h1>What you need to know about Sequencing
Sequencing is a method used in molecular biology to determine the nucleotide alignment and composition of a DNA or RNA fragment of interest. Alternative protocols, referred to as "Genotyping", are also used to compare genetic specificities for various species or individuals.
Sequencing takes advantage of engineered DNA polymerases to perform primer extension, incorporating nucleotides in the 5'-3' direction while using a denatured single-stranded DNA as template. Along the way, the polymerase randomly incorporates dideoxy nucleotides, which prevent the elongation to go further. This results in a mix of fragments for which the length difference is as low as one nucleotide long. This is enough to observe a variation in the electrophoresis pattern, which in turn can be recorded on an autoradiography film or an automated sequencer, given the fragments have respectively been labeled with radioactivity or fluorescent labels.
Four steps to successful results
Sequencing protocols are usually made of 4 steps:
- Template preparation
- Template purification
- Sequencing reaction
- Sequence analysis
1. Template preparation
Low amount of starting materials for sequencing can be amplified
2. Template purification
3. Sequencing reaction
4. Sequence analysis
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