Question

The X-ray autoradiogram of one strand of a fragment of DNA sequenced by the Sanger method was obtained. The 5 '-cnd of the nucleotide was labeled with \(^{32}\) P. Numbering from bottom to top of the autoradiogram, the lane from the ddG tube showed bands at positions 4 and \(11 ;\) ddC tube had bands at \(1,2,7,8,9,\) and \(13 ;\) ddA at \(5,10,\) and \(15 ;\) and \(\mathrm{dd} \mathrm{d} \mathrm{T}\) at \(3,6,12,\) and \(14 .\) What is the sequence of the fragment? Construct the autoradiogram pattern of the complementary sequence.

Short answer

Answer: The DNA sequence of the fragment is CCTGATCCCCAGTCTA. The pattern of the autoradiogram for its complementary sequence is as follows: - ddG tube: positions 1, 2, 7, 11 - ddC tube: positions 3, 9, 13 - ddA tube: positions 4, 10, 14 - ddT tube: positions 5, 6, 8, 12, 15

Step-by-step solution

Identify the bands corresponding to each nucleotide

Based on the positions given in the problem, we can list the bands for each ddNTP as follows: - ddG: positions 4, 11 - ddC: positions 1, 2, 7, 8, 9, 13 - ddA: positions 5, 10, 15 - ddT: positions 3, 6, 12, 14

Determine the DNA sequence

Starting with the lowest position, we identify which nucleotide is in the corresponding band and write down the sequence step-by-step. 1. ddC 2. ddC 3. ddT 4. ddG 5. ddA 6. ddT 7. ddC 8. ddC 9. ddC 10. ddA 11. ddG 12. ddT 13. ddC 14. ddT 15. ddA The sequence of the fragment is: CCTGATCCCCAGTCTA

Determine the complementary sequence

Now, we will find the complementary sequence by switching the letters of the original sequence with their complementary base pairs: - A <-> T - C <-> G The complementary sequence is: GGCATAGGGGTCAGAT

Construct the autoradiogram pattern of the complementary sequence

Using the complementary sequence, we will construct the pattern of autoradiogram for the complementary sequence on a new gel: - ddG: GGCATAGGGGTCAGAT -> positions: 1, 2, 7, 11 - ddC: GGCATAGGGGTCAGAT -> positions: 3, 9, 13 - ddA: GGCATAGGGGTCAGAT -> positions: 4, 10, 14 - ddT: GGCATAGGGGTCAGAT -> positions: 5, 6, 8, 12, 15 The autoradiogram pattern of the complementary sequence is: - ddG tube: positions 1, 2, 7, 11 - ddC tube: positions 3, 9, 13 - ddA tube: positions 4, 10, 14 - ddT tube: positions 5, 6, 8, 12, 15

Key concepts

Sanger method

The Sanger method, also known as chain termination method, is a technique used for DNA sequencing developed by Frederick Sanger. This method involves the synthesis of new DNA strands from a single-stranded template. During the synthesis, a mixture of normal deoxynucleotide triphosphates (dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs) is used.
Each ddNTP lacks a hydroxyl group at the 3' end, preventing further extension of the DNA strand once it is incorporated. The inclusion of all four ddNTPs (ddATP, ddTTP, ddCTP, ddGTP) in separate reactions results in DNA fragments of varying lengths, each ending with the respective nucleotide. The DNA fragments are then separated by capillary electrophoresis, with shorter fragments moving faster, creating a sequence of bands that correspond to the DNA sequence.

Autoradiogram

An autoradiogram is a physical representation of radioactive decay used for visualizing the DNA sequencing results obtained from the Sanger method. In the context of DNA sequencing, radioactive or fluorescent labels are attached to the ddNTPs. After the fragments are separated by electrophoresis, the bands are visualized on an X-ray film for radioactive labels or by a laser for fluorescent labels.
The pattern of bands is a direct reflection of the DNA sequence, with the smallest fragments (and thus earliest terminating ddNTPs) appearing at the bottom of the autoradiogram and the longest at the top. Autoradiography provides a snapshot that can be analyzed to determine the nucleotide sequence of the DNA.

Nucleotide sequence analysis

Nucleotide sequence analysis refers to the examination and interpretation of the DNA sequence data acquired from methods such as the Sanger method. This analysis involves identifying the order of the nucleotides - adenine (A), guanine (G), cytosine (C), and thymine (T) - which are the building blocks of DNA.
By examining the autoradiogram, scientists can determine the original DNA sequence. By reading the pattern of bands from the bottom to the top (representing the sequence from the 5' to 3' end of the synthesized strand), the sequence can be constructed nucleotide by nucleotide. This sequence information is crucial for understanding genetic information, diagnosing genetic disorders, and advancing genetic research.

Complementary DNA sequence

Complementary DNA sequence refers to the sequence of nucleotides that pairs with the given DNA strand following the base pairing rules: adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
In DNA sequencing, once the sequence of one strand is determined, the sequence of the complementary strand can be predicted because of this pairing rule. The process of reverse complementation is crucial for various molecular biology applications, including cloning and polymerase chain reaction (PCR) where understanding of the complementary sequence is necessary. The complementary sequence is also of great importance in predicting the autoradiogram pattern of the opposite strand during DNA sequencing analysis.