15. The fragments obtained from a Sanger sequencing experiment are as follows:
5´ – GAATTA – 3´
5´ – GAATTAT – 3´
5´ – GAATTATC – 3´
5´ – GAATTATCA – 3´
5´ – GAATTATCAC – 3´
Please identify the template sequence from the above given data:
Options:-
(a) 3´ – CTTAATAGTG – 5´,
(b) 3´ – GAATTATCAC – 5´,
(c) 5´ – CACTATTAAG – 3´ ,
(d) 5´ – GAATTATCAC – 3´,

Introduction to Sanger Sequencing

Sanger sequencing, also known as chain-termination sequencing, is a method used to determine the nucleotide sequence of DNA. The process involves the generation of DNA fragments of varying lengths, which are then analyzed to infer the sequence of the template strand. In this article, we will demonstrate how to identify the template sequence given a series of Sanger sequencing fragments.

The Sanger Sequencing Data

The fragments obtained from the Sanger sequencing experiment are:

1. 5´ – GAATTA – 3´

2. 5´ – GAATTAT – 3´

3. 5´ – GAATTATC – 3´

4. 5´ – GAATTATCA – 3´

5. 5´ – GAATTATCAC – 3´

These fragments represent the 3´ ends of the synthesized strands, each of which is complementary to the template DNA.

Understanding the Process

Sanger sequencing uses a method in which the DNA template is copied by incorporating modified nucleotides. These fragments represent sequences that were synthesized from the template, where the chain is terminated at specific bases. The challenge here is to identify the correct template strand, which serves as the source for these fragments.

The key is to understand that the fragments reflect the complementary sequence of the template strand. Thus, we must determine the complementary template sequence to match these fragments.

How to Determine the Template Sequence

To identify the template sequence, you can follow these steps:

1. Identify the end of the sequence: Since these are 3´ ends of fragments, we need to write the complementary bases for the 5´ to 3´ direction of the template strand.

2. Compare the fragments: The fragments share a common sequence that can help deduce the continuous template strand.

Breaking Down the Fragments

Let’s break down the first fragment, “GAATTA”:

• The complementary base pairs would be:

  • G (guanine) pairs with C (cytosine)

  • A (adenine) pairs with T (thymine)

  • T (thymine) pairs with A (adenine)

  • The sequence from the template strand for this would be “CTTAAT.”

Continuing this logic for the other fragments:

• “GAATTAT” would pair with “CTTAATA.”

• “GAATTATC” would pair with “CTTAATG.”

• “GAATTATCA” would pair with “CTTAATGA.”

• “GAATTATCAC” would pair with “CTTAATGAC.”

Answer to the Question

From the options provided, the correct template sequence is the one that complements the longest fragment sequence, which is GAATTATCAC.

• The corresponding template sequence for this is 3´ – GAATTATCAC – 5´.

Conclusion

The template sequence corresponding to the Sanger sequencing fragments provided is 3´ – GAATTATCAC – 5´. Understanding the process of Sanger sequencing and interpreting fragment sequences correctly allows you to identify the original template strand with accuracy.

Key Takeaway: To identify the template sequence in Sanger sequencing, it’s crucial to reverse complement the given fragment sequences and match them to the correct strand.