Question

Next-Generation Sequencing In reversible terminator sequencing, how would the sequencing process be affected if the \(3^{\prime}\)-end-blocking group of each nucleotide were replaced with the \(3^{\prime}\)-H present in the dideoxynucleotides used in Sanger sequencing?

Short answer

Replacing the blocking group with the 3'-H of ddNTPs would cause permanent termination, stopping sequencing after the first addition.

Step-by-step solution

Understand the Role of the Blocking Group

In reversible terminator sequencing, each nucleotide has a removable blocking group at the 3'-end. This group prevents further extension of the DNA strand, allowing the addition of only one nucleotide at a time. This controlled addition is essential for sequencing accuracy.

Compare With Sanger Sequencing

In Sanger sequencing, dideoxynucleotides (ddNTPs) are used to terminate DNA synthesis. These nucleotides lack a 3'-OH group, having a 3'-H instead, preventing further nucleotides from being added.

Analyze the Impact of Replacing the Blocking Group

If the reversible terminator sequencing process used ddNTPs with a 3'-H in place of the usual blocking group, DNA synthesis would permanently terminate. This is because the lack of a 3'-OH cannot be reversed, stopping the sequencing after the first nucleotide addition.

Explain the Consequences for Sequencing

Using ddNTPs would make further extension impossible after including the first nucleotide, disrupting the sequential addition process critical for high-throughput sequencing. This would make it impossible to read the entire sequence, as the chain would prematurely terminate at each incorporation point.

Key concepts

Reversible Terminator Sequencing

Reversible terminator sequencing is an advanced method used to determine the sequence of nucleotides in a DNA sample. This technique relies on special nucleotides, each tagged with a fluorescent label and a removable blocking group at the 3'-end. The key feature here is the use of a reversible blocking group, which temporarily halts the extension of the DNA strand.
This allows for the controlled addition of a single nucleotide at a time during sequencing.

• Each nucleotide added is detected by its unique fluorescent signal.
• The blocking group is then removed, permitting the next nucleotide to be added.
• This process repeats until the entire DNA sequence is determined.

Without the reversible nature of these blocking groups, as mentioned in the exercise, sequencing cannot proceed beyond the initial nucleotide addition.

Sanger Sequencing

Sanger sequencing, introduced by Frederick Sanger in the 1970s, is a foundational DNA sequencing method. It uses dideoxynucleotides (ddNTPs) that terminate DNA chain elongation. These ddNTPs are instrumental due to their lack of a 3'-OH group, replaced with a 3'-H.
This modification prevents further nucleotide incorporation, effectively marking the end of the sequence at that point.

• The process begins with strand synthesis using regular nucleotides (dNTPs) as well as a small proportion of ddNTPs.
• When a ddNTP is incorporated, the chain terminates.
• By running these DNA fragments through gel electrophoresis, the order of nucleotides can be determined based on fragment length.

This method, while slower and requiring more input material compared to modern techniques, provides precise sequence data beneficial for certain applications.

Dideoxynucleotides

Dideoxynucleotides are a critical component in Sanger sequencing due to their unique structure. Unlike regular deoxynucleotides, dideoxynucleotides are missing both a hydroxyl group at the 2'- and 3'-positions. This absence is what makes them so effective as chain terminators in the sequencing process, bringing the DNA strand synthesis to a halt.
Another difference lies in their ability to act as selective markers during sequencing.

• Each type of ddNTP (A, T, C, G) is labeled with a different fluorescent dye.
• This enables the identification of the terminating base at each position of the DNA strand.
• Once incorporated, the absence of a 3' hydroxyl group ensures no further nucleotide can be added, highlighting the sequence stops.

Their use in Sanger sequencing illustrates how small molecular changes can significantly influence DNA sequencing technologies.