Difference between revisions of "Ligation and Sequencing"
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==Sequencing== | ==Sequencing== | ||
Once the DNA sample is purified to remove any remaining nucleotides or enzyme, the circularized DNA can be sent for sequencing by a commercial sequencing service. The primer used to sequence the DNA should be a reverse primer taken from ~200 nt downstream of the 5' end of the original linear starting DNA. The sequencing results should reveal which bases were added and give a rough quantification of addition efficiency. | Once the DNA sample is purified to remove any remaining nucleotides or enzyme, the circularized DNA can be sent for sequencing by a commercial sequencing service. The primer used to sequence the DNA should be a reverse primer taken from ~200 nt downstream of the 5' end of the original linear starting DNA. The sequencing results should reveal which bases were added and give a rough quantification of addition efficiency. | ||
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Latest revision as of 07:41, 18 September 2017
Overview
Ligation and Sequencing is a method to detect single or multiple base addition to a single strand of DNA by Enzymatic Synthesis Methods. This detection method involves sequencing the added bases with a commercial sequencing service. The segment to be sequenced is effectively put in the middle of the starting DNA to avoid poor quality sequencing reads usually encountered at the ends of linear DNA.
Starting DNA
Instead of adding to a DNA oligonucleotide (~15 nt long), the addition will be performed on a single linear DNA strand with a known sequence that is long enough to be circularized (~1 kb). Long single-stranded DNA can be obtained by cutting a single-stranded DNA plasmid with restriction enzymes. Single-stranded DNA cannot be amplified by PCR.
Ligation
Once multiple bases have been added to the linear single strand of DNA, the DNA will be circularized with single-stranded DNA ligase. If the DNA is not circularized, the extended 3' end will be ligated to the 5' end of another linear piece of DNA, resulting in the same desired sequence.
Sequencing
Once the DNA sample is purified to remove any remaining nucleotides or enzyme, the circularized DNA can be sent for sequencing by a commercial sequencing service. The primer used to sequence the DNA should be a reverse primer taken from ~200 nt downstream of the 5' end of the original linear starting DNA. The sequencing results should reveal which bases were added and give a rough quantification of addition efficiency.