Sequencing by ligation
Encyclopedia
Sequencing by ligation is a DNA sequencing
method that uses the enzyme DNA ligase
to identify the nucleotide
present at a given position in a DNA sequence. Unlike most currently popular DNA sequencing methods, this method does not use a DNA polymerase to create a second strand. Instead, the mismatch sensitivity of a DNA ligase enzyme is used to determine the underlying sequence of the target DNA molecule.
that joins together ends of DNA molecules. Although commonly represented as joining two pairs of ends at once, as in the ligation of restriction enzyme
fragments, ligase can also join the ends on only one of the two strands (for example, when the other strand is already continuous or lacks a terminal phosphate
necessary for ligation). DNA ligase is sensitive to the structure of DNA and has very low efficiency when there are mismatches between the bases of the two strands.
Sequencing by ligation relies upon the sensitivity of DNA ligase for base-pairing mismatches. The target molecule to be sequenced is a single strand of unknown DNA sequence, flanked on at least one end by a known sequence. A short "anchor" strand is brought in to bind the known sequence.
A mixed pool of probe oligonucleotide
s is then brought in (eight or nine bases long), labeled (typically with fluorescent dyes) according to the position that will be sequenced. These molecules hybridize to the target DNA sequence, next to the anchor sequence, and DNA ligase preferentially joins the molecule to the anchor when its bases match the unknown DNA sequence. Based on the fluorescence produced by the molecule, one can infer the identity of the nucleotide at this position in the unknown sequence.
The oligonucleotide probes may also be constructed with cleavable linkages which can be cleaved after identifying the label. This will both remove the label and regenerate a 5' phosphate on the end of the ligated probe, preparing the system for another round of ligation. This cycle can be repeated several times to read longer sequences. This sequences every Nth base, where N is the length of the probe left behind after cleavage.
To sequence the skipped positions, the anchor and ligated oligonucleotides may be stripped off the target DNA sequence, and another round of sequencing by ligation started with an anchor one or more bases shorter.
A simpler, albeit more limited, technique is to do repeated rounds of a single ligation where the label corresponds to different position in the probe, followed by stripping the anchor and ligated probe.
Sequencing by ligation can proceed in either direction (either 5'-3' or 3'-5') depending on which end of the probe oligonucleotides are blocked by the label. The 3'-5' direction is more efficient for doing multiple cycles of ligation. Note that this is the opposite direction to polymerase based sequencing methods.
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
method that uses the enzyme DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
to identify the nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
present at a given position in a DNA sequence. Unlike most currently popular DNA sequencing methods, this method does not use a DNA polymerase to create a second strand. Instead, the mismatch sensitivity of a DNA ligase enzyme is used to determine the underlying sequence of the target DNA molecule.
Process
DNA ligase is an enzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that joins together ends of DNA molecules. Although commonly represented as joining two pairs of ends at once, as in the ligation of restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
fragments, ligase can also join the ends on only one of the two strands (for example, when the other strand is already continuous or lacks a terminal phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
necessary for ligation). DNA ligase is sensitive to the structure of DNA and has very low efficiency when there are mismatches between the bases of the two strands.
Sequencing by ligation relies upon the sensitivity of DNA ligase for base-pairing mismatches. The target molecule to be sequenced is a single strand of unknown DNA sequence, flanked on at least one end by a known sequence. A short "anchor" strand is brought in to bind the known sequence.
A mixed pool of probe oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s is then brought in (eight or nine bases long), labeled (typically with fluorescent dyes) according to the position that will be sequenced. These molecules hybridize to the target DNA sequence, next to the anchor sequence, and DNA ligase preferentially joins the molecule to the anchor when its bases match the unknown DNA sequence. Based on the fluorescence produced by the molecule, one can infer the identity of the nucleotide at this position in the unknown sequence.
The oligonucleotide probes may also be constructed with cleavable linkages which can be cleaved after identifying the label. This will both remove the label and regenerate a 5' phosphate on the end of the ligated probe, preparing the system for another round of ligation. This cycle can be repeated several times to read longer sequences. This sequences every Nth base, where N is the length of the probe left behind after cleavage.
To sequence the skipped positions, the anchor and ligated oligonucleotides may be stripped off the target DNA sequence, and another round of sequencing by ligation started with an anchor one or more bases shorter.
A simpler, albeit more limited, technique is to do repeated rounds of a single ligation where the label corresponds to different position in the probe, followed by stripping the anchor and ligated probe.
Sequencing by ligation can proceed in either direction (either 5'-3' or 3'-5') depending on which end of the probe oligonucleotides are blocked by the label. The 3'-5' direction is more efficient for doing multiple cycles of ligation. Note that this is the opposite direction to polymerase based sequencing methods.