Question

In Sanger Sequencing, which was used to generate the sequence waveform you analyzed, the amount of ddNTPs in the reaction is roughly 1%. What is the % of ddNTPs in the first step of the Next Gen sequencing?

Option 1: 1%
Option 2: 10%
Option 3: 25%
Option 4: 50%

Answer 1

Next Generation Sequencing does not use a fixed percentage of ddNTPs like in Sanger Sequencing; instead, NGS techniques often employ different methods that may not involve chain-terminating nucleotides, making the provided options non-representative of actual NGS protocols.

Step-by-step explanation:

In Next Generation Sequencing (NGS), the proportion of dideoxynucleotides (ddNTPs) used in the initial steps is significantly higher than in Sanger Sequencing. Unlike Sanger's method, NGS does not work with a set percentage of ddNTPs, as the NGS techniques often rely on different methods of DNA synthesis and sequencing by synthesis without chain-terminating nucleotides, or they use reversible terminators that allow the sequencing to continue after detection. Therefore, the options provided do not accurately represent the initial ddNTPs percentage in NGS, as the sequencing chemistry differs markedly.

In Sanger Sequencing, the ddNTPs terminate the DNA strand elongation and their ratio in the reaction mix is approximately 1%. This precise ratio of dNTPs to ddNTPs allows for the generation of DNA fragments of differing lengths for subsequent electrophoretic separation and fluorescence detection. Sanger's method, although accurate, is more time-consuming than NGS technologies, which have revolutionized sequencing by enabling the processing of multiple DNA strands in parallel with different and more advanced techniques.