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DNA
DNA. Source: Unsplash

Traditional DNA Sequencing: The Chain Terminator Method

Until recent years, the most widely used technique for sequencing DNA was the chain-terminator method, which was devised by Frederick Sanger. The first step in this procedure is to obtain single polynucleotide strands. Complementary DNA strands can be separated by heating, which breaks the hydrogen bonds between bases. Next, polynucleotide fragments that terminate at positions corresponding to each of the four nucleotides are generated. Finally, the fragments are separated and detected.

DNA Polymerase Copies a Template Strand

The chain-terminator method (also called the dideoxy method) uses an E. coli enzyme to make complementary copies of the single-stranded DNA being sequenced. The enzym is a fragment of DNA polymerase I, one of the

Action of DNA polymerase I.
Action of DNA polymerase I. source: Fundamentals of Biochemistry,5th edition, Voet.et.al

enzymes that participates in replication of bacterial DNA (Section 25-2A). Using the single DNA strand as a template, DNA polymerase I assembles the four deoxynucleoside triphosphates (dNTPs), dATP, dCTP, dGTP, and dTTP, into a complementary polynucleotide chain that it elongates in the 5′ → 3′ direction.

DNA polymerase I

can sequentially add deoxynucleotides only to the 3′ end of a polynucleotide that is base-paired to the template strand. Replication is initiated in the presence of a short polynucleotide (a primer) that is complementary to the 3′ end of the template DNA, and thus becomes the 5′ end of the newstrand. If the DNA being sequenced is a restriction fragment, as it usually is, it begins and ends with a restriction site. The primer can therefore be a short DNA segment with the sequence of this restriction site.

DNA Synthesis Terminates after Specific Bases

source: Fundamentals of Biochemistry,5th edition, Voet.et.al

In the chain-terminator technique), the DNA to be sequenced is incubated with DNA poly- merase I, a suitable primer, and the four dNTP substrates (reactants in enzymatic reactions) for the polymerization reaction. The key component of the reaction mixture is a small amount of a

2′,3′-dideoxynucleoside triphosphate

(ddNTP), which lacks the 3′-OH group of deoxynucleotides. When the dideoxy analog is incorporated into the growing polynucleotide in place of the corresponding normal nucleotide, chain growth is terminated because addition of the next nucleotide requires a free 3′-OH group.

Automated DNA sequencing by the chain-terminator method.
Automated DNA sequencing by the chain-terminator method. source: Fundamentals of Biochemistry,5th edition, Voet.et.al

By using only a small amount of the ddNTP, a series of truncated chains is generated, each of which ends with the dideoxy analog at one of the positions occupied by the corresponding base. Each ddNTP bears a different fluorescent “tag” so that the products of the polymerase reaction can be readily detected. Gel electrophoresis separates the newly synthesized DNA segments, which differ in size by one nucleotide. Thus, the sequence of the replicated strand can be directly read from the gel. Note that the sequence obtained by the chain-terminator method is complementary to the DNA strand being sequenced. The most advanced sequencing devices that employ the chain-terminator method identify each DNA fragment as it exits the bottom of a capillary electrophoresis tube, so the sequence data take the form of a series of peaks. Sample preparation and data analysis are fully automated, and sequences of DNA (called reads) of up to 1000 nucleotides can be obtained from a single reaction mixture before cumulative errors reduce the confidence of identification to unacceptable levels. Moreover, these systems contain arrays of up to 384 capillary tubes and hence can simultaneously sequence as many as 384 DNA segments.

The many reads that have been sequenced must be correctly assembled into the far longer strand of DNA from which they originated. This is done by comparing reads with overlapping sequences as is schematically indicated in. The overlapping sets of DNA fragments are generated by separately cleaving the DNA with at least two restriction endonucleases that have different sequence specificities. Alternatively, fragments may be generated by

DNA sequence data.
DNA sequence data. source: Fundamentals of Biochemistry,5th edition, Voet.et.al

subjecting a solution of the stiff double-stranded DNA to high-frequency sound waves, a process called sonication, thereby mechanically breaking (shearing) the DNA at random sites. For even relatively small chromosomes (the E. coli genome has 4600 kb; the average human chromosome has ∼125,000 kb), assembly is a highly computationally intensive process. In addition, the chain-terminator method has an error rate of ∼0.1% (which would lead to ∼125,000 mistakes in a 125,000-kb chromosome). To minimize these errors, the DNA must be independently sequenced multiple times—normally with at least a 10-fold redundancy.

About Fahmida Akter Bristi

I am currently doing my Bachelor degree. I love to write by exploring knowledge that is new to me. Hope this effort of mine benefits you all. Right now, I am the head of Project R. Franklin & Project Waksman in Society & Science Foundation. Knock me anytime. Email: fahmidabristi683@gmail.com

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Abulais Shomrat
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Abulais Shomrat
5 months ago

Good job. Want to see more from you.

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