Thus far we have seen methods that add a single base per cycle, known as sequencing by synthesis. In contrast, sequencing by ligation uses short segments of DNA called oligonucleotides instead of single bases to sequence DNA. Take a look at the diagram below to see the difference:
Since this is ligation, we use the enzyme DNA ligase rather than DNA polymerase. This enzyme joins together ends of DNA molecules.
Note that ligation is performed in the 3'-5' direction for multiple cycles, which is the opposite of how polymerase works.
Because DNA ligase has a low efficiency when there are mismatches between bases, we can be sure that only the oligonucleotides that match are ligated.
There are five main steps to sequencing by ligation, as outlined below.
A known sequence is flanked onto the target DNA strand. A short anchor sequence is then brought in to bind to this known sequence.
Oligonucleotides are short segments of DNA, and are characterized by a number of features, as outlined below.
The oligonucleotides have either lengths 8 (octamer) or 9 (nonamer).
The oligonucleotides are partially degenerate, meaning that at one of their positions they have a known nucleotide. For example, one oligonucleotide can have a known query position at 1, but unknown positions for 2-9. Another nucleotide can have a known position at 4, but unknown nucleotides at 1-3 and 5-9.
For our example, let's assume we have a nonamer whose known position is at query position 1.
Each oligonucleotide is tagged at the 3' ends with a fluorescent dye. The colors vary depending on the known query position.
The pool of oligonucleotides are mixed in with the target DNA and allowed to hybridize with target DNA sequence.
DNA ligase joins the molecule to the anchor when its bases match the unknown DNA sequence. based on what color light is emitted, we are able to see the nucleotide at the position of the unknown sequence.
The fluorescent labels are cleaved away, regenerating at 5'-phosphate group on the ends of the ligated probes. This will allow the next oligonucleotides to be ligated onto the rest of the unknown sequences.
This process in steps 4 and 5 are repeated until the nonamers have reached the end of the unknown DNA sequence. After this, the anchor sequence is reduced by one nucleotide, and the process is repeated.
A downside to this method is its limitation to short reads, and the time it takes to ligate oligonucleotides on and off. Additionally, there have been problems sequencing palindromic strands.
The positives to this method is that it is easy to implement with off-the-shelf reagents.
Linux for Beginners doesn't make any assumptions about your background or knowledge of Linux. You need no prior knowledge to benefit from this book. You will be guided step by step using a logical and systematic approach. As new concepts, commands, or jargon are encountered they are explained in plain language, making it easy for anyone to understand.$ Check price
This book doesn't make any assumptions about your background or knowledge of Python or computer programming. You will be guided step by step using a logical and systematic approach. As new concepts, commands, or jargon are encountered they are explained in plain language, making it easy for anyone to understand.$ Check price