|Maxam-Gilbert sequencing}}
[[Allan Maxam]] and [[Walter Gilbert]] published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases.<ref name=Maxam77>{{cite journal | vauthors = Maxam AM, Gilbert W | title = A new method for sequencing DNA | journal = Proc. Natl. Acad. Sci. USA | volume = 74 | issue = 2 | pages = 560–64 | date = February 1977 | pmid = 265521 | pmc = 392330 | doi = 10.1073/pnas.74.2.560 | bibcode = 1977PNAS...74..560M | doi-access = free }}</ref> Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning.  This method's use of radioactive labeling and its technical complexity discouraged extensive use after refinements in the Sanger methods had been made.

Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing [[acrylamide]] gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.<ref name=Maxam77 />

This method is mostly obsolete as of 2023.<ref name="PubMed m584">{{cite web | title=maxam gilbert sequencing | website=PubMed | url=https://pubmed.ncbi.nlm.nih.gov/?term=maxam+gilbert+sequencing&filter=years.2013-2023&sort=pubdate}}</ref>

=== Chain-termination methods ===
{{Main|Sanger sequencing}}
The [[Sanger sequencing|chain-termination method]] developed by [[Frederick Sanger]] and coworkers in 1977 soon became the method of choice, owing to its relative ease and reliability.<ref name="Sanger1977">{{cite journal | vauthors = Sanger F, Nicklen S, Coulson AR | title = DNA sequencing with chain-terminating inhibitors | journal = Proc. Natl. Acad. Sci. USA | volume = 74 | issue = 12 | pages = 5463–77 | date = December 1977 | pmid = 271968 | pmc = 431765 | doi = 10.1073/pnas.74.12.5463 | bibcode = 1977PNAS...74.5463S | doi-access = free }}</ref><ref name=Sanger75>{{cite journal | vauthors = Sanger F, Coulson AR | title = A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase | journal = J. Mol. Biol. | volume = 94 | issue = 3 | pages = 441–48 | date = May 1975 | pmid = 1100841 | doi = 10.1016/0022-2836(75)90213-2 }}</ref> When invented, the chain-terminator method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method.  Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of [[DNA sequencer]]s.

Sanger sequencing is the method which prevailed from the 1980s until the mid-2000s. Over that period, great advances were made in the technique, such as fluorescent labelling, capillary electrophoresis, and general automation. These developments allowed much more efficient sequencing, leading to lower costs. The Sanger method, in mass production form, is the technology which produced the [[Human Genome Project|first human genome]] in 2001, ushering in the age of [[genomics]]. However, later in the decade, radically different approaches reached the market, bringing the cost per genome down from $100 million in 2001 to $10,000 in 2011.<ref>{{cite web |last=Wetterstrand |first=Kris |title=DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) |publisher=[[National Human Genome Research Institute]] |access-date=30 May 2013 |url=https://www.genome.gov/sequencingcosts }}</ref>

=== Sequencing by synthesis ===
The objective for sequential sequencing by synthesis (SBS) is to determine the sequencing of a [[DNA]] sample by detecting the incorporation of a [[nucleotide]] by a [[DNA polymerase]]. An engineered polymerase is used to synthesize a copy of a single strand of DNA and the incorporation of each nucleotide is monitored. The principle of real-time sequencing by synthesis was first described in 1993<ref>{{cite journal |last1=Nyren |first1=P. |last2=Pettersson |first2=B. |last3=Uhlen |first3=M. |title=Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay |journal=Analytical Biochemistry |date=January 1993 |volume=208 |issue=1 |pages=171–175 |doi=10.1006/abio.1993.1024 |pmid=8382019 }}</ref> with improvements published some years later.<ref>{{cite journal |last1=Ronaghi |first1=Mostafa |last2=Uhlén |first2=Mathias |last3=Nyrén |first3=Pål |title=A Sequencing Method Based on Real-Time Pyrophosphate |journal=Science |date=17 July 1998 |volume=281 |issue=5375 |pages=363–365 |doi=10.1126/science.281.5375.363 |pmid=9705713 |s2cid=26331871 }}</ref> The key parts are highly similar for all embodiments of SBS and includes (1) [[Solid phase sequencing|amplification of DNA]] (to enhance the subsequent signal) and attach the DNA to be sequenced to a solid support, (2) generation of single stranded DNA on the solid support, (3) incorporation of nucleotides using an engineered polymerase and (4) real-time detection of the incorporation of nucleotide The steps 3-4 are repeated and the sequence is assembled from the signals obtained in step 4. This principle of real-time sequencing-by-synthesis has been used for almost all [[massive parallel sequencing]] instruments, including [[454 Life Sciences|454]], [[Pacific Biosciences|PacBio]], [[Ion Torrent|IonTorrent]], [[Illumina, Inc.|Illumina]] and [[MGI (company)|MGI]].