A primer used in dideoxy DNA sequencing is 20 nucleotides long and has the following sequence: 5’-GGATCCATGACTAGTCCGAC-3’. A segment of DNA is cloned into a vector and then the vector DNA is denatured and subjected to dideoxy DNA sequencing method. Below is the DNA sequence from a region of the vector. The primer-annealing site is shown in bold and underlined.

3-CCCGATCGGCCTAGGTACTGATCAGGCTGAATGACT-5’

Based on the sequence above, what would be the size(s) of the band(s) (i.e., the number of nucleotides in each band) in which dideoxyT had been added to the sequencing reaction?

Question

A primer used in dideoxy DNA sequencing is 20 nucleotides long and has the following sequence:  5’-GGATCCATGACTAGTCCGAC-3’. A segment of DNA is cloned into a vector and then the vector DNA is denatured and subjected to dideoxy DNA sequencing method. Below is the DNA sequence from a region of the vector. The primer-annealing site is shown in bold and underlined.

3-CCCGATCGGCCTAGGTACTGATCAGGCTGAATGACT-5’

Based on the sequence above, what would be the size(s) of the band(s) (i.e., the number of nucleotides in each band) in which dideoxyT had been added to the sequencing reaction?

BlackTom AI · Accepted Answer

ddT (ddTTP) causes chain termination when a thymidine would be incorporated into the growing strand — that is, when the polymerase encounters an adenine (A) on the template (because T pairs with A).

1) Relevant knowledge points
- In Sanger sequencing a labeled primer is extended from its 3′ end; fragment length on the gel = primer length + number of nucleotides added before termination (including the terminating ddNTP).
- ddT terminates when a T would be added, i.e., opposite an A in the template.
- The primer sequence (5′→3′) anneals antiparallel and complementary to the indicated template region; extension proceeds from the primer 3′ end into the nearby downstream template bases.

2) Alignment and reasoning (step by step)
- The primer is 20 nt long (5′-GGATCCATGACTAGTCCGAC-3′). The given template strand (written 3′→5′) contains the complementary, underlined annealing site so the primer’s 3′ end is positioned immediately adjacent to the first base to be copied.
- From that primer 3′ end, the polymerase will add bases complementary to the template bases that follow. Scanning the template bases immediately downstream of the primer, the adenines that will pair with incorporated thymidines appear at the 3rd and 7th positions from the primer 3′ end (i.e., the 3rd and 7th bases to be added).

3) Calculation of fragment sizes (all intermediate steps)
- Primer length = 20 nt.
- If ddT causes termination at the 3rd added base: fragment length = 20 + 3 = 23 nt.
- If ddT causes termination at the 7th added base: fragment length = 20 + 7 = 27 nt.

4) Why the correct answer is correct
- Because ddT termination events correspond exactly to incorporation opposite template A residues found at the 3rd and 7th positions downstream of the primer 3′ end, the Sanger ddT lane will show two bands whose lengths equal primer (20) plus 3 and plus 7 bases: 23 and 27 nt.

5) Why the other options are incorrect
- "1, 2, and 5": would require As at the 1st, 2nd and 5th positions downstream of the primer — not the pattern present.
- "21, 22, and 25": corresponds to terminations after 1, 2, and 5 added bases (20+1, +2, +5); those distances do not match the positions of As relative to the primer 3′ end.
- "3 and 7": (this option alone is not in the list as numeric fragments) would be counts of added bases, not fragment sizes; the correct reported fragment sizes are 23 and 27.

6) Conclusion / final confirmation
- Therefore the ddT sequencing lane will produce bands of 23 nt and 27 nt — confirming the provided correct answer: 23 and 27.

类似问题

2. How do dideoxynucleotide triphosphates (diNTPs) in the Sanger method facilitate DNA sequencing?

What is the role of primers in Sanger sequencing?

Dideoxynucleotide triphosphates (ddNTPs) are used in manual Sanger sequencing and in many automated methods of sequencing. What is the purpose of using dideoxynucleotides in DNA sequencing? Select ALL that apply. The figure below may be helpful.

Dideoxynucleotide triphosphates (ddNTPs) are used in manual Sanger sequencing and in many automated methods of sequencing. What is the purpose of using dideoxynucleotides in DNA sequencing? Select ALL that apply. The figure below may be helpful.

True or False? The Sanger method of DNA sequencing requires chemical modification of DNA and subsequent cleavage of nucleotide bases to facilitate chain termination.

更多留学生实用工具

智能学习助手

风格化写作助手

论文查重助手

文献引用助手

课堂转译助手

课堂笔记助手

Quiz搜索助手

学校历年真题

智能刷题助手

智能匹配练习

希望你的学习变得更简单